View larger version (16K): [in this window] [in a new window]   Figure 1. Flow cytometry analysis of in vitro expression of the scFv. Binding of Seam 3 mAb to permeabilized COS-7 cells transfected with an expression plasmid containing the scFv G1 gene (bottom, pS.scFvG1) or the empty vector pCI-neo (top). After being transiently transfected, cells were permeabilized and exposed to the Seam 3 mAb. FITC-conjugated anti–mouse IgG was used to detect Seam 3 binding. Horizontal lines indicate the gates that were defined to exclude background binding.

View larger version (16K): [in this window] [in a new window]   Figure 2. Bactericidal activity in sera from scFv-immunized mice. (A) Serum bactericidal activity in mice immunized with different doses (25, 75, and 150 µg) of an expression plasmid containing the scFv G1 gene fused to a signal peptide sequence (pS.scFvG1) or with 150 µg of the G1 protein (G1 scFv–KLH). Sera from mice administered with the empty vector (pCI-neo) were used as controls. Arrows indicate immunization times. Data represent geometric means + SD of eight determinations, with each conducted in duplicate on a different animal. *, P < 0.05 compared with empty vector sera as determined by ANOVA and Student-Newman-Keuls test. (B) Bactericidal titers in sera from mice immunized with various types of G1 scFv gene–containing plasmids (for a description see Table I). 10 animals per group were immunized on days 0, 21, and 42 with 150 µg DNA in PBS i.m. or, for comparison, with N-Pr MenB–TT (4 µg sialic acid) s.c. in CFA (day 0) and IFA (days 21 and 42). Sera were collected at day 56. Each marker indicates data from an individual mouse with mean geometric titers represented by a horizontal bar. P < 0.05, as determined by ANOVA and Student-Newman-Keuls test.

Specificity of the antibodies induced by DNA vaccination
Next, we verified that the antibodies induced by scFv gene immunization were directed against their intended target, that is the MenB CP. In these experiments, we focused on sera obtained from pT.scFvG1-immunized animals because these sera showed the highest titers (Fig. 2 B). The bactericidal activity of such sera was inhibited, in a dose-dependent fashion, by purified N-Pr MenB CP or the G1 scFv protein but not by a control, irrelevant scFv (designated H6; Fig. 3 A). Moreover, bactericidal activity was observed only with encapsulated MenB strains, but not with serogroups MenA or MenC (Fig. 3 B). These data indicated that vaccination with the G1 scFv gene induced anti-scFv antibodies that specifically cross reacted against the MenB capsule, thereby producing bacterial killing. Although we have previously shown that the antibodies induced by our antiidiotypic protein did not cross react with human PSA (13), it was of interest to verify that this also occurred after DNA immunization, especially in consideration that the latter can broaden the repertoire of recognized epitopes (20). Therefore, sera from pT.scFvG1-immunized animals were tested against neuraminidase-treated or untreated cells from the CHP 212 human cell line expressing high levels of PSA. The Seam 26 mAb, which is known to react with both human and MenB PSA, was used as a positive control. As expected, this mAb showed strong reactivity against untreated, but not neuraminidase-treated, cells (Fig. 3 C). Immune sera were totally devoid of reactivity against either untreated or neuraminidase-treated CHP 212 cells, indicating that human PSA cross-reactive antibodies were not induced by scFv gene immunization.

View larger version (18K): [in this window] [in a new window]   Figure 3. Immunization with the scFv gene induces capsule-specific antibodies. (A) Inhibition of bactericidal activity by increasing doses of purified N-Pr MenB CP (MenB CP) or the G1 scFv protein. An irrelevant scFv (designated H6) was used as a control. A serum sample (with a bactericidal titer of 576) from an animal immunized with the pT.scFvG1 plasmid was mixed with the inhibitors and tested in the bactericidal assay at a final dilution of 1:300. Data represent CFU counted after completion of the assay. This experiment is representative of three, with each conducted on a different serum sample. (B) Bactericidal activity against different meningococcal strains of serum samples obtained from pT.scFvG1-immunized animals. The indicated MenB, MenA, and MenC strains were tested. Data represent geometric means and SD from five determinations, with each conducted on a different animal. (C) Reactivity against human PSA of sera from pT.scFvG1-immunized animals. Neuraminidase-treated or untreated cells from the PSA-rich CHP 212 human neuroblastoma cell line were fixed in the wells of microtiter plates. Sera were diluted 1:20 and tested for binding to cell-coated plates by ELISA. Seam 3 and Seam 26 mAbs were used, respectively, as negative and positive controls at a concentration of 5 µg/ml. Data represent means + SD of five determinations conducted with the same serum samples used in B. Preimmune and immune samples were collected, respectively, on days 0 and 56 after the first immunization.

