Tropomyosin is a well conserved protein among different species and different phyla. Bioinformatic analysis () showed extremely close alignments of filarial tropomyosins from O. volvulus, A. vitae, and B. malayi (partial sequence based on expressed sequence tags) with tropomyosins from other helminths (e.g., A. lumbricoides, A. simplex) and those from mites, cockroaches, and crustaceans. Homology at the amino acid level among the different tropomyosins in which full-length sequence was available varied from 67–98% in identity (e-values ranging from 10−97 to 10−167). The conserved N-terminal DAIKK () and the tropomyosin signature LKEAExRAE were also present in all tropomyosins analyzed (). Filarial tropomyosins also clustered with those tropomyosins known to be allergenic (Ani s 3, Der p 10, Bla g 7, Pen a 1, and others) when phylogenetic trees were constructed (). Of note, the identity between OvTrop and Der p 10 was 72%, (with a similarity of 87% and an e-value of 3 × 10−167), thereby showing a high level of sequence conservation. Moreover, 3-D modeling predictions of Der p 10 and OvTrop were found to be indistinguishable () with both proteins with length sizes of 414.2 Angstroms.
FIG 1 A, Protein sequences of tropomyosin of helminths, mites, cockroaches, and crustaceans were aligned. Identical amino acids have one-letter codes and are black. Conserved amino acid groups are gray; nonconserved amino acids are non-shaded dots. Red letters (more ...)
To address the crossreactivity between filarial tropomyosin and HDM tropomyosin, sera from Fil+ and Fil− individuals were defined as being from atopic or non-atopic donors based on a positive response to a pool of common allergens (Phadiatop®). Positive individuals were screened further for specific reactivity to D. pteronyssinus, and four groups were defined with respect to HDM allergy: 1) Filaria−, non-Atopic or Ni-NA; 2) Filaria−, Atopic or Ni-A; 3) Filaria-infected, Non-Atopic or Fil-NA; and 4) Filaria-infected, Atopic or Fil-A.
Levels of Der p 10-specific and OvTrop-specific IgE, IgG, and IgG4 were measured (; ). The prevalence of IgE and IgG antibody to Der p 10 was increased in the Fil-A group when compared with Ni-A, with 16/49 Fil-A having antigen-specific IgE antibodies and 17/47 having antigen-specific IgG compared with 3/37 in the Ni-A group for IgE and 2/37 for IgG (P < 0.004 for both comparisons). When the RR and OR were calculated comparing the two HDM atopic groups (Ni-A and Fil-A), both analyses showed that Fil+ individuals had significantly increased likelihoods of having increased levels of Der p 10-specific IgE and IgG antibodies (). Although IgG4 anti-Der p 10 antibodies showed a similar trend in the Fil+ group, the RR/OR did not reach statistical significance. Antibody levels to OvTrop had the same pattern seen for antibody levels against Der p 10 ().
FIG 2 Increased prevalence of anti-Der p 10 antibodies in Fil+ subjects. Anti-OvTrop and -Der p 10 antibodies were measured in Ni-NA (Fil−non-atopic), Ni-A (Fil−atopic), Fil-NA (Fil+ non-atopic), and Fil-A (Fil+ atopic). Dots, individual OD (more ...)
Prevalence of IgE and IgG to tropomyosin are increased in filaria-infected populations
When the antibody levels obtained for Der p 10 and OvTrop by ELISA were compared, a strong relationship between levels of the IgE, IgG, and IgG4 anti-OvTrop antibodies and the respective isotypes of anti-Der p 10 (with r > 0.79 and P < 0.0001 for each antibody class tested; ) was seen. To test the crossreactivity between the two tropomyosins, depletion ELISA was performed (). As shown, pre-incubation of Der p 10 positive sera with OvTrop was able to deplete Der p 10-specific IgE, IgG, and IgG4 to levels comparable to sera pre-incubated with Der p 10. Pre-incubation with a recombinant nonhomologous protein (OvGST, used as negative control) showed no depletion of Der p 10-specific antibodies. In addition, sera from mice vaccinated with Der p 10 developed antibodies that not only recognized native tropomyosin in mite extract but also could be inhibited by Der p 10, Ovtrop, mite extract, and crude parasite extract (data not shown). To investigate the functionality of the crossreactive antibody further, we used sera of three individuals (two Fil-A and one Ni-A) positive for anti-tropomyosin (Der p 10 or OvTrop) to passively sensitize basophils. Basophils sensitized with IgE from these three individuals responded indistinguishably to increasing amounts of either Der p 10 or OvTrop antigens by releasing histamine ().
FIG 3 A, Correlation between anti-OvTrop (y-axis) and anti-Der p 10 (x-axis) IgE (left panel), IgG (middle panel), and IgG4 (right panel); each point represents an individual. B, Results of depletion by Der p 10 (closed circles), OvTrop (open circles), or control (more ...)
FIG 4 Der p 10 and OvTrop are functionally indistinguishable for eliciting IgE-mediated responses. Basophils sensitized with sera of atopic and Fil+ (#1 and #2) or atopic but Fil− (#3) individuals were stimulated with increasing concentrations of Der (more ...)
To demonstrate unequivocally that crossreactive antitropomyosin antibodies are induced by filarial infection, sera of nonhuman primates experimentally-infected with L. loa
, one of the few human filarial pathogens that have a nonhuman permissive host, were analyzed for Der p 10-specific IgE. As expected, Fil+
animals developed IgE specific to filarial antigens (BMA) (), while the control animals did not. In the first month following infection, each monkey infected with L. loa
displayed increased levels of IgE anti-Der p 10, with an approximate increase of 50% over pre-infection levels. The peak, however, was reached at the latest time points evaluated, 24–30 months post infection, with increases over 5 times above the pre-infection levels being observed. Not surprisingly, the responses to Der p 10 were paralleled by those to OvTrop. Interestingly, the kinetics of development of the IgE anti-OvTrop or Der p 10 responses were somewhat different from those to BMA. To exclude the possibility of the IgE anti-tropomyosin being a result of polyclonal IgE activation of B cells (as has been postulated based on early animal models),29–31
IgE to timothy was assessed in the sera of infected and uninfected monkeys. Neither the Fil+
nor the control group altered their anti-timothy IgE levels from background levels over the entire course of the infection ().
FIG 5 Crossreactive IgE develops in nonhuman primates infected with Loa loa. Infected monkeys (n = 4) were bled before infection and at various time points after infection; uninfected control monkeys (n = 3) were bled at the same time points. Left panel, pre-infection (more ...)