Plasmodium falciparum in Aotus nancymaae: A New Model for Placental Malaria

Abstract

Plasmodium falciparum-infected erythrocytes that display the variant surface antigen VAR2CSA bind chondroitin sulfate A (CSA) to sequester in placental intervillous spaces, causing severe sequelae for mother and offspring. Here, we establish a placental malaria (PM) monkey model. Pregnant Aotus infected with CSA-binding P. falciparum CS2 parasites during the third trimester developed pronounced sequestration of late-stage parasites in placental intervillous spaces that express VAR2CSA and bind specifically to CSA. Similar to immune multigravid women, a monkey infected with P. falciparum CS2 parasites over successive pregnancies acquired antibodies against VAR2CSA, with potent functional activity that was boosted upon subsequent pregnancy infections. Aotus also developed functional antibodies after multiple acute PM episodes and subsequent VAR2CSA immunization. In summary, P. falciparum infections in pregnant Aotus monkeys recapitulate all the prominent features of human PM infection and immunity, and this model can be useful for basic mechanistic studies and preclinical studies to qualify candidate PM vaccines. Clinical Trials Registration: NCT02471378.

Keywords: Aotus monkey; Plasmodium falciparum; VAR2CSA; animal model; placental malaria.

Figure 1.

Evidence of Plasmodium falciparum sequestration in the placenta of pregnant Aotus monkeys. A, Schematic of study design and pregnancy timeline. B, Parasite density of P. falciparum clone CS2 (Pf-CS2) in the peripheral and placental blood from pregnant Aotus monkeys after inoculation with Pf-CS2 parasites. Symbols indicate one unique animal while fills indicate different pregnancies in one animal. Asterisk indicates ring-stage placental parasites in one animal (solid hexagon). Peripheral parasitemia was compared to placental parasitemia by Wilcoxon signed rank test. C, Representative peripheral and placental blood smears (top) and placental tissue (bottom) stained with Giemsa. The arrows indicate Giemsa-stained parasites. The thin arrow indicates a ring-stage parasite in the peripheral blood smear, and the thick arrow indicates a mature-stage parasite in the placental blood smear. Images of stained placental tissues demonstrating the sequestration of Pf-CS2–infected erythrocytes in the intervillous spaces are shown.

Figure 2.

Placental IEs bind to CSA and express VAR2CSA on the surface. A, IE binding to CSA (reported as % of total parasites bound to CSA or to CD36) for parasites collected from peripheral versus placental blood. Images show representative Giemsa-stained binding assay spots of IE isolated from infected placenta. B, Surface expression of VAR2CSA from peripheral blood of pregnant Aotus was assessed by flow cytometry using anti-DBL3X antibody (right-most red shaded curve), compared to unstained IE (left-most green curve), or secondary antibody only (middle black curve). Abbreviations: BSA, bovine serum albumin; CSA, chondroitin sulfate A; FITC, fluorescein isothiocyanate; FSC, forward scatter; IE, infected erythrocyte.

Figure 3.

Pf-CS2 recrudescent infection induces broadly neutralizing antibodies. A, Course of infection in a recrudescent animal through first and second pregnancies. B, Surface expression of VAR2CSA by recrudescent Pf-CS2 IEs collected from pregnancy AO1-G2 was assessed by flow cytometry using anti-DBL3X antibody (right-most red data and curve) compared to secondary antibody only (left-most black data and curve) presented as a scattergram (upper panel) and a histogram (lower panel); similar results with anti-DBL4ε and anti-DBL5ε antibody were observed. Number represents the proportion of Pf-CS2 IEs that bound the VAR2CSA antibody. C, Plasma collected from AO1-G2 at delivery was analyzed for parasite binding inhibitory activity against the homologous parasite as well as 6 culture-adapted maternal isolates. The proportion of binding inhibition activity relative to the control well (phosphate-buffered saline) is reported for each parasite. Inhibition activity of plasma from the first pregnancy (AO1-G1) is shown as reference. Data represent mean ± SD of 3 independent assays with duplicate wells in each assay. Abbreviations: C delivery, cesarean delivery; FSC, forward scatter; P, pregnancy; Pf-CS2, Plasmodium falciparum clone CS2.

Figure 4.

VAR2CSA antibody acquisition and functional activity in AO1. ELISA of serum IgG recognizing FCR3-allele (homologous) VAR2CSA domains (left axis). Functional antibodies measured by binding inhibition activity of Aotus plasma (right axis). Data represent mean ± SD. Arrows indicate Plasmodium falciparum exposure via recrudescence or infection. X-axis labels indicate pregnancy number (1P, 2P, 3P, 4P) or no pregnancy (NP); and indicate parasite isolates (CAMP, CS2, and maternal isolates M920 and M710). Abbreviations: BI, binding inhibition; CAMP, Malayan Camp; ELISA, enzyme-linked immunosorbent assay; OD, optical density; Pf-CS2, P. falciparum clone CS2.

Figure 5.

Functional serum activity in 3 PM-experienced and vaccinated Aotus. Plasma samples were collected from 3 placental malaria-experienced monkeys 40 weeks postimmunization with DNA vaccine encoding full-length VAR2CSA ectodomain, under protocol AN200218AS. Plasma were analyzed for parasite binding inhibitory activity against 5 fresh maternal Plasmodium falciparum isolates in Mali. Data represent mean ± SEM. The proportion of binding inhibition activity in immune sera is reported relative to pooled naive sera from the monkeys.