View larger version (25K): [in this window] [in a new window]   Figure 4. Measurement of anti-scFv and anti-MenB antibodies in sera from scFv gene–immunized mice. Serum samples obtained from mice immunized with the scFv protein (A) or the pT.scFvG1 plasmid (B) were tested at a 1:300 dilution for antibody binding to plates sensitized with the G1 scFv. Binding was measured by ELISA using IgM- and IgG-specific reagents. For each group, five serum samples, each obtained from a different animal, were tested. (C and D) Isotype distribution of anti-MenB antibodies in sera from animals immunized with the G1 scFv gene. Sera from pT.scFvG1-immunized animals were tested at a 1:200 (C) or a 1:100 (D) dilution for antibody binding to bacteria-coated plates. Antibody binding was detected by ELISA using isotype-specific reagents. Data represent means and SD from five determinations, with each conducted on a different animal. Preimmune and immune samples were collected, respectively, on days 0 and 56 after the first immunization.

Protective effects of passively administered antibodies
To completely assess the functional properties of the antibody response induced by scFv gene immunization, we ascertained the ability of immune sera to passively protect infant rats against meningococcal bacteremia. In these experiments we measured the number of blood CFU in pups inoculated with pools of sera obtained before or after pT.scfvG1 immunization and challenged i.p. with MenB strain 2996. As positive controls, groups of pups were treated with a pool of sera from N-Pr MenB–TT–immunized animals or with the Seam 3 mAb. Pretreatment with the pT.scfvG1 immune serum pool (diluted up to 1:8) (P < 0.05) significantly protected pups from bacteremia (Table II). These effects were similar to those observed with the serum pool from N-Pr MenB–TT–immunized animals. In contrast, animals inoculated with a preimmune serum pool or with a pool obtained after immunization with the empty plasmid were not protected (Table II). These data indicated that immunization with pT.scFvG1 induced serum antibodies capable of affording passive protection against systemic spreading of MenB.

	DISCUSSION

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After exploring different strategies in this paper, it was possible to consistently induce bactericidal and protective activity in the sera of animals immunized with the gene encoding for our scFv mimic. Serum bactericidal activity was totally accounted for by scFv-specific antibodies that cross reacted with the MenB capsule, as shown by complete abrogation of killing by competing G1 scFv or MenB CP. Moreover, immune sera could not kill meningococci with MenA or MenC capsules. The antibodies induced by scFv gene immunization were predominantly of the IgG2a isotype, which is typical of a Th1 response. Finally, the induced antibodies were non–cross-reactive with human PSA.

The importance, in terms of protection in humans, of the bactericidal responses observed in our experiments after scFv gene vaccination remains, of course, to be established. It should be noted, in this respect, that bactericidal activity was tested using rabbit complement, which produces considerably higher bactericidal titers than human complement (22). Nevertheless, it is encouraging that the antibodies induced by scFv gene immunization were protective in a well characterized in vivo model, such as the infant rat model.

This paper illustrates the potential of DNA vaccination–based strategies to augment antibody responses against protein mimetics. The versatility and ease of manipulation of gene vaccination allowed us to quickly and efficiently screen several approaches, including the addition of T helper epitopes (23, 24). Unexpectedly, the most successful strategy that resulted in markedly augmented bactericidal titers involved deletion of the secretory signal peptide sequence initially placed before the scFv (Fig. 2 B). In DNA vaccination, homologous or heterologous signal sequences are generally used to direct the antigen into the endoplasmic reticulum of the transfected cell, thus leading to secretion, with the rationale of augmenting availability of the immunogen for uptake by antigen-presenting cells. However, there are few data on the relationships between the cellular localization of the antigen and the type and extent of the immune response. In a comparative study, two plasmids either containing or lacking a signal sequence produced similar antibody levels despite differential intracellular targeting of the encoded antigen (25). In another study, cytoplasmic ovalbumin induced lower IgG1 but higher IgG2a than secreted ovalbumin (26). Interestingly, mouse immunization with a plasmid encoding a modified version of carcinoembryonic antigen (CEA), devoid of its signal peptide and fused to a T helper epitope, resulted in higher anti-CEA antibody levels relative to those observed using the wild-type CEA plasmid (27). Further studies will be necessary to clarify whether the mechanisms underlying the effects reported in these papers, as well as in the present one, involve differential antigen uptake and/or processing by antigen-presenting cells. Irrespective of the mechanisms involved, our data strongly indicate that manipulation of secretory signals deserves further exploration in the challenging task of optimizing antiinfectious DNA vaccines. In our hands, removal of secretory signals recently proved highly effective in increasing the immunogenicity of two additional unrelated mimics (unpublished data). Thus, this strategy may be widely applicable in the field of peptide mimotopes or recombinant antiidiotypes.

