Characterization of human immunoglobulin (Ig) isotype and IgG subclass response to Bartonella henselae infection

Abstract

Serologic parameters of cat scratch disease (CSD) were evaluated by Western blot analysis. Sera from patients with serologically confirmed CSD antigen were screened for immunoglobulin (Ig) isotype-specific as well as IgG subclass-specific reactivity against Bartonella henselae whole-cell antigen. Bartonella-negative control sera were used to determine baseline antibody activity. Heterogeneous B. henselae-specific IgG reactivity with numerous protein bands, ranging from >150 to <17 kDa, was observed. Though individual banding patterns were variable, one approximately 83-kDa B. henselae protein (Bh83) was immunoreactive with all CSD sera tested, suggesting it is a conserved antigen during infection. Bh83 was not recognized by reference human antisera against Rickettsia rickettsii, Chlamydia group positive, Treponema pallidum, Orientia tsutsugamushi, Fransciscella tularensis, Ehrlichia chaffeensis, Mycoplasma pneumoniae, and Escherichia coli, although other cross-reactive proteins were evident. Significantly, CSD sera failed to recognize the 83-kDa protein when tested against Bartonella quintana antigen, though sera from B. quintana-infected patients did react to Bh83. This cross-reactivity suggests epitope conservation during infection with B. henselae or B. quintana. Western blot analysis further revealed similar banding patterns when B. henselae was reacted against the Ig isotypes IgG and IgG1 and both secretory and alpha chains of IgA. Neither IgM nor IgE reacted significantly to Bartonella antigen by our Western blot analysis. Dissection of the antibody response at the IgG subclass level indicated that prominent antigen recognition was limited to IgG1. These observations provide insight into induced immunity during CSD and provide evidence for conserved epitope expression during infection with B. henselae or B. quintana.

FIG. 1

(a) Western blot analysis of serum IgG (heavy and light chains) activity reacting with SDS-PAGE-separated proteins of B. henselae whole-cell antigen cocultivated with Vero cells. Lane numbers are indicated at the top; serum reference numbers are provided below, and corresponding IFA results are listed in Table 1. Lanes 1, 2, 6, and 10, IgG activity in control sera from patients with negative Bartonella IFA titers (sera no. S1, S2, S6, S10 respectively). Lanes 3, 4, 5, 7, 8, 9, IgG recognition of antigenic proteins by serum from patients with IFA titers positive for CSD (sera no. S3, S4, S5, S7, S8, S9 respectively). (b) IgG Western blot reacting with B. henselae whole-cell antigen prepared on rabbit blood agar plates. Lanes 1 and 9, antibody activity in seronegative control specimens (sera no. S11 and S17 respectively). Lanes 2 through 8, antigen recognition revealed by CSD-positive patient serum IgG (sera no. S12, S13, S14, S15, S8, S16, S9 respectively). Positions of molecular size standards (in kilodaltons) are indicated at the right. An arrow in each panel denotes the location of Bh83, recognized exclusively by all CSD IFA-positive sera tested.

FIG. 2

Western blot analysis of IgG (heavy and light chains) of various sera reacting with other bacterial pathogens for reactivity to B. henselae agar-derived antigen. Lanes 1, 3, 5, 7, 9, and 11, IFA-positive CSD sera; lane 2, R. rickettsii; lane 4, F. tularensis; lane 6, F. tularensis negative control; lane 8, T. pallidum; lane 10, Chlamydia; lane 12, Rickettsia prowazekii. Positions of molecular size standards (in kilodaltons) are indicated at the right. An arrow denotes the location of Bh83.

FIG. 3

Western blot analysis of IgG (heavy and light chains) of IFA-positive CSD sera (lanes 1 to 7) reacted against B. henselae (a) and B. quintana (b) antigens. Lanes 1 to 4 contain sera from patients with PCR-confirmed B. quintana infection. Positions of molecular size standards (in kilodaltons) are indicated at the right. An arrow in each panel denotes the location of Bh83.