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Comparison of monoclonal and polyclonal antibodies for the detection of canine IgG1 and IgG2, and associations with infection outcome in Leishmania infantum naturally infected dogs

https://doi.org/10.1016/j.vetimm.2009.07.017Get rights and content

Abstract

In murine models of leishmaniasis, IgG subclass expression is a proxy measure for Th1/Th2 cellular immune response bias. However, in dogs, the reservoir of zoonotic visceral leishmaniasis, no consistent association has been described between IgG subclass ratios and disease resistance. Inconsistent results may reflect lack of specificity of commonly used commercial antibodies. Our aim was to measure IgG1 and IgG2 responses to crude Leishmania antigen using commercial polyclonal antibodies for comparison with a panel of commercially unavailable monoclonal antibodies, in a cohort of 60 naturally infected dogs, and to compare associations between subclass responses and clinical or parasitological outcomes. IgG1 and IgG2, measured by both antibodies, were higher in clinically symptomatic than in asymptomatic dogs (P  0.03), reflecting general upregulation of IgG in infected dogs. Unlike the murine model, canine IgG2:IgG1 ratios were not predictive of clinical or parasitological outcomes of infection. Associations between subclass levels and positivity by bone marrow culture and PCR were not consistent when measured with different antibodies. Further research is needed to re-evaluate the specificity of commercially available IgG subclass antibodies.

Introduction

Zoonotic visceral leishmaniasis (ZVL), a vector-borne disease caused by the protozoan parasite Leishmania infantum, results in significant mortality and morbidity in the reservoir host (the domestic dog) and represents a serious public health problem in endemic areas (the Mediterranean basin, Latin America, and parts of central and eastern Asia). Vaccination of the canine reservoir host has been proposed as the most effective control strategy (Dye, 1996), with increasing numbers of experimental ZVL vaccines entering clinical trials in dogs. To validate vaccine immunogenicity, an essential step in vaccine development, there is a need for diagnostic tools to characterize immunological correlates of protective and non-protective canine immune responses to infection and vaccination. In the murine model of Leishmania major, the profile of IgG subclass titres against crude Leishmania antigen (CLA) has been well validated as a proxy measure of T-cell activation, and has been used in evaluation of the immunogenicity of experimental vaccines against the parasite in mice. In particular, a high ratio of specific IgG2a:IgG1 has been associated with a Th1-type pro-inflammatory cytokine response (production of IFN-γ by CD4+ helper T lymphocytes), which in turn promotes resistance to disease caused by L. major infection. Conversely, a comparatively low IgG2a:IgG1 ratio results from preferential activation of a non-protective Th2 response involving production of IL-4 and progression to clinical disease (Snapper and Paul, 1987, Heinzel et al., 1989, Coffman et al., 1993, Germann et al., 1995). Measurement of IgG subclass expression as a proxy for Th1 response is highly attractive as an additional measure of ZVL vaccine immunogenicity in dogs, the target species for vaccination. However, the interpretation of IgG subclass profiles in relation to clinical outcomes in dogs is currently unclear: some studies show greater elevations in IgG2 than IgG1 level in dogs with asymptomatic compared to symptomatic infection (Deplazes et al., 1995, Nieto et al., 1999, Iniesta et al., 2005, Carrillo et al., 2007, Ramos et al., 2008), whilst others show the opposite association (Cordeiro-da-Silva et al., 2003, Reis et al., 2006, Cardoso et al., 2007, Iniesta et al., 2007). One reason for these inconsistent results may be a lack of specificity of the commercially available polyclonal conjugate antibodies used in those studies (Day, 2007). For example, four subfractions of IgG from normal canine serum were found to cross-react with both IgG1 and IgG2 polyclonal antibodies (Mazza et al., 1993). It was suggested that higher specificity for canine IgG subclasses might be achieved using a previously described panel of monoclonal antibodies (Mazza et al., 1994a, Mazza et al., 1994b, Day, 2007). Only two studies to date have employed these monoclonal antibodies to measure IgG subtypes in dogs with ZVL and both have involved cohorts of naturally exposed outbred dogs in Brazil. In the first of these investigations, the IgG2:IgG1 ratio was lower in clinically ill and PCR positive dogs, but signs of disease were associated with generalized elevation in all four IgG subclasses, and great variation between individual dogs meant that relative IgG subclass levels were not a useful marker of resistance or susceptibility (Quinnell et al., 2003). In the second investigation a similar broad elevation in all four subclasses was found in seropositive, parasite positive and symptomatic dogs, but seropositive and asymptomatic dogs had a serological response dominated by IgG1 to the exclusion of IgG2. Moreover, this second study included a group of naïve dogs vaccinated with a commercially available product and these animals had a striking serological response dominated by IgG2 (Oliveira et al., 2009).

