Williams, B. G.; King, L. D. W.; Pulido, D.; Quinker, D.; Lias, A. M.; Silk, S. E.; Ragotte, R. J.; Davies, H.; Barrett, J. R.; McHugh, K.; Rigby, C. A.; Alanine, D. G. W.; Barfod, L.; Shea, M. W.; Cowley, L. A.; Dabbs, R. A.; Pattinson, D. J.; Douglas, A. D.; Lyth, O. R.; Illingworth, J. K.; Jin, J.; Carnot, C.; Kotraiah, V.; Christen, J. M.; Noe, A. R.; MacGill, R. S.; King, C. R.; Birkett, A. J.; Soisson, L. A.; Skinner, K.; Miura, K.; Long, C. A.; Higgins, M. K.; Draper, S. J.

In recent years, reticulocyte-binding protein homologue 5 (RH5) has emerged as a leading blood-stage Plasmodium falciparum malaria vaccine antigen. The most advanced blood-stage vaccine candidate in a Phase 2b clinical trial, RH5.1/Matrix-M[TM], is based on a full-length soluble protein-with-adjuvant formulation. RH5 interacts with cysteine-rich protective antigen (CyRPA) and RH5-interacting protein (RIPR) to form an essential heterotrimeric RCR-complex. Here, we investigated whether a vaccine candidate based on the ternary RCR-complex could substantially improve upon the leading clinical candidate RH5.1/Matrix-M[TM] in preclinical studies. Using a panel of monoclonal antibodies (mAbs) we confirm that parasite growth-inhibitory epitopes on each antigen are exposed on the surface of the RCR-complex and that mAb pairs binding to different antigens can function additively or synergistically to mediate parasite growth inhibition activity (GIA) in vitro. However, immunisation of rats with the RCR-complex consistently fails to outperform RH5.1 alone. We show this is due to immuno-dominance of RIPR coupled with the inferior potency of anti-full length RIPR polyclonal IgG antibodies as compared to the anti-RH5 and anti-CyRPA response. To address this, we identified the growth-inhibitory antibody epitopes of RIPR are clustered within C-terminal EGF-like domains of RIPR. A fusion of these EGF domains to CyRPA, called R78C, combined with RH5.1, provided a new vaccination strategy that improves upon the levels of in vitro GIA seen with RH5.1 alone. Superiority of the combination antigen vaccine candidate was achieved by the induction of a quantitatively higher, but qualitatively similar, polyclonal antibody response that demonstrated additive GIA across the three antigen targets. These preclinical data justified the advancement of the RH5.1+R78C/Matrix-M[TM] combination vaccine to a Phase 1 clinical trial.