Optimization of a Plasmodium falciparum circumsporozoite protein repeat vaccine using the tobacco mosaic virus platform

Abstract

Plasmodium falciparum vaccine RTS,S/AS01 is based on the major NPNA repeat and the C-terminal region of the circumsporozoite protein (CSP). RTS,S-induced NPNA-specific antibody titer and avidity have been associated with high-level protection in naïve subjects, but efficacy and longevity in target populations is relatively low. In an effort to improve upon RTS,S, a minimal repeat-only, epitope-focused, protective, malaria vaccine was designed. Repeat antigen copy number and flexibility was optimized using the tobacco mosaic virus (TMV) display platform. Comparing antigenicity of TMV displaying 3 to 20 copies of NPNA revealed that low copy number can reduce the abundance of low-affinity monoclonal antibody (mAb) epitopes while retaining high-affinity mAb epitopes. TMV presentation improved titer and avidity of repeat-specific Abs compared to a nearly full-length protein vaccine (FL-CSP). NPNAx5 antigen displayed as a loop on the TMV particle was found to be most optimal and its efficacy could be further augmented by combination with a human-use adjuvant ALFQ that contains immune-stimulators. These data were confirmed in rhesus macaques where a low dose of TMV-NPNAx5 elicited Abs that persisted at functional levels for up to 11 mo. We show here a complex association between NPNA copy number, flexibility, antigenicity, immunogenicity, and efficacy of CSP-based vaccines. We hypothesize that designing minimal epitope CSP vaccines could confer better and more durable protection against malaria. Preclinical data presented here supports the evaluation of TMV-NPNAx5/ALFQ in human trials.

Keywords: CSP; antigenicity; immunogenicity; malaria; vaccines.

Fig. 1.

Predicted TMV capsid structures. (A) The native fold of the TMV monomer displaying NPNAx3 (circled) at the N terminus. (B) Circular permutant of TMV, where the N and C termini have been repositioned to the pore and an exposed loop was used to display NPNAx3. (C) Model of a TMV disk showing the exposed loops with NPNAx3 (based on 3KML crystal structure). All structures were predicted using Rosetta prediction server and pictures generated using PyMOL software.

Fig. 2.

Design and production of TMV particles. (A) Rosetta structure predictions showing poly-NPNA of various lengths on the exposed loop (NPNAx3 circled). (B) Overlaid, minimum energy structures of NPNAx5 antigen (arrows) on the exposed loop of the circular permutant (Loop) or on the N terminal (NT) and C terminal (CT) of the native TMV fold, as predicted by Rosetta protein prediction server. Shown below each structure is the electron micrograph (40,000×) of the respective vaccine preparation.

Fig. 3.

(A and C) ELISA titration curves of mAb 580, mAb 317 and CIS43 against TMV antigens expressing various copy numbers of poly-NPNA or the NPNAx5 displayed on the Loop, NT, or CT of the TMV monomer. All antigens were coated at 100 ng/mL. (B and D) Relative potency (OD = 1 mAb concentration) for ELISA curves shown in A and C, respectively.

Fig. 4.

Optimization of immunogenicity of TMV vaccines/AddaVax in C57BL/6 mouse challenge model. Mice (n = 10) were vaccinated thrice with 1.25 µg TMV-NPNAx3, -x4, -x5, or FL-CSP vaccines (A–C) or with 2.5 µg TMV-NPNAx5, -x7, -x10, and -x20 vaccines (D–F) or with 1.25 µg TMV-NPNAx5-Loop, -NT, -CT, or FL-CSP (G–I) using AddaVax adjuvant. All vaccines were given at 3-wk intervals ELISA and challenge was at 2 wk after the third dose. (A, D, and G) Geometric mean ELISA titer using (NANP)₆ peptide coat. (B, E, and H) Mean avidity index using either a repeat peptide coat (Left) or FL-CSP coat (Right). (C, F, and I) Survival curves of mice postchallenge. (J and K) Mean OD₄₁₅ ratio for NPNAx3/NPNAx20 coated plates for mouse sera tested at 1:8,000 dilution. Error bars are ±SEM; red symbols are protected mice and asterisks indicate the level of statistical significance. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05.

Fig. 5.

TMV-NPNAx5/ALFQ immunogenicity in C57BL/6 mouse challenge model. Mice (n = 10) were immunized 3× 2.5 µg dose of NPNAx5, FL-CSP in ALFQ (A–D) or with 3× 2.5 µg dose of NPNAx5, -x7, -x10, and -x20 vaccines in ALFQ (E–J). At 2 wk after the third dose, ELISA was conducted and mice were challenged. (A and E) Geometric mean NPNA titers. (B and F) Mean avidity index using repeat peptide coat. (C and G) Survival curve of mice following challenge. (D) Relative titers against FL-CSP and repeat peptide coat for NPNAx5/ALFQ and FL-CSP/ALFQ vaccinated mice. (H) The mice that survived the challenge were bled again at week 11 and week 15 and their repeat ELISA titers were plotted. (I) Results of rechallenge of mice at 15 wk after the third dose. (J) Subclass analysis of mice by ELISA showing the ratio of IgG2c: IgG1 titer. Error bars are ±SEM; red symbols are protected mice; ****P < 0.0001; *P < 0.05.

Fig. 6.

Rhesus model immunogenicity. (A–E) Rhesus of Indian origin (n = 6) vaccinated three times with 20 µg or 40 µg NPNAx5 or 40 µg FL-CSP in ALFQ adjuvant. (F–H) Rhesus of Chinese origin vaccinated thrice with 40 µg of NPNAx5 (n = 6), 40 µg NPNAx20 (n = 4), or 40 µg FL-CSP (n = 6) in ALFQ. (A and F) Geometric mean ELISA titer at 2 wk after the third dose; (B and G) Mean repeat peptide avidity index; (C and H) Mean ILSDA at 1:200 or 1:300 serum dilution. (D) Repeat Ab geometric mean titer over 47 wk after the third dose; the time of each vaccination is marked as V1, V2, V3. The 47WP3 time-point has data from a subset of animals; 20 µg NPNAx5 (n = 3); both 40-µg dose groups (n = 2 each). Dotted line indicates historical geometrical mean values at 3 wk after the dose in RTS,S/AS01 vaccinated humans vaccinated on a 0- to 1–2-mo schedule (28). (E) ILSDA assay at 1:80 pooled serum dilution at four time-points weeks 4, 12, 24, and 47 after the third dose. ****P < 0.0001; **P < 0.01.