Antibody isotype analysis of malaria-nematode co-infection: problems and solutions associated with cross-reactivity

Abstract

Background: Antibody isotype responses can be useful as indicators of immune bias during infection. In studies of parasite co-infection however, interpretation of immune bias is complicated by the occurrence of cross-reactive antibodies. To confidently attribute shifts in immune bias to the presence of a co-infecting parasite, we suggest practical approaches to account for antibody cross-reactivity. The potential for cross-reactive antibodies to influence disease outcome is also discussed.

Results: Utilising two murine models of malaria-helminth co-infection we analysed antibody responses of mice singly- or co-infected with Plasmodium chabaudi chabaudi and Nippostrongylus brasiliensis or Litomosoides sigmodontis. We observed cross-reactive antibody responses that recognised antigens from both pathogens irrespective of whether crude parasite antigen preparations or purified recombinant proteins were used in ELISA. These responses were not apparent in control mice. The relative strength of cross-reactive versus antigen-specific responses was determined by calculating antibody titre. In addition, we analysed antibody binding to periodate-treated antigens, to distinguish responses targeted to protein versus carbohydrate moieties. Periodate treatment affected both antigen-specific and cross-reactive responses. For example, malaria-induced cross-reactive IgG1 responses were found to target the carbohydrate component of the helminth antigen, as they were not detected following periodate treatment. Interestingly, periodate treatment of recombinant malaria antigen Merozoite Surface Protein-119 (MSP-119) resulted in increased detection of antigen-specific IgG2a responses in malaria-infected mice. This suggests that glycosylation may have been masking protein epitopes and that periodate-treated MSP-119 may more closely reflect the natural non-glycosylated antigen seen during infection.

Conclusions: In order to utilize antibody isotypes as a measure of immune bias during co-infection studies, it is important to dissect antigen-specific from cross-reactive antibody responses. Calculating antibody titre, rather than using a single dilution of serum, as a measure of the relative strength of the response, largely accomplished this. Elimination of the carbohydrate moiety of an antigen that can often be the target of cross-reactive antibodies also proved useful.

Figure 1

Antibody isotype responses in infection and co-infection with Litomosoides sigmodontis and malaria. Mice were infected with 25 Ls L3 larvae on day 0 and/or 10⁶ Pcc-infected RBCs on day 60 post-Ls infection. Serum antibody responses measured by ELISA at day 80 post-Ls infection to (A) Ls antigen (LsA) and (B) crude Pcc infected RBC antigen (pRBC). Th2 isotype IgG1 is shown in (Ai) and (Bi) whilst the Th1 associated isotype IgG2a is shown in (Aii) and (Bii). Grey bars represent control mice, black bars represent Pcc mice, white bars the Ls mice and the chequered bars co-infected mice (Pcc-Ls). The letter X highlights those responses that are cross-reactive. Groups not connected by the same letter denote pairs that are significantly different according to Tukey's Pairwise analysis while those that share the same letter do not differ significantly. (C) Polyclonal IgE responses. Black squares represent Ls mice, white squares represent Pcc mice and grey squares represent co-infected (Pcc-Ls) mice. White circles indicate control animals. Data shown in A & B are compiled from 3 experiments (control mice n = 13, Ls mice n = 37, Pcc mice n = 14, Pcc-Ls mice n = 41).

Figure 2

Antibody isotype responses in infection and co-infection with Nippostrongylus brasiliensis and malaria. Mice were infected with 200 Nb L3 larvae and/or 10⁵ Pcc-infected RBCs on day 0. Serum antibody titres (A) IgG1 (B) IgG2a to recombinant Pcc antigen MSP-1₁₉ (i), crude Pcc antigen (pRBC) (ii) and crude Nb antigen (NbA) (iii) were measured at day 20 post-infection for 8 mice per infection group. Black bars represent the Pcc mice, white bars the Nb mice and the chequered bars the co-infected mice (Pcc-Nb). Antibody titres are shown on the y-axis and represent the reciprocal of the greatest dilution at which O.D was greater than the mean plus 3 standard deviations of the O.D values observed for control mouse sera at a 1/200 dilution. The letter X highlights those responses that are cross-reactive. Groups not connected by the same letter denote pairs that are significantly different according to Tukey's Pairwise analysis.

Figure 3

Antibody responses to protein epitopes in infection and co-infection with Nippostrongylus brasiliensis and malaria. The parasite antigens used in ELISA were pre-treated with periodate to disrupt carbohydrate epitopes, in order to determine if cross-reactive responses target the carbohydrate or protein moiety of an antigen. Mice were infected with 200 Nb L3 larvae and/or 10⁵ Pcc-infected RBCs on day 0. Serum antibody titres (y-axis) were measured at day 20 post-infection for 8 mice per infection group and represent the reciprocal of the greatest dilution at which O.D was greater than the mean plus 3 standard deviations of the O.D values observed for control mouse sera at 1/200 dilution. Antibody isotypes (A) IgG1 (B) IgG2a (C) IgG3 to recombinant Pcc antigen MSP-1₁₉ (i), crude Pcc antigen (pRBC) (ii) and crude Nb antigen (NbA) (iii) before (-) and after (+) treatment with periodate are shown. Black bars represent Pcc mice, white bars the Nb mice and the chequered bars the co-infected (Pcc-Nb) mice. (*) denotes pairs which are significantly different according to Tukey's pairwise analysis.