Interestingly, in this study immunization with the G1 scFv gene induced total anti–G1 scFv antibody levels that were similar, by ELISA, to those induced by the corresponding protein (Fig. 4, A and B). Yet, as discussed above, bactericidal activity was markedly higher after gene vaccination. This suggests that (a) a heterogeneous response is induced by scFv immunization, in which only a portion of the induced antibodies is cross-reactive with the MenB CP, and (b) MenB cross-reactive antibodies are preferentially induced by gene, not protein, scFv immunization. One of the interesting features of genetic immunization, which may perhaps explain these findings, is its ability to change the hierarchy of immunodominance of the recognized epitopes relative to that induced by conventional immunization. For example, DNA encoding for the mycobacterial antigen Ag85 increased the number of recognized Ag85 T cell epitopes over those recognized after immunization with live bacteria and changed the hierarchy of immunodominance in favor of the newly recognized epitopes (20).

Studies are underway to determine the epitope specificity and relative frequency of B cell and T cell subpopulations activated by scFv gene, compared with protein, immunization. We are also testing the hypothesis that the increased functional activity of the antibodies induced by gene vaccination is related to the adjuvant-like properties of the immunizing plasmids (23). Interestingly, major adjuvant-dependent differences have been documented in the ability of N-Pr MenB, the antigen mimicked by our scFv, to induce bactericidal activity. For example, N-Pr MenB–TT induced bactericidal activity when given in Freund's adjuvant (8, 13) but not in alum (28). In contrast, a conjugate of N-Pr MenB with porin B, a protein with adjuvant-like properties (29), could induce bactericidal activity using either adjuvant (28).

Our data are in general agreement with previous reports dealing with immunization with minigenes encoding for peptide mimotopes. DNA vaccination was recently used to redirect the immune system from a Th2 to a more effective Th1 response (30). Moreover, a similar approach was successful in inducing serum bactericidal activity against MenC CP (31) and antibodies directed against the type 4 pneumococcal CP (32).

Collectively, these data indicate that DNA immunization offers new ways of stimulating the immune system and suggest that these features can be exploited in the prevention of diseases caused by encapsulated bacteria. This is especially relevant for infections, such as those caused by MenB, for which no vaccine is available because of the failure of conventional approaches. Moreover, despite considerable success, existing conjugate vaccines are not without problems, including complexities in polysaccharide production and conjugation. These difficulties can become particularly challenging with vaccines consisting of multiple conjugates. In contrast, it seems relatively easy to clone different mimics in a single vector for vaccinating against pathogens with multiple serotypes. Thus, the global use of vaccines directed against encapsulated bacteria would be facilitated by the development of effective DNA vaccines, especially in consideration of some additional advantages such as their low cost and independence from the cold chain.

	MATERIALS AND METHODS

Top Abstract RESULTS DISCUSSION MATERIALS AND METHODS References

 
Bacterial strains and reagents.
Meningococcal strains 2996, 8047, and MC58 (MenB), F8238 and A1 (MenA), and C11 (MenC) were provided by M.M. Giuliani (Chiron Corp., Siena, Italy). Purified N-Pr MenB CP and the Seam 3 and Seam 26 mAbs were provided, respectively, by A. Bartoloni and M. Mariani (Chiron Corp., Siena, Italy). The G1 and the irrelevant H6 scFvs were expressed recombinantly in Escherichia coli and purified as previously described (13, 18, 33). For immunization experiments, the G1 scFv–KLH and the N-Pr MenB–TT conjugates were prepared as previously described (13).

DNA constructs and expression analysis.
To generate plasmids for DNA vaccination (Table I), we used pCI-neo, a mammalian expression vector (Promega). The G1 scFv–encoding sequence was PCR-amplified as previously described (13–18) and ligated into the multiple cloning site of pCI-neo, generating pscFvG1. In the design of pS.scFvG1, the leader sequence was inserted at the beginning (i.e., at the 5' NheI/EcoRI flanking site) of the G1 scFv gene. To produce the pT.scFvG1, a tetanus toxin universal T helper cell epitope (19) was inserted at the beginning of the scFv. Finally, a plasmid (pST.scFvG1) was generated containing both the secretory sequence and the T helper cell epitope before the scFv gene.

