CORRELATION OF HIGH LEVELS OF ANTIBODIES TO MULTIPLE PRE-ERYTHROCYTIC PLASMODIUM FALCIPARUM ANTIGENS AND PROTECTION FROM INFECTION CHANDY C. JOHN,* ANN M. MOORMANN, DANIEL C. PREGIBON, PETER ODADA SUMBA, MARILYN M. MCHUGH, DAVID L. NARUM, DAVID E. LANAR, MARK D. SCHLUCHTER, AND JAMES W. KAZURA Center for Global Health and Diseases, Rainbow Center for International Child Health, Division of Pediatric Infectious Diseases, Division of Clinical Epidemiology, Rainbow Babies and Children’s Hospital, Case Western Reserve University, Cleveland, Ohio; Kenya Medical Research Institute, Kisian, Kenya; National Institutes of Health, Bethesda, Maryland; Walter Reed Army Institute for Research, Washington, District of Columbia Abstract. High levels of antibodies to multiple antigens may be more strongly associated with protection from infection than antibodies to a single antigen. Antibody-associated protection against Plasmodium falciparum infection was assessed in a cohort of 68 adults living in an area of holoendemic malaria in Kenya. Antibodies to the pre- erythrocytic antigens circumsporozoite protein (CSP), liver-stage antigen-1 (LSA-1), thrombospondin-related adhesive protein (TRAP), and blood-stage antigens apical membrane antigen-1 (AMA-1), erythrocyte binding antigen-175 (EBA-175), and merozoite surface protein 1 (MSP-1) were tested. Peptides were used for CSP (NANP repeat) and LSA-1 (central repeat), and recombinant antigens were used for TRAP (aa D 48 –K 394 ), AMA-1 (ectodomain, non- glycosylated), EBA-175 (non-glycosylated), and MSP-1 (MSP-1 19 ). Weekly microscopy testing for P. falciparum infec- tion was performed over a 12-week period after drug-mediated clearance of P. falciparum parasitemia. Individuals with high levels of IgG antibodies (> 2 arbitrary units) to CSP, LSA-1, and TRAP had a 57% decrease in the risk of infection (95% confidence interval  20–77%, P  0.016). This decreased risk remained significant after adjustment for age, prior parasitemia, bed net use, sickle cell trait, and village of residence. In contrast, protection against infection did not correlate with high levels of IgG antibodies to blood-stage antigens or IgM antibodies to pre-erythrocytic or blood-stage antigens. High levels of IgG antibodies to CSP, LSA-1, and TRAP may be useful immune correlates of protection against P. falciparum infection in malaria-endemic populations. INTRODUCTION Identification of reliable and reproducible immune corre- lates of protection against Plasmodium falciparum infection will be important in the development and testing of malaria vaccines. Results from studies of rodent malaria models and humans with partial immunity induced by irradiated sporo- zoites or repeated natural infections suggest that both anti- body and cytokine responses to pre-erythrocytic antigens con- tribute to or correlate with protection against P. falciparum infection. 1–7 However, neither antibody nor cytokine re- sponses to circumsporozoite protein (CSP) correlated with protection against P. falciparum infection in malaria-naive volunteers immunized with the RTS,S CSP vaccine. 8,9 The identification of immune correlates of protection that can be used to evaluate vaccine immunogenicity and efficacy in populations where malaria is endemic presents additional challenges. First, most individuals will have pre-existing im- mune responses to multiple pre-erythrocytic antigens. Sec- ond, the time at which and number of sporozoites to which an individual has been exposed cannot be controlled. Third, im- mune responses and partial resistance to blood-stage para- sites co-exists in persons who have been repeatedly infected, thus complicating interpretation of endpoints such as the appearance of asexual parasitemia. Despite these con- straints, some studies have shown that cytokine responses to pre-erythrocytic antigens such as liver-stage antigen-1 (LSA- 1) 10–14 are associated with protection from P. falciparum in- fection in malaria-endemic areas. In addition, a recent study of the RTS,S CSP vaccine in individuals living in a malaria- endemic area demonstrated that CD4-mediated interferon- (IFN-) responses to CSP were associated with protection from infection. 15 In contrast, observations made in some of the same populations have failed to document a correlation of protection with antibodies to LSA-1 10,16 and circumsporozo- ite protein (CSP), 16 although a trend toward protection was seen with antibodies to CSP in the RTS,S vaccine study. 15 Given that persons living in malaria-holoendemic areas have experienced repeated natural infections since childhood and develop antibody responses to multiple pre-erythrocytic an- tigens, we hypothesized that the presence of high levels of antibodies to multiple as opposed to single antigens may be a more robust correlate of protection from infection. In contrast, antibodies to blood-stage antigens, which act primarily on the growth of parasites undergoing growth in red blood cells, would more likely be associated with protection from high-density parasitemia and morbidity. To test this hy- pothesis, we measured IgG and IgM antibodies to six P. fal- ciparum vaccine candidate antigens in adults residing in a malaria-holoendemic area of Kenya. High levels of antibodies to these antigens individually and in combination were com- pared with time and risk of re-infection after clearance of pre-existing blood-stage parasitemia. This study was per- formed in adults because the high levels of immunity in this population would allow us the best chance to identify robust immune correlates of protection from infection. Knowledge of likely correlates would be very useful in planning such a study in children, who are at the highest risk of complications from malaria. Antigens to which antibodies were tested in- cluded the pre-erythrocytic antigens CSP, LSA-1 and thro- mobospondin related adhesive protein (TRAP) and the blood-stage antigens apical-membrane antigen-1 (AMA-1), erythrocyte-binding antigen-175 (EBA-175), and merozoite surface protein-1 (MSP-1). * Address correspondence to Chandy C. John, Rainbow Center for International Child Health, Rainbow Babies and Children’s Hospital, RBC 487, Case Western Reserve University, 11100 Euclid Avenue, MS6008, Cleveland, OH 44106. E-mail: chandy.john@case.edu Am. J. Trop. Med. Hyg., 73(1), 2005, pp. 222–228 Copyright © 2005 by The American Society of Tropical Medicine and Hygiene 222