Validation of A Cytometric Multiplex Assay and Examination Of Antibody Responses to Plasmodium Falciparum Antigens in the Highlands of Western Kenya During a Period of Low Malaria Transmission

Abstract

Malaria is one of the world's most deadly diseases. Even though it is highly preventable and treatable, it is a leading cause of morbidity and mortality in the tropics. As the campaign for malaria eradication widens, more areas will achieve low transmission of Plasmodium falciparum. However, little data exists on how absence of transmission will affect antibody responsesto malaria antigens. The current knowledge of immunological responses to malaria, a major tropical disease, is insufficient, and a better understanding of these responses would supportvaccine development. The aim of this study was to validate and standardize a cytometric multiplex assay for simultaneous detection of antibodies to malaria vaccine candidates and examineantibody responses to P. falciparum antigens in the highland regions of western Kenya. Antibody frequencies and levels to P. falciparum vaccine candidate antigens and a VCA-p18, non-malarialantigen were measured, just before interruption of transmission and fifteen months later. One thousand randomly selected individuals from two study sites (Kipsamoite and Kapsisiywa) prone to epidemics in western Kenya were tested for antibody reactivity. The degreeof association between median fluorescence intensities (MFI) or optical densities (OD) was assessed using Pearson's correlation (r). Chi-square analysis was used to compare the antibody frequencies at the two sites while paired antibody frequencies were evaluated by McNemartest. Differences in antibody levels between the two sites were compared using Mann Whitneytest. Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88- 0.99, P<O.OOOI- 0.004), and antibodies to variants of the same antigen were accurately distinguishedwithin a multiplex reaction. MFI values were essentially identical for all antigens tested at 1000 beads/analyte/well versus 5000 beads/analyte/well (all r~ 0.99, all P < 0.0(01). Correlationsbetween CBA and ELISA were strong (AMA-1-3D7 r > 0.55, AMA-l-FVO r> 0.37,EBA-175 r> 0.8, MSP-1-19 r> 0.92, MSP-1-42 r> 0.89, MSP-3 r> 0.70, GLURP-ROr> 0.82,GLURP-R2 r> 0.90 (P < 0.05) for all antigens except AMA-l-FVO. Antibody levels and frequenciesat both sites increased across age groups (P < 0.0001). IgG levels to all P.falciparum antigens decreased at both study sites over the fifteen-month period but not for Epstein Barr VirusVCA-p18 antigen. IgG levels for most antigens correlated significantly with each other for both sites during the first and second surveys. These results show that CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample, and has greater range of values in positive samples and lower background readings for blank samples than ELISA. Further, the rapid decline in median antibody levels to all vaccine candidates indicates that these populations will be at risk of epidemics. This study gives us an opportunity to further understand correlates of humoral immunity in an epidemiological context that informs vaccine development strategy in unstableseasonal transmission areas experiencing zero transmission