Weak Anamnestic Responses of Inbred Mice to Yersinia F1 Genetic Vaccine Are Overcome by Boosting with F1 Polypeptide While Outbred Mice Remain Nonresponsive¹

In many countries, Yersinia pestis is an endemic pathogen that occasionally infects domestic animals and humans. Enteropathogenic Yersinia and the plague-producing Y. pestis both initiate infections in lymphatic tissues. However, contrasting pathologies are presented (1). The orally transmitted Yersinia pseudotuberculosis and Yersinia enterocolitica generally cause self-limited disease, characterized by abscess formation, mesenteric lymphadenitis, and ileitis (2, 3). In contrast, Y. pestis is transmitted by the bite of an infected flea or by inhalating infectious aerosol (4). These two routes of exposure give rise to either regional lymphadenitis or pneumonia. Rapid extracellular expansion of Y. pestis, unchecked by the immune system, leads to septicemia, lung colonization, and organ failure, with necrotic focal lesions of visceral organs.

The Yersinia adherence molecule invasin binds to β integrins on the surface of a variety of cells. The interaction of β integrin with invasin is required for Yersinia to attach to mammalian cells and promotes cellular internalization (5). However, contact with the mammalian cell membrane stimulates the expression and cytoplasmic translocation of Yersinia outer proteins (Yops)³ into the cytosol to block phagocytosis (6). The activity of YopH, a protein-tyrosine phosphatase, prevents transduction of a cellular internalization signaling event (6, 7, 8, 9), YopE indirectly disrupts filamentous actin polymerization (6), and YpkA, a serine/threonine kinase (10), may interfere with signal transduction.

Yops are encoded by a plasmid that is homologous in all pathogenic Yersinia. However, Y. pestis does not synthesize either invasin or YadA, another adhesin produced by Y. enterocolitica and Y. pseudotuberculosis. Consequently, no established adhesin has been described for Y. pestis, although the pH 6 Ag has been suggested to play such a role (11). Translocation of Yops by Y. pestis in cultured mammalian cells required introduction of YadA in some reports (8), while the results of another study (12) showed translocation by unmodified Y. pestis. Thus, it remains unclear whether cytosolic delivery of Yops occurs in vivo during Y. pestis infection. An IS200-like element inserted into the chromosomal gene (13) results in defective expression of invasin by Y. pestis. Infection with enteropathogenic Yersinia initiates class I-dependent CTL responses (14). It remains a possibility that the host immune response to Y. pestis may be deficient without the immune stimulation provided by adherence to cellular substratum and cytosolic delivery of bacterial Ags. Adhesin-independent infections may promote systemic dissemination by preventing CTL-mediated control of primary foci. To test this possibility, we used a genetic vaccine, consisting of sequences encoding the fraction 1 (F1) capsule Ag, to simulate cytosolic insertion of pathogen-derived Ags and examined Ab and T cell immunity resulting from intracellular expression of Y. pestis proteins.

Recombinant F1 polypeptide (rF1) was prepared as previously described (15). Endotoxin was removed by passing rF1 over a column of immobilized polymyxin B (Detoxi-Gel, Pierce, Rockfort, IL) as previously described (15). The F1 gene was isolated from a genomic clone of the caf operon (16) using PCR and synthetic oligonucleotides of sequences flanking the gene. The plasmid pREF1 was made by ligating the F1-coding fragment into the mammalian cell expression vector pREP4 (Invitrogen, Carlsbad, CA) downstream of a Rous sarcoma virus promoter. A hygromycin resistance gene is also contained within pREF1. Plasmids were propagated in Escherichia coli strain DH5α (Invitrogen) and were isolated by alkaline lysis, followed by anion exchange chromatography (Qiagen, Valencia, CA). Plasmids were dialyzed extensively against PBS (pH 7.4) to remove residual endotoxin. The final preparations of plasmid or rF1 contained <1 U of endotoxin/injection, as determined by a Limulus lysate assay (BioWhittaker, Walkersville, MD).

