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.

The Circumsporozoite Protein (CSP) of Plasmodium falciparum contains an N-terminal region, a conserved Region I (RI), a junctional region, 25–42 copies of major (NPNA) and minor repeats followed by a C-terminal domain. The recently approved malaria vaccine, RTS,S/AS01 contains NPNAx19 and the C-terminal region of CSP. The efficacy of RTS,S against natural infection is low and short-lived, and mapping epitopes of inhibitory monoclonal antibodies may allow for rational improvement of CSP vaccines. Tobacco Mosaic Virus (TMV) was used here to display the junctional epitope (mAb CIS43), Region I (mAb 5D5), NPNAx5, and NPNAx20 epitope of CSP (mAbs 317 and 580). Protection studies in mice revealed that Region I did not elicit protective antibodies, and polyclonal antibodies against the junctional epitope showed equivalent protection to NPNAx5. Combining the junctional and NPNAx5 epitopes reduced immunogenicity and efficacy, and increasing the repeat valency to NPNAx20 did not improve upon NPNAx5. TMV was confirmed as a versatile vaccine platform for displaying small epitopes defined by neutralizing mAbs. We show that polyclonal antibodies against engineered VLPs can recapitulate the binding specificity of the mAbs and immune-focusing by reducing the structural complexity of an epitope may be superior to immune-broadening as a vaccine design approach. Most importantly the junctional and restricted valency NPNA epitopes can be the basis for developing highly effective second-generation malaria vaccine candidates.