The current study tests the association between IgG1 and IgG2 subclasses and clinical/parasitological outcomes using the commercially available polyclonal antibodies on archived samples from the study described above (Quinnell et al., 2003) which were previously tested using monoclonal antibodies. This study is important to establish (1) whether IgG1 and IgG2 subclasses measured by polyclonal antibodies are related to clinical/parasitological outcomes in this population, and if so, (2) whether choice of antibodies will bias associations with these outcomes. Analysis was performed to detect associations between absolute levels and ratios of IgG1 and IgG2 subclasses, and a range of infection outcome measures (clinical signs of leishmaniasis, bone marrow PCR, and parasitological culture). This study represents the first comparison of IgG subclass levels in naturally infected dogs measured with both polyclonal and monoclonal antibodies.

Section snippets

Sample selection

Samples were generated during a longitudinal study comprising a cohort of naturally exposed Brazilian dogs which were followed up in the field for 2 years and subjected to repeated bi-monthly clinical examination, serology (crude parasite ELISA) parasitological culture and bone marrow PCR, as described previously (Quinnell et al., 1997). In that study, Leishmania-specific IgG subclasses were quantified by ELISA using monoclonal antibodies prepared from cell culture supernatants: mAb B6 for IgG1

Results and discussion

The values of Pearson's correlation coefficient, calculated for pairwise comparisons of IgG subclass level measurements, are shown in Table 2. IgG1 units measured by polyclonal and monoclonal antibodies were positively correlated, though the r2 value was not particularly high (Pearson's correlation coefficient = 0.75; r2 = 0.56, P < 0.001). IgG2 units measured by the two antibodies were also significantly correlated, but with a lower r2 value (Pearson's correlation coefficient = 0.54; r2 = 0.29, P < 

Acknowledgements

We thank Dr. E. Nancy Miller at the Cambridge Institute for Medical Research for supplying CLA antigen used in this study. C.C. is supported by a BBSRC/Pfizer doctoral training grant. The work was also supported by the Wellcome Trust.

References (26)

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    Some studies associated asymptomatic infection with higher IgG2 levels compared to IgG1 (Deplazes et al., 1995; Iniesta et al., 2005), while others observed the opposite during active clinical infections (Bourdoiseau et al., 1997; Nieto et al., 1999; Boceta et al., 2000; Almeida et al., 2005; Reis et al., 2006; Cardoso et al., 2007; Teixeira Neto et al., 2010). Carson et al. (2010) described that the levels of both IgG subclasses are higher in SD whereas in a more recent study Lima et al. (2017) reported higher IgG1 levels for SD but no differences in those of IgG2 between AD and SD. It is in this context we aimed the present study to (i) analyze the profile of specific IgG1 and IgG2 antibodies in a cohort of domestic dogs living in Tunisian endemic areas for L. infantum transmission, in order to check whether IgG1 or IgG2 may characterize dog’s clinical status and (ii) investigate whether the parasite-induced production of autoantibodies played a role in the conflicting data reported in the literature.

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