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Lyme disease (20), or Lyme borreliosis, is a tick-borne illness of humans and domestic animals caused by at least three antigenically diverse species of spirochetes (Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii) classified collectively as B. burgdorferi sensu lato. Recent clinical trials showed that monovalent recombinant subunit vaccines composed of the B. burgdorferi outer surface protein A (OspA) lipoprotein were efficacious through two Lyme disease transmission seasons (19, 21).

At the start of feeding, B. burgdorferi spirochetes in ticks are highly vulnerable to OspA antibodies imbibed from immunized hosts (9), but after adaptation to the mammalian host environment following natural or experimental inoculation, most spirochetes down-regulate OspA expression and become resistant to OspA antibodies (3, 5, 9, 14). Protection from tick-borne transmission of B. burgdorferi appears to depend on OspA immunization achieving a critical threshold level of circulating antibodies prior to the tick bite (8).

The addition of mammalian-host-stage antigens may extend the duration, or enhance the level, of protective efficacy of transmission-blocking OspA vaccines against tick-borne Lyme borreliosis. Decorin binding protein A (DbpA) is another B. burgdorferi surface-exposed lipoprotein that has shown vaccine efficacy against experimental infection in the mouse model (5, 10, 13, 14). B. burgdorferi continues to express DbpA, but not OspA, after dermal inoculation and remains vulnerable to DbpA antibodies during the early stages of local and disseminating infection in mice (5, 14); additionally, DbpA is immunogenic during human Lyme disease (6). OspC (25) and other antigens (1, 11) on mammalian-host-adapted B. burgdorferi also represent potential targets for protective or disease-resolving antibodies. We compared the protective efficacies of DbpA and OspA, singly and in combination, against dermal challenge of mice as a first step in the evaluation of second-generation DbpA-OspA combination vaccines for Lyme disease.

The recombinant fusion lipoproteins Lpp2:OspA_N40 (OspA_N40) and Lpp2:DbpA_N40(His)₆ (DbpA_N40) were used as vaccine antigens and had been previously described (5, 14). A detergent extract of Escherichia coli membrane proteins (5) was used as a negative-control antigen preparation.

In one vaccine experiment, four groups of 20 female 7-week-old C3H/HeJ mice (The Jackson Laboratory, Bar Harbor, Maine) were immunized by intraperitoneal injection of 10 µg of DbpA_N40, 10 µg of OspA_N40, 5 µg of DbpA_N40 plus 5 µg of OspA_N40, or 2.5 µg of E. coli protein extract with complete Freund's adjuvant and then, 4 weeks later, were given a second immunization of protein in incomplete Freund's adjuvant. At week 6, five of the mice in each immunization group were challenged by subcutaneous injection, into the dorsolateral thorax, of cloned B. burgdorferi N40 (2) from an exponentially growing culture diluted with BSKII medium (14) to give escalating doses of 10³, 10⁴, 10⁵, or 10⁶ spirochetes in 0.1-ml volumes. Other experiments used vaccines prepared by adsorbing antigens to the aluminum hydroxide adjuvant Alhydrogel (Superfos Biosector, Kvistgård, Denmark). Mice were immunized by subcutaneous injection of 0.1 ml of vaccine at weeks 0, 4, and 8 and challenged with spirochetes at week 10. A challenge dose of 10⁴ was used for B. burgdorferi N40 and Sh-2-82; a dose of 10⁵ was used for B. garinii G25 and B. afzelii IPF. The median infective doses for these isolates were determined to be approximately 3 × 10² for N40 (14), 6 × 10² for Sh-2-82 (14), 3 × 10³ for G25, and 2 × 10⁴ for IPF. Two weeks after challenge, the mice were killed by CO₂ asphyxiation and samples of the inoculation site skin, blood, ear, urinary bladder, and both tibiotarsal joints were cultured in BSKII plus antibiotics to detect spirochetal infection (14).

An enzyme-linked immunosorbent assay was used as previously described (14) to determine the prechallenge DbpA and OspA immunoglobulin G (IgG) endpoint titers of antisera from individual mice and antisera pooled from mice within each immunization group. The borreliacidal activity of the prechallenge antisera was determined, in a microtiter plate format, as the dilution of antiserum from each individual mouse giving 50% growth inhibition by a [³H]adenine metabolic labeling method (15) or as the dilution of pooled antiserum giving a >90% reduction in spirochete numbers (14).

Antigen expression by the spirochetes was evaluated by a direct immunofluorescence assay using combined DbpA_N40- and OspA_B31 (14)-specific purified rabbit polyclonal IgG antibodies conjugated with the fluorochromes Alexa 546 and Alexa 488, respectively, according to the manufacturer's protocol (Molecular Probes, Inc., Eugene, Oreg.). Double-labeled slides were viewed at 1,000× magnification, using a Nikon E600 epifluorescence microscope (Nikon, Melville, N.Y.), and images were acquired with a Sony DKC-5000 digital photo camera (Sony Electronics, Inc., Park Ridge, N.J.).