The mouse cell line P815 was obtained from American Type Culture Collection (Manassas, VA). Cells that constitutively expressed the F1 polypeptide were produced by transfecting P815 with pREF1 and selecting stably transformed cells by hygromycin resistance using methods previously described (17). Plasmid DNA (5 μg) was mixed with 15 μl of Lipofectin (Life Sciences, Gaithersburg, MD) in 200 μl of serum-free medium (Eagle’s MEM) and added to the cells for 12 h (37°C, CO₂ incubator). The transfected cells were subcultured into 4 ml of Eagle’s MEM containing 5% FBS and 400 U/ml of hygromycin (Calbiochem, San Diego, CA). Colonies appearing in 10 to 14 days were maintained in culture medium containing hygromycin (400 U/ml).

Female BALB/c mice (4 mo old; Harlan Sprague-Dawley, Frederick, MD) were injected i.m. (rectus femoris muscle) with 10 μg of pREF1, plasmid without gene insert (pREP9) in 50 μl of PBS (50 mM Na₂HPO₄ and 140 mM NaCl, pH 7.4), or with 10 μg of purified rF1 in PBS resuspended in alhydrogel adjuvant (E. M. Sergeant Co., Nutley, NJ). A total of three injections were given, 2 wk apart, unless indicated otherwise. Sera were collected from tail veins for Ab analyses before and 1 wk after each injection.

In conducting research using animals, the investigators adhered to the Guide for the Care and Use of Laboratory Animals, prepared by the committee on care and use of laboratory animals of the Institute of Laboratory Animal Resources, National Research Council.

Purified rF1 (1 μg/well in 100 μl of PBS) was used to coat the wells (37°C, 2 h) of 96-well plastic plates (Immulon, San Francisco, CA). The plates were then blocked (37°C, 4 h) with 100 μl of 0.2% casein in PBS and washed with PBS containing 0.1% Tween-20 (Sigma, St. Louis, MO). Preimmune or immune mouse sera were diluted in PBS containing 0.02% casein, and 100 μl of each dilution was added to duplicate wells (37°C, 2 h). The plates were washed with PBS and incubated (37°C, 1 h) with a 1/2000 dilution of peroxidase-conjugated goat anti-mouse Ab (Pierce). The plates were washed, and 100 μl of ABTS (Kirkegaard Perry, Gaithersburg, MD) was added. After 20 min, 100 μl of 1% SDS was added to stop the reaction. Peroxidase levels were detected by measuring the absorbance at 405 nm.

Immunized and control mice were administered 400 to 800 × LD₅₀ of Y. pestis CO92 (LD₅₀ = 2.3 × 10⁴ CFU) by nasal aerosol, as previously described (15). All infected animals were monitored daily until 28 days postchallenge.

Spleens were removed from mice, and mononuclear cell preparations were prepared as previously described (18). The mononuclear cells (5 × 10⁶/well) were cultured with syngeneic dendritic cells (5 × 10⁵/well), obtained as previously described (18), in 24-well plates containing a 20 μg/ml final concentration of purified rF1. Culture medium consisted of RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 10 μM HEPES, 50 μM 2-ME, 100 IU/ml penicillin, and 100 μg/ml streptomycin. The mononuclear cell cultures were harvested after 5 days of incubation (37°C, 5% CO₂/95% humid air) for lymphocyte cytotoxicity assays.

To measure Ag-specific CTL activity, 4-h ⁵¹Cr release assays were performed. Briefly, target cells were labeled with 300 μCi of ⁵¹CrO₄Na₂ (Amersham, Arlington Heights, IL) for 90 min before assay. The ⁵¹Cr-labeled target cells (1 × 10⁴ in 100 μl) were washed, resuspended (2 × 10⁵/ml), and added to 96-well U-bottom plates. Mononuclear effector cells were added, and the wells were incubated for 4 h. Supernatant fluids (200 μl) were harvested and measured in a radiation counter. Percent specific lysis was calculated as follows: 100 × [(experimental release − spontaneous release)/(maximum release − spontaneous release)].

Values for lysis of untreated cells were subtracted to obtain specific lysis. Spontaneous release was determined by incubating ⁵¹Cr-labeled target cells in medium alone, and maximum release was determined by incubating cells in 1% SDS. Assays were performed in triplicate wells. Spontaneous release of ⁵¹Cr from target cells for data shown was <15%.

