Robust antibody and CD8+ T-cell responses induced by P. falciparum CSP adsorbed to cationic liposomal adjuvant CAF09 confer sterilizing immunity against experimental rodent malaria infection

Abstract

Despite several decades of extensive research, the development of a highly efficacious malaria vaccine has yet to be accomplished. While the RTS,S malaria vaccine candidate shows the potential to prevent a substantial number of clinical malaria cases, significant improvements in protective efficacy are still needed. Multiple studies have shown that RTS,S induces protective antibody and CD4⁺ T-cell responses, but limited or negligible CD8⁺ T cells. In this study, we evaluated the immunogenicity and protective capacity of full-length recombinant P. falciparum circumsporozoite protein (CSP) administered with the novel cationic liposomal adjuvant system CAF09. Using newly developed transgenic rodent malaria parasites expressing the full-length P. falciparum CSP, we demonstrate that this liposome-based protein-in-adjuvant formulation is capable of inducing robust antibody and CD8⁺ T-cell responses that strongly inhibit parasite infection and development of liver stages, conferring durable sterilizing immunity. These findings underscore the potential of liposome-based adjuvants for inducing robust humoral and CD8⁺ T-cell responses and warrant further studies toward the development of novel subunit vaccine formulations with this adjuvant system.

Keywords: CD8+ T cells; P. falciparum; adjuvant; antibodies; circumsporozoite protein; malaria; vaccine.

Fig. 1

IgG titers against the N-terminal region, repeat region and C-terminal region of P. falciparum CSP. Mice immunized two or three times (n = 12 per group) with Pf rCSP-CAF09 developed comparable antibody titers against the P. falciparum CSP N-terminal peptide (left) and the repeat region peptide (center). However, three immunizations resulted in significantly higher titers against the C-terminal region (right). Titers are reported as the corresponding sera dilution at which OD₄₀₅ was 1.0. Horizontal lines represent the geometric mean titer of the group. Abbreviations: ns, not significant; Pf rCSP, P. falciparum recombinant circumsporozoite protein; OD, optical density

Fig. 2

Antigen-specific CD8⁺ T-cell responses induced by Pf rCSP-CAF09 immunizations. CD8⁺ T-cell responses were assessed in spleens of mice 10 days after the last immunization with either two or three doses of Pf rCSP-CAF09 (n = 3 per group). Cellular responses were compared to those induced by a prime-boost regimen using recombinant influenza (FluPf) and vaccinia (VacPf) viruses expressing selected sequences of the P. falciparum CSP (n = 3). Mice immunized with CAF09 (3×) alone did not produce measurable T-cell responses. The production of IFN-γ by CD8⁺ T cells was assessed in ex vivo stimulation assays using LM1 cells pulsed with the synthetic peptide DYENDIEKKI. Bars represent means ± SEM. Abbreviations: ns, not significant; IFN-γ, interferon-gamma; SEM, standard error of the mean

Fig. 3

P.b.-P.f. CSP-FL CD8CT parasite liver burdens in mice immunized with Pf rCSP-CAF09. Mice immunized a two times or b three times with Pf rCSP-CAF09 significantly inhibited sporozoite infection compared to adjuvant-only (CAF09) or naïve controls. Geometric mean with 95% confidence interval; n = 6 per group. Abbreviations: Pf rCSP-CAF09, P. falciparum recombinant circumsporozoite protein in adjuvant CAF09; P.b.-P.f. CSP-FL CD8CT, P. berghei–P. falciparum CSP full-length CD8 epitope C-terminus transgenic parasite; rRNA, ribosomal RNA; RT-qPCR reverse-transcription quantitative real-time polymerase chain reaction

Fig. 4

Assessment of sterile protection in Pf rCSP-CAF09-immunized mice against sporozoite infection. a Mice immunized three times with Pf rCSP-CAF09 were challenged with P.b.-P.f. CSP-FL CD8CT chimeric sporozoites delivered by five infectious mosquito bites. Four days after challenge, daily blood smears were taken and analyzed under a microscope until day 14. b Nine out of ten mice (90%) developed sterile immunity when challenged 2 weeks after the last immunization (n = 10). c Five weeks later, the nine mice that were sterilely protected in b were challenged again, with none of the animals developing blood-stage parasitemia. Kaplan–Meier plots show the time to detection of parasites in the blood for each group after the challenge (n = 10 for Naïve group). Data were analyzed for statistical significance using Log-rank (Mantel-Cox) test **P < 0.01; ****P < 0.0001. d Antibody titers against different regions of CSP were evaluated in mice immunized three times with Pf rCSP-CAF09 before and after sporozoite challenge (Test bleed 1 and Test bleed 2, respectively). IgG levels against the N-terminal region, repeat region and C-terminal region of CSP did not change significantly in sterilely protected mice during the 5-week interval. Titers are reported as the corresponding sera dilution at which OD405 was 1.0 and compared for significance using a paired Student’s t-test; n = 9. Abbreviations: ns, not significant; Pf rCSP-CAF09, P. falciparum recombinant circumsporozoite protein in adjuvant CAF09; OD, optical density

Fig. 5

P.b.-P.f. CD8CT transgenic sporozoite liver infection in T-cell depleted Pf rCSP-CAF09-immunized mice. Mice immunized three times with Pf rCSP-CAF09 had a dramatically lower parasite liver burden than those vaccinated with adjuvant only (CAF09 3×). The depletion of CD8⁺ T cells significantly impaired their capacity to inhibit parasite liver stages compared to mice treated with an isotype control antibody or with a CD4⁺ T-cell depleting antibody. Geometric mean with 95% confidence interval; n = 6/group. Abbreviations: Pf rCSP-CAF09 P. falciparum recombinant circumsporozoite protein in adjuvant CAF09; P.b.-P.f. CSP-FL CD8CT, P. berghei–P. falciparum CSP full-length CD8 epitope C-terminus transgenic parasite; rRNA, ribosomal RNA; RT-qPCR reverse-transcription quantitative real-time polymerase chain reaction; α-CD4 mAb, anti-CD4 monoclonal antibody; α-CD8 mAb anti-CD8 monoclonal antibody