DbpA and OspA in combination protect against higher B. burgdorferi challenge doses than single-antigen vaccines. The possibility that dual immunity to both DbpA and OspA could provide more effective protection than either single antigen alone was addressed in two complementary ways. First, we attempted to exceed the B. burgdorferi challenge dose at which the single antigens provided protection, and second, we asked whether DbpA-OspA combinations provided protection at a lower vaccine dose that was ineffective for either single immunogen. Nearly all mice immunized with 10 µg of either DbpA_N40 or OspA_N40 were protected from challenge with 10³ or 10⁴ spirochetes (Table 1), as expected from our earlier observations (14). At a challenge dose of 10⁵ or 10⁶ spirochetes, mice immunized with DbpA_N40 or OspA_N40 alone were protected only partially or not at all. In contrast, all mice immunized with the combined DbpA_N40-OspA_N40 vaccine (5 µg of each antigen) were protected against even the highest challenge dose. The 10⁶ challenge inoculum is at least 3,000 times higher than the median infectious dose for this B. burgdorferi strain (14). At each challenge dose, all mice vaccinated with the E. coli extract were infected, with at least three of the five tissues tested being culture positive for B. burgdorferi. The antisera from all mice immunized with either DbpA_N40 or OspA_N40, or the combination of both proteins, inhibited the in vitro growth of B. burgdorferi N40, and antisera from E. coli-immunized mice were not borreliacidal. The in vitro killing potency of the antisera from DbpA_N40-immunized mice was about 20-fold lower than that of OspA_N40, probably because DbpA is expressed at much lower levels than OspA in vitro (5, 14). Interestingly, the potency of OspA_N40 antisera was not significantly different from that of the combined vaccine (P = 0.43, Student's two-tailed t test) in this in vitro assay. Our observations clearly showed that the in vitro and in vivo potencies of DbpA_N40 and OspA_N40 antibodies were divergent and that surrogate in vitro assays were inadequate to predict the relative effectiveness of single-antigen and combined-antigen vaccines. The enhanced in vivo activity of DbpA_N40 antibodies is likely due to prolonged or increased vulnerability of the spirochetes to DbpA_N40 antibodies (5, 14), the potentiating effects of immune effector functions toward DbpA_N40 but not OspA_N40 antibodies, or a combination of the two effects.

DbpA and OspA in combination are more effective than single-antigen vaccines against heterologous B. burgdorferi sensu lato isolates. Next, mice were immunized with single-antigen or DbpA_N40-OspA_N40 combination vaccines formulated with Alhydrogel, an adjuvant approved for use in humans. Antisera from mice immunized with either 1.0- or 10-µg doses inhibited the in vitro growth of B. burgdorferi N40, and again, DbpA_N40 antiserum was less potent for killing in vitro than antisera against OspA_N40 or the DbpA_N40-OspA_N40 combination vaccine (Table 2). Mice were well protected against challenge with a 10⁴ dose of the homologous B. burgdorferi N40 strain when immunized with the 10-µg dose regimen of DbpA_N40 (9 of 10), OspA_N40 (10 of 10), or the DbpA_N40-OspA_N40 combination vaccine (10 of 10). However, the DbpA_N40-OspA_N40 combination vaccine also elicited significant protection (8 of 10) at the 1.0-µg dose, a level at which the single-antigen vaccines were only partially protective. Nearly identical results were also obtained with a 0.1-µg dose and with adjuvant-free formulations of these antigens, demonstrating the intrinsic immunogenicity of the recombinant lipoproteins (data not shown).

Heterogeneity of OspA and DbpA expression by B. burgdorferi may limit efficacy of single-antigen vaccines. Cultures of cloned B. burgdorferi N40 and uncloned B. burgdorferi Sh-2-82 were found to be heterogeneous for DbpA and OspA expression when double labeled and examined by direct immunofluorescence microscopy (Fig. 1). One percent of B. burgdorferi N40 spirochetes appeared to express little or no OspA, and 2 to 3% expressed little or no DbpA. For B. burgdorferi Sh-2-82, the OspA and DbpA variants represented approximately 9 and 11% of the population, respectively. It is likely that phenotypic heterogeneity in the inoculum contributed to the limited efficacy of the single-antigen vaccines (Tables 1 and 2), particularly for B. burgdorferi Sh-2-82. This phenotypic heterogeneity may be relevant to tick-transmitted B. burgdorferi, since one recent study reported that spirochetes in salivary glands of feeding ticks expressed host-stage OspC predominantly but some still expressed OspA (7).

View larger version (82K): [in this window] [in a new window]   FIG. 1.   Detection of B. burgdorferi phenotypic variants deficient in DbpA or OspA by direct immunofluorescence assay. B. burgdorferi N40 and Sh-2-82 spirochetes from standard cultures were fixed to microscope slides and double labeled with a combination of two fluorochrome-conjugated IgGs, prepared against DbpA and OspA. Staining of a representative microscopic field for B. burgdorferi N40 (A to C) and for B. burgdorferi Sh-2-82 (D to F) is shown. Images were acquired alternately with the green filter (A and D) (DbpA labeling), the blue filter (B and E) (OspA labeling), or the triple-band-pass filter (C and F) (DbpA and OspA labeling). Leftward-pointing arrows indicate representative DbpA-deficient spirochetes, and rightward-pointing arrows indicate representative OspA-deficient spirochetes.