IFN-γ was measured by a commercially available kit (Genzyme, Cambridge, MA), according to the manufacturer’s instructions. Mononuclear cells from vaccinated and control mice were collected as described above and cultured (7 days, 37°C, 5% CO₂) with rF1 (10–50 μg/ml) in 24-well cell culture plates. Culture supernatants (300 μl) were used for cytokine analyses.

Mouse serum Abs were isotyped by using a commercially available kit (Bio-Rad, Richmond, CA). The rF1 (1 μg/well) was adsorbed onto 96-well Immulon plates (30 min, 37°C), then washed with PBS. The wells were washed with PBS, blocked with 1% BSA in PBS (22°C, 30 min), and washed with PBS containing 0.2% Tween-20 (Sigma). Dilutions (1/100) of mouse sera were added, and plates were incubated for 30 min (37°C) and washed. Rabbit anti-mouse Ig (isotype specific) were added (100 μl) to the appropriate wells (37°C, 30 min), the plates were washed with PBS, and peroxidase-conjugated goat anti-rabbit was added (1/2000 dilution, 30 min, 37°C). The plates were washed with PBS to remove unbound reagents, and chromogenic substrate (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)) was added. Absorbance (405 nm) was measured after a 20-min incubation.

The F1 Ag is encoded within an operon of a 100-kb plasmid (19) that includes accessory gene products required for optimal expression of the F1 Ag in bacteria. The F1 polypeptide forms a high molecular mass aggregate (>300 kDa; data not shown) when expressed in E. coli or mammalian cells. No detectable F1 was released into the culture medium, suggesting that most of the Ag remained within the transfected cells. Mice (BALB/c) were injected i.m. with pREF1 in PBS or with purified rF1 adsorbed to alhydrogel. Serum Ab titers were measured 1 wk after each of three injections. Significant Ab responses were apparent for both plasmid and recombinant polypeptide injections (Fig. 1, A and B). However, the maximum levels of Ab elicited by rF1 immunizations were greater than those obtained with plasmid (Fig. 1, A and B). The genetic vaccination provided no protection from high dose challenges (400–800 LD₅₀) with live bacteria, whereas mice injected with rF1 were protected (Fig. 2). Because it was possible that the plasmid was not efficiently taken up by the appropriate APCs, we also injected mice with plasmid encapsulated in cationic liposomes. The circulating Ab levels of animals that received plasmid in liposomes were equivalent to those obtained from mice injected with plasmid in buffer (Fig. 3).

Ab responses of additional mouse strains were examined to detect differences in the immune recognition of plasmid-expressed F1 Ag that were dependent on H-2 haplotype. Serum Ab responses were examined 1 wk after the third injection of plasmids or rF1. Although there were some individual variations, the responses of BALB/c (H-2^d), C3H (H-2^k), and SJL (H-2^s) mice to rF1 and pREF1 were comparable (Fig. 4), presenting Ab titers of >100,000 and >50,000, respectively. Slight increases in background nonspecific Ab levels were detected in sera from all mice injected with the control plasmid (Fig. 4). We next examined Ab responses of outbred Swiss-Webster mice. Serum Ab responses of the outbred mice to rF1 were equivalent in magnitude to those of BALB/c mice. In contrast to the results obtained with inbred mice, there were no detectable Abs induced by immunization with pREF1. It was also noted that the Swiss-Webster mice did not exhibit a nonspecific response to the control plasmid.