Plasmid preparation.
Plasmids for in vitro transfection or mouse immunization were grown in E. coli DH5 and purified using EndoFree Plasmid Maxi or Giga kits (QIAGEN). Each lot of plasmid DNA had a A₂₆₀/A₂₈₀ ratio 1.8 (as determined by UV spectrophotometry), endotoxin content 0.1 EU/µg DNA (as determined by Limulus Amebocyte Lysate assay kit; Associates of Cape Cod Inc.), and a predominantly supercoiled form.

FACS analysis.
The ability of engineered DNA constructs to express functional G1 scFv was analyzed by flow cytometry. A subconfluent monolayer of COS-7 cells (CCL-70, a monkey kidney fibroblast cell line; American Type Culture Collection) was transiently transfected with 2.5 µg of vector DNA per 10⁶ cells in a synthetic cationic lipid solution (TransFast; Promega). The pCI-neo mammalian vector (empty vector) was used as a control. After 48 h, the transfected cells were washed in PBS (0.01 M phosphate, 0.15 M NaCl, pH 7.2), trypsin treated, and fixed overnight with 1 ml paraformaldehyde (2.5 mg/ml). The cells were then permeabilized with 1 ml PBS containing 0.2% (vol/vol) Tween 20 (PBS-Tween) and incubated with the Seam 3 mAb (4 µg/ml in PBS) for 2 h at 37°C. 10 µg/ml FITC-labeled rabbit anti–mouse IgG (Abcam Ltd.) was used to detect bound Seam 3.

Immunizations.
For DNA immunization, 6–8-wk-old BALB/c mice (Charles River Laboratories) were injected in the quadriceps muscle with purified DNA at the doses indicated in the figures in 50 µl of PBS. Mice were immunized on days 0, 21, and 42 with equal plasmid doses, and tail veins were bled on days 0, 36, and 56 to obtain sera. Moreover, groups of mice were immunized with scFv G1–KLH or with N-Pr MenB–TT conjugates in Freund's adjuvant, as previously described (13).

Bactericidal assay.
The bactericidal assay was performed as previously described (13) with minor modifications. In brief, bacteria were grown to the early stationary phase and mixed in equal volumes with serially diluted (ranging from 1:3 to 1:768) heat-inactivated serum and undiluted baby rabbit complement (Cederlane). The reciprocal of the highest final serum dilution causing (50% killing of the inoculum was recorded as the bactericidal titer. Because the lowest final serum dilution tested was 1:9, the lower limit of detection of the assay was a titer of 9. To assess inhibition of bactericidal activity, mixtures of twofold serial dilutions of inhibitor and diluted serum were incubated for 20 min at 37°C. After adding complement and bacteria, the test was completed as described above.

ELISA tests.
To determine the isotype distribution of anti–MenB antibodies, we used a whole bacteria ELISA (13). Anti–human PSA antibodies were detected by ELISA, using untreated or neuroaminidase-treated neuroblastoma CHP 212 cells, which express high levels of PSA. Both of these assays were performed exactly as previously described (13). Binding of serum antibodies to the scFv G1 was measured by an identical ELISA test, with the exception that plates were sensitized with 2 µg/ml of purified scFv.

Infant rat model of passive protection.
To study the protective effects of sera obtained from immunized animals, we used an infant rat model, exactly as described previously (34). In brief, 5-d-old Wistar rats (Charles River Laboratories) were inoculated i.p. with serially diluted mouse sera and, 2 h later, challenged i.p. with 8 x 10³ CFU MenB (strain 2996). Blood samples were obtained at 18 h after challenge, serially diluted, and plated onto chocolate agar (100 µl/plate). Because the lowest plated dilution was 1:10, the lower detection limit of the assay was 100 CFU/ml of blood. Pups were considered protected from bacteremia in the presence of sterile blood cultures. All animal experiments reported in this paper were approved by the Department of Pathology and Experimental Microbiology Committee for Animal Studies and Istituto Superiore di Sanità .

Data expression and statistical analysis.
Bactericidal titers were converted to log₂ titer values to calculate means and SDs and to assess statistical significance using one-way analysis of variance (ANOVA) and Student-Keuls-Newman test. For the purpose of calculating means and SDs, sera with bactericidal activities below the detection threshold (i.e., with a titer <9) were given an arbitrary titer of 4.5 (i.e., half the lower detection limit). Differences in the frequency of protected animals were analyzed using Fisher's exact test.