Poly I:C elicits broader and stronger humoral and cellular responses to a Plasmodium vivax circumsporozoite protein malaria vaccine than Alhydrogel in mice

Abstract

Malaria remains a global health challenge, necessitating the development of effective vaccines. The RTS,S vaccination prevents Plasmodium falciparum (Pf) malaria but is ineffective against Plasmodium vivax (Pv) disease. Herein, we evaluated the murine immunogenicity of a recombinant PvCSP incorporating prevalent polymorphisms, adjuvanted with Alhydrogel or Poly I:C. Both formulations induced prolonged IgG responses, with IgG1 dominance by the Alhydrogel group and high titers of all IgG isotypes by the Poly I:C counterpart. Poly I:C-adjuvanted vaccination increased splenic plasma cells, terminally-differentiated memory cells (MBCs), and precursors relative to the Alhydrogel-combined immunization. Splenic B-cells from Poly I:C-vaccinated mice revealed an antibody-secreting cell- and MBC-differentiating gene expression profile. Biological processes such as antibody folding and secretion were highlighted by the Poly I:C-adjuvanted vaccination. These findings underscore the potential of Poly I:C to strengthen immune responses against Pv malaria.

Keywords: TLR3-ligand; adjuvant; antibody-secreting cells; malaria; memory B cells.

Figure 1

Adjuvanted-malaria vaccine specific to P. vivax circumsporozoite protein elicits long-lasting IgG responses in mice. (A) Outline of the blood draws and intramuscular vaccination with the recombinant yPvCSP-All_CT epitopes protein combined with Poly I:C (n=6) or Alhydrogel (n=6). (B) IgG titers specific to the vaccine antigen were measured before, during and after vaccination in plasma samples through ELISA. Dots and columns represent individual values detected for each mouse and median, respectively. Red and blue colors indicate animals immunized with yPvCSP-All_CT epitopes + Poly I:C or Alhydrogel, respectively. *p<0.05; **p<0.01; ****p<0.0005.

Figure 2

Poly I:C-adjuvanted malaria vaccine triggers a broader isotypic diversification of IgG responses via the intramuscular route in comparison to the Alhydrogel counterpart. Mice were immunized via intramuscular with the recombinant yPvCSP-All_CT epitopes protein combined with Poly I:C (n=6) or Alhydrogel (n=6). (A) IgG1; (B) IgG2b; (C) IgG2c; (D) IgG3) titers specific to the vaccine antigen were measured before, during and after vaccination in plasma samples through ELISA. Red and blue colors indicate animals immunized with yPvCSP-All_CT epitopes + Poly I:C or Alhydrogel, respectively. Dots and columns represent individual values detected for each mouse and median, respectively. *p<0.05; **p<0.01; ****p<0.0005.

Figure 3

Poly I:C-adjuvanted malaria vaccine induces a more potent antibody-secreting cell response in the mouse spleen via the intramuscular route than the Alhydrogel counterpart. (A) Outline of intramuscular vaccination with the recombinant yPvCSP-All_CT epitopes protein combined with Poly I:C (red - n=9) or Alhydrogel (blue - n=9) and tissue sampling (n=3 per group per time point). (B) Sequential gating strategy to enumerate plasma cells (PCs) through flow cytometry. (C) Percentage and (D) absolute number of splenic PCs at different time points upon vaccination through flow cytometry. (E) Representative images of the ELISPOT results for PvCSP-specific IgG-secreting cells at day 10 of the third vaccine dose (left panel). The numbers on top of each image indicate the quantity of spot-forming cells enumerated per well plated with 0.66 × 106 mouse splenocytes. (F) Magnitude of PvCSP-specific IgG-secreting cells per spleen of immunized mice (right panel). Dots and bars represent the totality of splenic PvCSP-specific IgG-secreting cells individually detected for each mouse and median, respectively. *p<0.05

Figure 4

Poly I:C-adjuvanted malaria vaccine induces a stronger memory B-cell response via the intramuscular route relative to the Alhydrogel counterpart. (A) Sequential gating strategy to enumerate follicular B cells and memory B cells through flow cytometry. Percentage (B, D) absolute number of cells (C, E) detected in the spleen of mice at different time points upon vaccination through flow cytometry. Red and blue colors indicate animals immunized with yPvCSP-All_CT epitopes + Poly I:C or Alhydrogel, respectively. Dots and columns represent individual values detected for each mouse and median, respectively. * p<0.05

Figure 5

Poly I:C-adjuvanted malaria vaccine elicits modifications in the transcriptome of splenic B cells, enhancing their differentiation into antibody-secreting or/and memory B cells. (A) Subcutaneous immunization with yPvCSP-All_CT epitopes + Poly I:C, yNLP-PvCSP_CT + Poly I:C, or Poly I:C alone, number of doses and their intervals, and euthanasia time for spleen excision, B-cell isolation and freezing for further RNA extraction. (B) Log fold-change (FC) of differential expressed genes (DEGs) exclusively induced by the yPvCSP-All_CT epitopes + Poly I:C vaccination or mutually induced by yPvCSP-All_CT epitopes + Poly I:C and one of the remaining immunizations. (C) Number of DEGs exclusively detected in splenic B cells of mice vaccinated with yPvCSP-All_CT epitopes + Poly I:C. (D) LogFC of DEGs associated with B-cell differentiation into antibody-secreting cells (PB/PC) or memory B cells (MBC) detected upon yPvCSP-All_CT epitopes + Poly I:C vaccination. (E) Major biological processes and (F) molecular functions of splenic B-cell DEGs derived from mice vaccinated with yPvCSP-All_CT epitopes + Poly I:C through Gene Ontology analyses. Interaction networks between B-cell-derived DEGs (red dots) elicited upon yPvCSP-All_CT epitopes + Poly I:C vaccination with transcription factors (G) and DEG-encoding protein with proteins (H). Dotted lines represent LogFC values ≥ -2 and ≤ 2. .