Immunization with virus-like particles conjugated to CIDRα1 domain of Plasmodium falciparum erythrocyte membrane protein 1 induces inhibitory antibodies

Abstract

Background: During the erythrocytic cycle, Plasmodium falciparum malaria parasites express P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) that anchor the infected erythrocytes (IE) to the vascular lining of the host. The CIDRα1 domain of PfEMP1 is responsible for binding host endothelial protein C receptor (EPCR), and increasing evidence support that this interaction triggers severe malaria, accounting for the majority of malaria-related deaths. In high transmission regions, children develop immunity to severe malaria after the first few infections. This immunity is believed to be mediated by antibodies targeting and inhibiting PfEMP1, causing infected erythrocytes to circulate and be cleared in the spleen. The development of immunity to malaria coincides with acquisition of broad antibody reactivity across the CIDRα1 protein family. Altogether, this identifies CIDRα1 as an important vaccine target. However, the antigenic diversity of the CIDRα1 domain family is a challenge for vaccine development.

Methods: Immune responses in mice vaccinated with Virus-Like Particles (VLP) presenting CIDRα1 antigens were investigated. Antibody reactivity was tested to a panel of recombinant CIDRα1 domains, and the antibodies ability to inhibit EPCR binding by the recombinant CIDRα1 domains was tested in Luminex-based multiplex assays.

Results: VLP-presented CIDRα1.4 antigens induced a rapid and strong IgG response capable of inhibiting EPCR-binding of multiple CIDRα1 domains mainly within the group A CIDRα1.4-7 subgroups.

Conclusions: The study observations mirror those from previous CIDRα1 vaccine studies using other vaccine constructs and platforms. This suggests that broad CIDRα1 antibody reactivity may be achieved through vaccination with a limited number of CIDRα1 variants. In addition, this study suggest that this may be achieved through vaccination with a human compatible VLP vaccine platform.

Keywords: Antigenic diversity; CIDRα1; Malaria; PfEMP1; Plasmodium falciparum; Vaccine; Virus-like particle.

Fig. 1

Characterization of VLP-antigen conjugated vaccines. a Dynamic light scattering (DLS) showing SpyT-VLP (blue) with an average size of 37 nm (12.18% polydispersity, Pd). After coupling VLP to catCIDRα1.4 (brown) or CIDRα1.4cat (green) the size increases to 56 nm (12.19%Pd) and 88 nm (27.83 %Pd), respectively. b SDS-gel loaded with catCIDRα1.4 or CIDRα1.4cat antigens (32kDa) mixed with SpyT-VLP (16kDa) or naked VLP (8kDa): 1. SpyT-VLP; 2. Naked VLP + catCIDRα1.4; 3. SpyT-VLP + catCIDRα1.4; 4. Naked VLP + CIDRα1.4cat; 5. SpyT-VLP + CIDRα1.4cat

Fig. 2

Anti-CIDRα1.4 IgG. ELISA titers elicited in mice vaccinated with catCIDRα1.4 or CIDRα1.4cat. Each line represents the titration of serum from a mouse. Black lines indicate serum from mice immunized with CIDRα1.4-VLP in aluminum hydroxide. Red lines indicate mice immunized with soluble CIDRα1.4 in Freund’s incomplete adjuvant. Table shows titers calculated as area under the curve for each vaccine group (mean ± standard deviation), P values from t-tests

Fig. 3

Antibody reactivity and EPCR-binding inhibition in Luminex. a Anti-HB3var03 CIDRα1.4 IgG levels in serum. b EPCR-binding inhibition of pooled, purified IgG in  %. c Serum-IgG levels to 42 CIDRα1 protein variants not including the immunogen variant HB3var03 measured in 3–5 mice. d EPCR-binding inhibition of 33 EPCR-binding CIDRα1 protein variants (also excluding HB3var03). All for mice immunized with catCIDRα1.4 or CIDRα1.4cat on VLP or as soluble antigen in Freund’s incomplete adjuvant. Box plots showing median reactivity with 25th and 75th percentiles, upper and lower adjacent values and outliers. *Indicate statistically significant difference, P = 0.03 and P = 0.05 for catCIDRα1.4 and CIDRα1.4cat, respectively using Wilcoxon rank-sum test

Fig. 4

Inhibition of EPCR-binding of specific CIDRα1 domain variants. EPCR-binding inhibition (%) of 34 recombinant CIDRα1 domains by purified IgG pooled from mice immunized with VLP-catCIDRα1.4, catCIDRα1.4, CIDRα1.4cat-VLP or CIDRα1.4cat. Each bar represents one specific recombinant CIDRα1 domain variant annotated according to its domain subtype. Arrows indicate the HB3var03 variant used as immunogen. Order of CIDRα1 protein variants tested is shown as listed in Fig. 6a

Fig. 5

Correlation between anti-CIDRα1 IgG levels and EPCR-binding inhibitory capacity of CIDRα1 domains in pooled IgG purified from mice immunized with VLP-catCIDRα1.4. Each dot represents one EPCR-binding CIDRα1 domain and colors represent each of the four immunization schemes (34 domains × 4 immunization groups): VLP-catCIDRα1.4 (red), catCIDRα1.4 (orange), CIDRα1.4cat-VLP (blue), and CIDRα1.4cat (green). The immunogen CIDRα1.4 variant is outlined. The box includes indicates data from three CIDRα1.4_DD2var32 and one CIDRα1.4_1983-5 domain

Fig. 6

Sequence analysis of CIDRα1 domains. a Pairwise sequence similarity (19 kDa sequences) of the assayed CIDRα1 domains to HB3var03 CIDRα1.4. Blue (immunogen variant) and red asterisks (*) mark CIDRα1 domain variants inhibited > 40%. b Maximum likelihood tree (key bootstrap [N = 50] values are indicated on branches) of 885 CIDRα1 sequences (30 kDa) (generated in [26]). The 34 CIDRα1 variants tested for EPCR-binding inhibition (marked with boxes) are red if inhibited > 40% by total purified IgG from animals immunized with VLP-catCIDRα1.4 or for the cognate immunogen, blue. CIDRα1 variants not inhibited are green