The Pennsylvania State University The Graduate School College of Medicine COMPLEMENT AND ANTIBODY-MEDIATED ENHANCEMENT OF RED BLOOD CELL INVASION BY PLASMODIUM FALCIPARUM A Dissertation in Microbiology and Immunology by Sergei Sergeievich Biryukov 2016 Sergei Sergeievich Biryukov Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2016 The dissertation of Sergei Sergeievich Biryukov was reviewed and approved* by the following: Jose Stoute Professor of Medicine, and Microbiology and Immunology Dissertation Advisor Chair of Committee David J. Spector Professor Emeritus of Microbiology and Immunology Todd Schell Associate Professor of Microbiology and Immunology Neil Christensen Professor of Pathology, and Microbiology and Immunology Channe Gowda Professor of Biochemistry and Molecular Biology Aron Lukacher Professor and Chair of Microbiology and Immunology *Signatures are on file in the Graduate School ii Abstract Plasmodium falciparum malaria is responsible for close to one million deaths worldwide each year. The Plasmodium life cycle involves invasion of hepatocytes and erythrocytes. The repeating cycle of erythrocyte invasion and destruction is the root cause of the morbidity and mortality brought about by the pathogen. Development of naturally acquired immunity against the parasite is an extremely slow process taking years to decades to develop, and it does not seem to result in sterile immunity. Attempts to develop a vaccine to block erythrocyte invasion have proven unsuccessful. Clinical data have demonstrated the development of a strong humoral immune response in vaccinated individuals. Although antibodies raised against vaccine antigens inhibit invasion in vitro in the presence of heat-inactivated (HI) serum, there is poor efficacy in vivo. The reasons for this discrepancy are unknown. A number of studies, in both humans and animal models, have shown that the complement cascade is activated during malaria infection. We hypothesize that complement activation and opsonization of merozoites enhances complement receptor 1 (CR1)-mediated invasion of erythrocytes. If we are correct, the parasite can hijack this arm of the innate immune response for its own benefit, avoiding or diminishing the efficacy of antibodies. We tested this hypothesis by studying the effect of mouse monoclonal anti-merozoite surface protein 1 (MSP-1) antibody 5.2 (mAb5.2) and antibodies from recipients of a merozoite vaccine (MSP142) for their ability to enhance or inhibit invasion in a complement-dependent manner. We carried out invasion assays with the above antibodies with appropriate controls in fresh or HI serum, or in complement-depleted or reconstituted serum. In addition, we tested iii the relevance of the CR1 pathway by inhibiting with sCR1. Invasion of erythrocytes was enhanced by fresh serum relative to 3 min, 5 min, or 30 min HI serum. Furthermore, addition of mAb5.2 to fresh serum increased the enhancement effect. Addition of C2 and Factor B to 3 min HI serum, but not to 30 min, rescued the enhancement of invasion in the presence of mAb5.2. Likewise, Compstatin, a C3 specific inhibitor, and sCR1 negated the enhancing affects of mAb5.2 in fresh serum but not that of fresh serum alone. Furthermore, soluble CR1 had invasion inhibitory activity in 3 min HI serum that was independent of antibody. Purified antibodies from MSP-1 vaccinees showed inhibitory activity in C3/C4 inactivated serum, but inhibition was drastically reduced in C3/C4 reconstituted serum. Passive transfer of anti-P. berghei antibodies into wild type mice enhanced parasitemia in an inverse dose-dependent manner. In addition, C3-deficient mice showed decreased parasite growth relative to wild type mice. In addition, we utilized erythrocytes from individuals with paroxysmal nocturnal hemoglobinuria (PNH), which results in the absence of glycosylphosphatidylinositol (GPI)-anchored receptors, in an effort to identify novel receptors utilized by the parasite for erythrocyte invasion. We found that PNH RBCs were invaded less effectively than wild type RBCs. Therefore, we carried out growth and invasion assays with anti-GPI erythrocyte receptor antibodies against six potential targets with appropriate controls in fresh or HI serum, or in complement-depleted or reconstituted serum. We identified Semaphorin-7A (SEMA7A) as a potential erythrocyte receptor that may interact with complement in promoting erythrocyte invasion by merozoites. iv Our results demonstrate that merozoites can highjack the complement system to their own advantage in a number of ways. Activation of the complement cascade via the classical pathway can allow merozoites to bind to CR1 and invade via this pathway. In addition, in the absence of antibodies, activation of the mannose binding lectin pathway by default may allow merozoites to bind to other complement receptors on the RBC that mediate invasion. Such mechanisms could explain or contribute to the low efficacy of vaccine-induced anti-merozoite antibodies to block invasion in vivo. These findings are of great importance to the efforts to develop a merozoite blocking vaccine. v Table of Contents List of Figures ................................................................................................................ viii Abbreviations .................................................................................................................. x Acknowledgments .......................................................................................................... xii Chapter I: Literature Review ............................................................................................ 1 Introduction to malaria .......................................................................................... 1 Malaria a Severe Public Health Problem ................................................... 1 Malaria and Plasmodium falciparum Lifecycle ........................................... 2 Plasmodium falciparum Erythrocyte Invasion Pathways ...................................... 5 Invasion Ligands Utilized by Merozoites ............................................................... 7 The Merozoite Surface Proteins ................................................................ 7 The P. falciparum Erythrocyte Binding Antigens and the P. falciparum Reticulocyte Binding Protein Homologues ................................................. 9 Characterization of Humoral Immune Response to Plasmodium Erythrocytic Stages ................................................................................................................ 12 Naturally acquired immunity ..................................................................... 12 Conflicting Data Between in vitro vs. in vivo Blood-stage Studies ........... 20 Common in vitro Testing Methods and Antibody Functions ..................... 23 The complement system .................................................................................... 25 Complement Activation and Malaria ........................................................ 27 Role of Complement in Cerebral Malaria ................................................. 32 Role of Complement in Placental Malaria ................................................ 36 Role of Complement in Severe Malarial Anemia ..................................... 40 Paroxysmal Nocturnal Hemoglobinuria Erythrocyte Invasion by P. falciparum .. 45 Merozoite-specific Thrombospondin-related Anonymous Protein and Semaphorin 7A ........................................................................................ 47 Concluding remarks ............................................................................................ 48 Chapter II: Materials and Methods ................................................................................ 51 Chapter III: Complement and Antibody-mediated Enhancement of Red Cell Invasion and Growth of Plasmodium falciparum .......................................................................... 63 Abstract: ............................................................................................................. 63 Significance: ....................................................................................................... 64 vi Introduction: ........................................................................................................ 65 Results: .............................................................................................................. 67 Discussion: ......................................................................................................... 73 Acknowledgments: ............................................................................................. 78 Figure Legends: .................................................................................................. 79 Figures: .............................................................................................................. 84 Chapter IV: Inhibition of erythrocyte invasion by Plasmodium falciparum due to Semaphorin 7A and complement interaction ................................................................. 98 Introduction: ........................................................................................................ 98 Results: ............................................................................................................ 100 Discussion: ....................................................................................................... 106