Identification and characterization of Loa loa antigens responsible for cross-reactivity with rapid diagnostic tests for lymphatic filariasis

Abstract

The Global Program to Eliminate Lymphatic Filariasis (LF) relies on rapid diagnostic tests (RDTs) to determine where annual mass drug administration for LF is required and when it can be stopped. These tests detect a Wuchereria bancrofti glycoprotein in the blood of infected persons via a carbohydrate moiety recognized by the monoclonal antibodies AD12 and DH6.5. Loiasis cross-reactivity with LF RDTs has recently been recognized as a serious obstacle to LF elimination in loiasis-endemic areas. To better understand the nature of this cross-reactivity, we used the DH6.5 antibody to immunoaffinity purify Loa loa antigens from the sera of individuals with a positive RDT due to loiasis. Immunoblot analysis revealed many circulating AD12/DH6.5-reactive antigens, and proteomic analysis identified multiple L. loa proteins in LF RDT-positive loiasis sera. These included both secreted and somatic proteins, suggesting that they may be released by dying L. loa adult worms and/or microfilariae. Unlike the single high molecular weight W. bancrofti circulating filarial antigen that is reliably present in the blood of persons with bancroftian filariasis, reactive L. loa antigens appeared to be only transiently present in the blood of a subset of persons with loiasis. These key differences between the circulating antigens of W. bancrofti and L. loa can be used to differentiate positive results generated by both species and may lead to improved diagnostic tests for LF and loiasis.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Field studies and samples tested.

(A) Central Cameroon field study. Because the three FTS positive samples were very weakly positive by FTS and negative by ICT, we chose not to examine them further. (B) East Region study. Participants who were ICT-positive on initial screening were visited the following week for venous blood collection. Nine venous blood samples were FTS-positive. Two of these tested negative for filarial antigen by ELISA; the other seven were tested further by ELISA and/or western blot as indicated. Gray shading indicates the origin of the samples used in the proteomic analysis.

Fig 2. Loiasis patient sera lack antibodies specific to the AD12 carbohydrate epitope.

Serum samples were tested for the ability to block AD12 binding to B. malayi antigens that contain the AD12 epitope. The horizontal line denotes the mean value for each group.

Fig 3. Loiasis sera positive by LF RDT contain multiple AD12 epitope-containing antigens.

(A) AD12 western blot showing reactive bands in pooled sera from 13 individuals with W. bancrofti infection (Wb CFA+) or 12 uninfected individuals (CFA-). (B) Antigens from sample P811355 (355), and pooled cross-reactive sera. Soluble L. loa antigen (Loa Ag, 0.5 μg) served as a positive control. Because the antigen level was much higher in sample P811355 than in the pooled sample, different chemiluminescent exposure times were used for the same blot (45 seconds for soluble L. loa antigen and P811355; 15 minutes for the pooled serum sample).

Fig 4. L. loa secretes glycoproteins reactive with monoclonal antibody AD12.

Western blot of AD12 glycoproteins immunoaffinity purified from 1mL of culture supernatant (ES products) from L. loa adult worms (L5) or microfilaria (Mf). Soluble L. loa antigen (Loa Ag, 0.5 μg) served as a positive control.