Ab responses were consistently obtained only from inbred mice immunized with pREF1. However, these responses were always less than those obtained from rF1 immunizations. It was conceivable that differences in the magnitude of the immune response to plasmid compared with that to rF1 were related to the introduction of the Ag into an intracellular compartment that circumvented efficient T cell priming of Ab responses. To examine this possibility more closely, BALB/c and Swiss-Webster mice were injected with 10 μg of pREF1 followed by one injection of 10 μg of rF1 2 mo later. Serum samples were collected at 1 and 3 wk following the polypeptide injection. With BALB/c mice, serum Ab titers were equal to or greater than those obtained from mice that had received three injections of recombinant polypeptide (Fig. 5,A). These results suggested that BALB/c mice generated a vigorous primary response to plasmid-expressed F1. In contrast, immunization of Swiss-Webster mice with pREF1 did not produce an anamnestic response to rF1 (Figs. 4 and 5,B). The Ab level induced by pREF1 priming and rF1 boost of outbred mice was less than or equal to the Ab response obtained from BALB/c mice by a single injection of rF1 (Fig. 5,B). Because three injections of pREF1 in Swiss-Webster mice produced little or no Ab response (Fig. 4), these data confirmed that the outbred mice did not respond to F1 when plasmid was used for immunization.

Our data indicated that the relative magnitude of the Ab response to plasmid-expressed F1 varied little between inbred mouse strains. We further dissected the immune response to F1 to detect any potential differences among mouse strains. Based on Ab isotype ratios (IgG2a:IgG1), plasmid-injected BALB/c mice had a greater Th1 propensity than mice immunized with rF1 (Fig. 6). In contrast, mice that were primed with pREF1 and boosted with rF1 exhibited a predominant Th2 response, similar to mice that had received three injections of rF1. The control plasmid also induced detectable levels of background Th1-like Abs, perhaps as a result of responses to CpG motifs within the bacterial DNA (20).

When additional mouse strains were examined, MHC-dependent immune responses to the plasmid-expressed F1 were apparent (Fig. 7). Mononuclear cells of all strains tested released equivalent levels of IFN-γ when the mice were immunized with rF1, and only H-2^d and H-2^s mice that were immunized with plasmid-expressed F1 appeared to secrete levels of IFN in vitro that were greater than those in controls (Fig. 7).

The Ab response to rF1 was presumably mediated by an endocytic, MHC class II-dependent route of presentation. By the same reasoning, the endogenous expression of Ag from pREF1 may be more effective in inducing CTL responses. To test this, P815 cells that stably expressed F1 from the transfected pREF1 vector were ⁵¹Cr labeled and used for targets. Lymphocytes for the CTL assays were obtained from spleens of BALB/c mice 12 wk after the last immunization. Although there was a greater level of CTL recognition of F1 resulting from cytosolic expression of the Ag (pREF1 immunization) compared with that after rF1 immunization, the responses were weak in general (Table I).

We examined serum F1-specific Ab levels of immunized mice at 2-wk intervals after the last injection. There was a slight decrease in Ab titers with extended time for mice immunized with pREF1 alone (Fig. 8). However, when mice were immunized with either rF1 alone or pREF1 followed by a boost with rF1, Ab titers stayed consistently high over the time intervals examined (Fig. 8). These results indicated that primary or secondary immunization by an exogenous route was necessary to maintain long lived Ab responses.

The systemic nature of Y. pestis replication contrasts with the localized pathology and self-limiting disease caused by enteropathogenic species and suggests that significant differences exist in the immune response to each. In a previous study (21), we calculated that there are 600 to 6000 Ag molecules expressed in each plasmid-transfected cell depending upon the APC used. Our current results indicate that cytosolic targeting of the capsular F1 polypeptide stimulates CTL recognition of cells that present F1 peptides. Moderate CTL responses were also noted following immunization with rF1. We conclude that CTL responses to F1 are not an important component of protective immunity to Y. pestis, but that cytosolic insertion of Yop Ags, such as by invasin-positive species, is likely to result in significant levels of T cell recognition. The role of specific T cell subsets in the primary response to endogenous F1 is at present unknown. For the case of Y. enterocolitica, immune recognition of MHC class I-presented peptides explicitly contributes to cellular immunity (14). In addition, increased Y. enterocolitica infection in spleens of a resistant mouse strain (C57BL/6 × DBA/2) results from blocking CD8⁺ T cells with mAbs, while anti-CD4 treatment prevents DTH (22). A further analysis of immune responses to Yersinia will be necessary to clarify whether CTL immunity plays an essential role in resistance to or recovery from enteric and nonenteric infections.

