Antibodies and malaria “always nice to receive a complement” John Penry March 24, 2015

1988 Ronald Reagan Seoul Games Die Hard, Rain man, Working girl Bon Jovi, Guns’n’Roses, George Michael, INXS

Veterinary Immunology ‘88 Mostly: B,T cells, monocytes, neutrophils, eosinophils, basophils, mast cells, T helpers “humoral” “call mediated” Some: complement, interleukin, interferon ELISA, W blots, AGID

Complement – the history Discovered in late 1800’s (Jules Bordet, Paul Ehrlich) – “complement”, “alexine” Opsonisation mediated effect discovered 1930’s (Stanley) Small proteins, circulating precursors

Complement’s roles Links the innate and adaptive arms Attraction by chemotaxis Opsonisation Cell lysis Agglutination – clustering and binding

Complement’s roles Nature.com

Activation pathways Classical Alternative Lectin (non-self recog.: PAMPS)

Cell research, 20, 2010

MAC (membrane attack complex)

Useful ref: Dunkelberger and Song, Cell Res (2010) 20: 34-50 National Cancer Institute Useful ref: Dunkelberger and Song, Cell Res (2010) 20: 34-50

Our paper Human antibodies fix complement to inhibit Plasmodium falciparum invasion of erythrocytes and are associated with protection again Malaria Boyle et al, Immunity 42, 2015

Our paper Human antibodies fix complement to inhibit Plasmodium falciparum invasion of erythrocytes and are associated with protection again Malaria Antibodies use complement to stop the Malaria organism invading RBCs

“The Age” Mar 18

Authors and labs

Authors and labs

Malaria Agent is 5 protozoal spp: Plasmodium Female Anopheles vector Uncomplicated to severe disease 250M cases/yr worldwide, 1.2M deaths

Research thrust “a lack of established immune correlates of protection severely hampers the evaluation and prioritization of vaccines” “……the role of complement in Ab-mediated protection has not been defined”

Toolkit Merozoite invasion assay (FC) Immunoblot Immuno electron microscope Immunofluorescence microscopy ELISA Flow cytometry Cohort study

IgG (exposed):C dependent inhibitory activity Aim: to assess the role of C in Ab inhibition of invasion NS: normal serum – active C HIS: heat-inactivated serum – inactive C

IgG (exposed):C dependent inhibitory activity MIA Fig 1 Immunoblot

IgG (exposed):C dependent inhibitory activity IF microscopy

IgG (exposed):C dependent inhibitory activity ELISA

Conclusion – part 1 Anti-malaria Abs enhance C deposition on merozoites Via C1q fixation leading to increased C3b deposition and MAC formation

C1q fixation, Ab-C’ inhibition Aim: what is the relative importance….activated “classical C cascade” vs the “alternative cascade” and: does MAC deposition (lysis) inhibite invasion?

C1q fixation, Ab-C’ inhibition Fig 2 MIA

C1q fixation, Ab-C’ inhibition Flow cytometry

Conclusion – part 2 C1q fixation alone was sufficient to mediate substantial Ab-C’ inhibition M lysis dependent on “classical cascade” – MAC deposition, lysis, not artifact of aglutination rapid

Ab-C’ inhibition predominant Aim: what is the importance of Ab-C’ inhibition in naturally acquired immunity

Ab-C’ inhibition predominant Fig 3

Ab-C’ correlation to cytophilic Mz Ab

C1q fixation..correlates..Ab-C’ inhibition Immunoblot

Conclusion – part 3 The majority of human Ab require C factors to inhibit Mz invasion Ab-C’ is the predominant mechanism – acquired in naturally exposed people Ab mediated C1q deposition on Mz correlated with Ab-C’ activity

Ab-C associated with Mal. protection Aim: gather epidemiologic evidence of the importance of Ab-C’ in acquired immunity to Malaria Longitudinal, cohort study Ab for C1q fixation Surveillance for parasitemia, clin. Malaria

Ab-C associated with Mal. protection Fig 4

Ab-C associated with Mal. protection

Ab-C associated with Mal. protection

Conclusion –part 4 Increase in Ab dependent C1q deposition seen ….increasing age and in parasitemic children Consistent with expected acquisition of immunity High C1q deposition associated with clinical disease protection

MSP1/MSP2 targets of Ab-C’ Aim: identify the Mz antigen targets of Ab-C’ inhibition

MSP1/MSP2 targets of Ab-C’ Fig 5

MSP1/MSP2 targets of Ab-C’

Conclusion – part 5 Ab to MSP1 and MSP2 inhibited invasion in the presence of NS, not HIS Similar for human purified anti MSP2 MSP Ab induced by vacc. able to inhibit invasion via Ab-C’

Final words Ab-C’ inhibition is a prominent mechanism targeting the Mz – naturally acquired immun. Human anti-Mal Ab have evolved to recruit C for functional activity An-C’ inhibition can be mediated by Ab through vaccination

Cell research, 20, 2010

Sir Frank Macfarlane Burnet