A VAR2CSA:CSP conjugate capable of inducing dual specificity antibody responses

Abstract

Background: Vaccine antigens targeting specific P. falciparum parasite stages are under pre-clinical and clinical development. It seems plausible that vaccine with multiple specificities will offer higher protection. With this hypothesis, we exploited the Spy-Tag/SpyCatcher conjugation system to make a, post expression, dual antigen conjugate vaccine, comprising two clinically tested antigen candidates (CSP and VAR2CSA).

Methods: The DBL1x-DBL2x-ID2a region of VAR2CSA was genetically fused with SpyTag at N-terminus. The full-length CSP antigen was genetically fused to C-terminal SpyCatcher peptide. The covalent interaction between SpyTag/SpyCatcher enables the formation of DBL1x-DBL2x-ID2a:CSP conjugate vaccine. Immunogenicity and quality of antibody responses induced by the conjugate vaccine, as well as a control CSP-SpyCatcher vaccine, was tested in BALB/c mice.

Results: Serum samples obtained from mice immunized with the conjugate vaccine were able to recognize both untagged DBL1x-DBL2x-ID2a as well as CSP antigen. Moreover, the geometric mean anti-CSP antibody titer was 1.9-fold higher in serum (at day 35 and 55 post-first immunization) from mice immunized with the conjugate vaccine, as compared to mice receiving the control vaccine.

Conclusion: The data obtained in this study serves as proof-of-concept for the simultaneous induction of antibodies directed against individual antigen components in a dual stage anti-malaria vaccine.

Keywords: CSP-SpyCatcher; Circumsporozoite protein; DBL1x-DBL2x-ID2a:CSP conjugate; Malaria vaccine; SpyTag-DBL1x-DBL2x-ID2a; VAR2CSA; bacterial superglue.

Fig. 1

SDS-PAGE analysis of SpyTag-DBL1-DBL2x-ID2a as well as CSP-SpyCatcher antigen. a Reduced (lane 1) and non-reduced (lane 2) SDS-PAGE gel of SpyTag-DBL1-DBL2x-ID2a antigen. The band shift indicate the disulfide bond formation, as well as the protein expressed well in baculovirus expression system. On the gel a total of 12 ul sample was loaded, and dilution buffer was used to adjust end concentration into 2 µg. The size of the band correspond to 118 kDa. b Non-reduced (lane 1) and reduced (lane 2) SDS-PAGE gel of CSP-SpyCatcher antigen. No band shift is seen for CSP-SpyCatcher as it contain fewer cycteines for the disulfide bonds as well as band shift formation. The protein expressed as soluble in the baculovirus expression system, and again the dilution buffer was used to adjust end concentration (2 µg) in the total of 12 ul sample loaded on the gel. The theoretical size of CSP-SpyCatcher is 52 kDa but on the SDS-gel it was seen as 72 kDa. C The DBL1-DBL2x-ID2:CSP conjugate formed after overnight incubation (at 4°C) of SpyTag-DBL1-DBL2x-ID2a and CSP-SpyCatcher antigen (lane 1 top). Equal molar amounts of antigens were calculated andmixed (to attain 5 µg final concentration). The excess unconjugated SpyTag-DBL1-DBL2x-ID2a antigen (lane 1 middle).

Fig. 2

ELISA showing the amount of anti-CSP IgG in sera from mice immunized with DBL1x-DBL2x-ID2a:CSP or CSP-SpyCatcher. A. Anti-CSP IgG titers in sera (n = 5) obtained from mice immunized (prime-boost-boost) with 5 µg of either DBL1x-DBL2x-ID2a:CSP (filled circles) or CSP-SpyCatcher (open squares), using Aluminum hydroxide as extrinsic adjuvant. B. Anti-DBL1x-DBL2x-ID2a IgG in sera from mice (n = 5) immunized (prime-boost-boost) with 5 µg of the DBL1x-DBL2x-ID2a:CSP conjugate vaccine. The OD cut off value of 0.2 was used to determine endpoints titres.

Fig. 3

Geometric mean titres. GMT of anti-CSP IgG antibodies in sera obtained from mice (n = 5) immunized at days 14, 35, 49 and 55 with 5 µg of DBL1x-DBL2x-ID2a:CSP (filled circles) or CSP-SpyCatcher (open triangles) formulated in aluminium hydroxide. Analyzed sera were obtained on days 14, 35, 49 and 55, respectively. The geometric mean titer of sera obtained on day 35 and 55 from the DBL1x-DBL2x-ID2a:CSP vaccination group (n = 5) was more than 1.9-fold higher than in sera obtained at the same time points from the CSP-SpyCatcher vaccination group. P-values based on Mann-Whitney Rank sum test were P=0.20 and P=0.27, respectively.

Fig. 4

Flow cytometry. Reactivity of sera obtained from mice (n = 5) immunized (prime-boost-boost) with 5 µg of DBL1x-DBL2x-ID2a: CSP (full circles) or CSP-SpyCatcher (open triangles) against native VAR2CSA (FCR3 genotype) expressed on the surface of infected red blood cells. Control sera (pre-bleeds) is shown as (open squares). The geometric mean recognition values for the DBL1x-DBL2x-ID2a:CSP group ranged between 7 and 37 whereas that of the CSP-SpyCatcher group ranged from 5 to 9, and the difference between the groups was statistically significant (p=0.0079). In comparison, the negative control (pre-bleed) sera had the lowest geometric mean recognition values of less than 5.