Details concerning the initiation of Y. pestis infections are not well understood. Enteropathogenic Yersinia bind specifically to β₁ integrin on the basolateral membrane of intestinal epithelial cells by means of the inv gene product invasin (5). Breaches in intercellular tight junctions caused by transepithelial migration of inflammatory neutrophils may allow transient access to these β integrin binding sites (23) and subsequent tissue invasion. Moreover, host immune responses to all Yersinia are restricted by a complex interplay of bacterial virulence factors that also control the development of lesions. The secreted V protein (24) and perhaps other Lcr plasmid-encoded products, such as YopB (25), may suppress the release of the proinflammatory cytokines TNF-α and IFNγ, while YopE is a contact-dependent cytotoxin (8). A potential superantigen has also been described for some strains of Y. pseudotuberculosis (26).

Vaccinations with the soluble V protein (27) or the capsular F1 Ag (15) provide protection from the most virulent strains of Y. pestis. Abs against the V polypeptide are postulated to neutralize the inhibition of TNF-α and IFN-γ secretion (28), thus restoring immune effector functions. Based on our results and previous reports (15), Ab neutralization is the most likely mechanism of protection induced by vaccination with recombinant F1 polypeptide. Although high levels of F1 polypeptide are expressed intracellularly, no detectable amounts of Ag are released extracellularly from transfected cells (our unpublished observations). However, significant Ab titers resulted from genetic immunization, suggesting that the cells that were initially transfected with plasmid were responsible in vivo for stimulating F1-specific Ab responses, and that Ag taken up by secondary APCs may be less important. Previous studies indicate that dendritic cells have a key role in presenting Ags from genetic immunizations in mice,⁴ and thus may be the primary APCs that were transfected with the F1 plasmid.

It was noteworthy that the maximum Ab levels obtained from intracellular targeting of the F1 Ag were 3 times less than those resulting from immunizations with polypeptide in adjuvant. Perhaps a careful study of immunization schedules may improve these results. It is also possible that internalization and endosomal transport of the polypeptide are critical for stimulating optimal Ab responses, and that intracellular expression of F1 results in a dominant MHC class I-associated presentation pathway. Alternatively, the induction of lower Ab levels may have resulted from the shift from a Th2 response, induced by polypeptide immunization, to a more Th1-like response to plasmid-expressed Ag. In addition, MHC class II-dependent Th cell activity may support the CTL responses to transfected F1, especially if the Ag is more limited in concentration than that resulting from injections of the recombinant polypeptide (29). Our results also suggest that a Th2-induced Ab response is more efficacious for immune protection against Y. pestis in mice.

All the homozygous H-2 mouse strains that were immunized with the F1 expression plasmid responded at roughly equivalent Ab levels, whereas no primary or secondary Ab stimulation was detected in the outbred Swiss-Webster mice. Although this phenomenon may be specific to the F1 Ag, no similar comparisons of outbred and inbred mouse responses to genetic vaccines have been reported. There was also considerable variability among the homozygous H-2 strains tested in secondary in vitro cytokine responses to F1. Previous studies postulated that mouse resistance to Y. enterocolitica is multigenic and is not controlled exclusively by H-2 loci (30) but depends on the production of IFN-γ (31). Recognition of endogenous peptides by CTLs is generally limited to Ags that bypass endosomes (32). Preliminary data suggest that CTL responses to F1 also vary for mice of differing H-2 background (our unpublished observations). Collectively, these results suggest that caution should be used in interpreting genetic immunization studies that rely only on data obtained from one strain of mice.

Finally, our results suggest a useful immunization strategy that combines the traditional polypeptide vaccine with an Ag expression plasmid. Following a primary injection with either plasmid or F1 polypeptide, all inbred mouse strains responded with equivalent Ab levels to a secondary stimulation by recombinant polypeptide. Because CTL activity was also generated by the genetic vaccine, and optimal Ab responses resulted from a boost with polypeptide in adjuvant, a combination plasmid/polypeptide vaccine approach may optimally promote both humoral and cellular mechanisms of immunity to Yersinia.

We appreciate the excellent technical assistance of Beverly Dyas.