Antigenic variation in Treponema pallidum: TprK sequence diversity accumulates in response to immune pressure during experimental syphilis

Abstract

Pathogens that cause chronic infections often employ antigenic variation to evade the immune response and persist in the host. In Treponema pallidum (T. pallidum), the causative agent of syphilis, the TprK Ag undergoes variation of seven V regions (V1-V7) by nonreciprocal recombination of silent donor cassettes with the tprK expression site. These V regions are the targets of the host humoral immune response during experimental infection. The present study addresses the causal role of the acquired immune response in the selection of TprK variants in two ways: 1) by investigating TprK variants arising in immunocompetent versus immunosuppressed hosts; and 2) by investigating the effect of prior specific immunization on selection of TprK variants during infection. V region diversity, particularly in V6, accumulates more rapidly in immunocompetent rabbits than in pharmacologically immunosuppressed rabbits (treated with weekly injections of methylprednisolone acetate). In a complementary experiment, rabbits preimmunized with V6 region synthetic peptides had more rapid accumulation of V6 variant treponemes than control rabbits. These studies demonstrate that the host immune response selects against specific TprK epitopes expressed on T. pallidum, resulting in immune selection of new TprK variants during infection, confirming a role for antigenic variation in syphilis.

Figure 1. Effectiveness of pharmacological treatment and tprK expression

Significant delay in development of VDRL antibody titers (Panel A), reduced IFN-γ expression (Panel B), and longer persistence of T. pallidum cells in lesions from suppressed rabbits (Panel C) confirmed the effectiveness of pharmacological immunosuppression. Pharmacological treatment did not alter tprK mRNA levels in treponemes from control and treated rabbits (Panel D).

Figure 2. Accumulation of diversity in tprK V regions determined by FLA analysis

RSDI (reciprocal of Simpson's diversity index) values show that, in immunocompetent rabbits infected with the Chicago C1 strain, sequence diversity tends to accumulate more rapidly in V6, V4, V5, and V7 than in treated rabbits (Panels D-G). No difference in diversity was seen for V1, V2, and V3 (Panels A-C) between the two groups of rabbits.

Figure 3. Accumulation of diversity in tprK V regions determined by sequence analysis

Diversity Score (DS) values for TprK V1-V7 (Panels A-G) at weeks 1-5 after infection with the Chicago C2 strain. The sequences of at least 10 TprK variants were determined for each lesion per time point. For each group of sequences, DS is calculated as the number of new V regions detected divided by the total number of sequences determined. When DS = 0, no new variants are detected compared to the inoculum, while a value of 1 indicates that none of the sequences is identical to the known inoculum sequences. DS values were found to be significantly higher (p<0.05) in controls with respect to suppressed rabbits for V6 (Panel F) at week 2 of infection.

Figure 4. Correlation of development of anti-V6 antibody with presence of V6 variants

In immunocompetent rabbits, development of antibodies against the predominant Chicago C1 V6 variant steadily increases during experimental infection (panel A, Δ Symbol). Anti-V6 antibody however, fails to develop until later in infection and is significantly lower in titer in treated rabbits than in controls (p<0.05) at week 3 and 4 after infection (Panel B, ▲ Symbol). In both groups, developing anti-V6 antibody parallels the accumulation of new V6 variants seen by FLA analysis (reported in Fig. 2F and here again for comparison purposes) and sequencing (Fig. 3F), suggesting a role for antibody in selection of new TprK variants in vivo.

Figure 5. Accumulation of sequence diversity in tprK V5 and V6 regions in pre-immunized rabbits

In Chicago C2-infected rabbits, V5 diversity is not significantly different in rabbits previously immunized with V5-KLH, V6-KLH, or KHL alone, or in unimmunized controls (Panel A). In contrast, V6 variants more readily are seen in rabbits previously immunized with V6-KLH, compared to V5-KLH- or KLH-immunized rabbits or in unimmunized controls (Panel B).