Chlamydia trachomatis polymorphic membrane protein D is a species-common pan-neutralizing antigen

Abstract

Infections caused by the obligate intracellular pathogen Chlamydia trachomatis have a marked impact on human health. C. trachomatis serovariants are the leading cause of bacterial sexually transmitted disease and infectious preventable blindness. Despite decades of effort, there is no practical vaccine against C. trachomatis diseases. Here we report that all C. trachomatis reference serotypes responsible for sexually transmitted disease and blinding trachoma synthesize a highly conserved surface-exposed antigen termed polymorphic membrane protein D (PmpD). We show that Ab specific to PmpD are neutralizing in vitro. We also present evidence that Ab against serovariable-neutralizing targets, such as the major outer membrane protein, block PmpD neutralization. This finding suggests that a decoy-like immune evasion strategy may be active in vivo whereby immunodominant type-specific surface antigens block the neutralizing ability of species-common PmpD Ab. Collectively, these results show that PmpD is a previously uncharacterized C. trachomatis species-common pan-neutralizing target. Moreover, a vaccine protocol using recombinant PmpD to elicit neutralizing Ab in the absence of immunodominant type-specific Ab might be highly efficacious and surpass the level of protection achieved through natural immunity.

Fig. 1.

Rabbit 155-kDa antiserum is specific for PmpD. (A) Protein gel of EB and rPmp E. coli lysates used in immunoblotting. (B) Immunoblot with absorbed 155-kDa antiserum. Lanes from left to right are E EB, partially purified rPmp polypeptides A, B, C, D1, D2, D3, E, F, G, H, and I. ∗, the 155-kDa antiserum recognized two polypeptides (≈80 and 42 kDa) in the EB lysate and all three rPmpD polypeptides. The marked polypeptides in A correspond to the polypeptides recognized by the 155-kDa antiserum in B.

Fig. 2.

PmpD is a species-common C. trachomatis Ag. HeLa cells infected with the 15 C. trachomatis (A–L3) serovars and C. muridarum (MoPn) were stained by indirect fluorescent Ab with anti-PmpD serum and anti-LPS. Inclusions for each of the C. trachomatis serovars (A–L3) reacted strongly with anti-PmpD. In contrast, inclusions of C. muridarum (MoPn) failed to react with anti-PmpD. Inclusions of all 16 strains reacted with anti-LPS.

Fig. 3.

PmpD is immunoaccessible by immunodot blot and scanning immunoelectron microscopy. (A) D EB were blotted onto nitrocellulose membrane and incubated with different mAb (Upper) and anti-PmpD and preimmune sera (Lower). MAb against MOMP and LPS reacted with EB, whereas mAb specific to HSP60 was nonreactive. Anti-PmpD reacted with EB, but preimmune serum was nonreactive. Protein concentrations are shown for mAb and reciprocal dilutions for anti-PmpD and preimmune sera. (B) L2 EB bound to paraformaldehyde-fixed HeLa cells were reacted with anti-PmpD (Left) or preimmune sera (Right), then probed with gold-conjugated anti-rabbit IgG. Immunogold labeling of PmpD is shown as bright spheres on the EB surface (Left). In contrast there was no association of gold with bound EB incubated with preimmune sera (Right). Left arrow indicates gold-labeled EB; right arrow indicates unlabeled EB. (Scale bar: 0.25 μm.)

Fig. 4.

PmpD is a target of neutralizing Ab. The neutralizing activity of anti-PmpD was assayed against C. trachomatis serovars A, Ba, C, D, E, F, G, K, L2, and C. muridarum (MoPn). Results are expressed as the reciprocal of the serum dilution resulting in 50% reduction in IFU (50% endpoints) in HaK cells. The experiment was repeated twice in triplicate; representative data from a single experiment are depicted. Anti-PmpD neutralized C. trachomatis serovars but failed to neutralize the infectivity of C. muridarum (MoPn).

Fig. 5.

Ab to MOMP and LPS inhibit anti-PmpD-mediated neutralization of chlamydial infectivity. D EB were incubated with different primary and secondary Ab, then plated onto HaK monolayers and assayed for IFU to determine percent neutralization. Both anti-PmpD and mAb to MOMP were neutralizing, whereas mAb to LPS was not neutralizing. Preincubation with mAb to LPS or MOMP significantly blocked the ability of anti-PmpD to neutralize infectivity when compared to anti-PmpD alone (∗, P < 0.05 by using two-tailed unpaired t test). Preincubation with anti-PmpD negated the blocking effect of mAb to LPS or MOMP.

Fig. 6.

Gene arrangement of the pmp gene family in the C. trachomatis and C. pneumoniae genomes. The sequenced C. trachomatis genomes are ≈1.04 Mb, whereas the C. pneumoniae genomes are ≈1.23 Mb in size. The gene families are identified via differing colors, as originally assigned by Grimwood and Stephens (40). pmpA, yellow; pmpB/C, green; pmpD, red; pmpE/F, orange; pmpG, blue; pmpH, purple; pmpI, black. The chromosomal distance between each pmp gene/gene cluster is indicated in kilobases (kb).