INTRODUCTION

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Orally administered live avirulent Salmonella vaccine strains colonize the gut-associated lymphoid tissue (Peyer's patches) and reach deep tissues, including the liver and spleen, via the circulatory system (8, 10, 30, 33). Avirulent cya crp asd Salmonella strains expressing foreign antigens from bacterial, viral, and parasitic pathogens have been constructed as live recombinant Salmonella-based antigen delivery systems for oral vaccinations (11, 27). The recombinant avirulent Salmonella strains, while eliciting anti-Salmonella immune responses, can also induce antigen-specific humoral, mucosal, and cellular immune responses to recombinant proteins expressed by the immunizing organism. This avirulent Salmonella technology offers prospects for developing multivalent vaccines (8, 11, 13, 14, 30, 33) that can be used to eventually develop safe, easy-to-use, and cost-effective oral vaccines for mass immunization against a wide variety of disease-causing pathogens.

Streptococcus pneumoniae causes life-threatening diseases, including pneumonia and meningitis. It is also associated with otitis media (ear infections) in young children and acute respiratory infections in humans of all age groups (1, 31). Ninety distinct capsular serotypes of S. pneumoniae have been associated with human infections (16). People with human immunodeficiency virus infection or AIDS have been shown to have invasive pneumococcal infections more frequently than the population at large (17). Pneumococcal diseases kill more people than any other infectious disease, claiming around 10 million lives yearly worldwide (29), including at least 1 million children with respiratory infections in developing countries. Pneumonia is the sixth leading cause of death in the United States. The estimated annual cost of pneumococcal morbidity and mortality in the United States is $23 billion (21). The emergence of penicillin resistance and multi-drug-resistant strains threatens the clinical management of pneumococcal disease (28, 36). The reservoir of pneumococci infecting humans is maintained largely by nasopharyngeal carriage, which is usually asymptomatic.

The present 23-valent capsular polysaccharide vaccine is only 60% effective against pneumococcal pneumonia in the elderly (35) and is not immunogenic enough in children under 2 years of age to warrant its use in that high-risk population (18). Chemical conjugates of capsular polysaccharides and proteins are being developed as immunogenic forms of the polysaccharides for immunization of children. Another approach that is being investigated is immunization with pneumococcal proteins that have been shown to elicit protective immunity in mice (6, 29). These proteins should be highly immunogenic in children and in the elderly, and they could be produced inexpensively enough for application in the developing world, where cost is a major factor in vaccine production and use. Protein antigens have the added advantage that they can be easily delivered through oral immunization with a live vaccine vector such as an avirulent Salmonella strain.

Pneumococcal surface protein A (PspA) is expressed on all pneumococci (5, 9) and has been shown to elicit protection against pneumococcal sepsis (25, 40) and carriage (42) in mice. The mature PspA from S. pneumoniae Rx1 has a molecular mass of 65 kDa and contains four distinct domains: an NH₂-terminal charged -helical coiled-coil domain, a proline-rich domain, 10 tandem-repeat regions, and a 17-amino-acid carboxy terminus (44). The repeat region of PspA forms a choline binding site which mediates the attachment of PspA to the cell surface lipoteichoic acids of pneumococci (46). The -helical domain comprises almost half of the protein and contains the protection-eliciting epitopes. PspA has been shown to exhibit serologic and molecular weight variability (9). However, in spite of this variability, many of the protection-eliciting epitopes of different PspAs are cross-reactive, and immunization with a single PspA can elicit protection against strains expressing different capsular polysaccharide types and serologically divergent PspAs (25, 40). As a result, any future PspA vaccine would probably require only a few different PspAs to elicit optimal protection (6).

In this report, we describe the construction and evaluation of a recombinant oral live Salmonella typhimurium vaccine strain which stably expresses a fragment of Streptococcus pneumoniae Rx1 PspA that includes its leader, -helical region, proline-rich region, and the first five repeats of the choline binding region. The DNA encoding this fragment was cloned into a high-copy-number Asd⁺ vector (pUC replicon based) in the avirulent cya crp asd S. typhimurium 4550. The immunogenicity and protective properties of the vaccine were evaluated in animals.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. Table 1 lists the bacterial strains and plasmids used in this work. S. typhimurium vaccine strains (cya crp asd mutants) were grown in Luria broth (L broth) or on Luria agar (L agar) containing diaminopimelic acid (DAP; 50 µg/ml) (Sigma, St. Louis, Mo.) (20). S. typhimurium 4550 (37) harboring the Asd⁺ vector pYA3148 or the recombinant plasmid pYA3193 was grown in L broth or on L agar with no DAP supplementation. All Salmonella strains were grown with aeration from a nonaerated static overnight culture. Buffered saline containing 1% gelatin was routinely used as a diluent. S. typhimurium vaccine clones were stored frozen at 70°C in 1% peptone containing 5% glycerol (12, 27). Escherichia coli DH1(pJY4347) (45) was grown in L broth containing erythromycin (200 µg/ml) and stored frozen at 70°C in L broth containing 10% glycerol. For challenge studies, virulent S. pneumoniae type 3 strain WU2 (4), stored at 70°C in Todd-Hewitt broth containing 20% glycerol, was grown at 37°C under anaerobic conditions in the BBL Gas Pack Plus anaerobic system (Becton Dickinson Microbiology Systems, Cockeysville, Md.) in Todd-Hewitt broth plus 0.5% yeast extract (4).

PCR. A fragment of the pspA gene was PCR amplified from plasmid DNA from E. coli DH1(pJY4347). The amplified fragment included the 5' region of the pspA gene from the ATG (nucleotides 127 to 129) start codon through the signal peptide leader sequence up to the end of the fifth tandem repeat in the choline binding region (Fig. 1 and 2). This fragment includes 1,503 bp and encodes the first 470 amino acids of S. pneumoniae Rx1 PspA. The PCR primer sequences were as follows: NH₂ primer (33 bp), 5' CAT GTC ATG AAT AAG AAA AAA ATG ATT TTA ACA 3'; and COOH primer (28 bp), 5' C GGG ATC CTA TGC CAT AGC GCC GTT AGC 3' (The Midland Certified Reagent Company, Midland, Tex.). The TC ATG A BspHI site was created on the NH₂ primer for ligation into the NcoI site of the Asd⁺ vector pYA3148. The C-terminal PCR primer has the BamHI site for ligation into the BamHI site of the Asd⁺ vector pYA3148. Vent polymerase and Vent buffer (New England Biolabs, Beverly, Mass.) were used in the PCR mixture. The PCR was carried out with 30 cycles of 95°C (1 min), 56°C (1 min), and 72°C (2 min) with the 480 Thermocycler (Perkin-Elmer Cetus, Calif.). The amplified PCR product of the 1.5-kb pspA gene was evaluated in a 1% Tris-acetate-EDTA (TAE)-agarose gel and purified by using a Gene Clean kit (BIO 101 Inc., La Jolla, Calif.).

View larger version (10K): [in this window] [in a new window]   FIG. 1.   PspA domains expressed by recombinant S. typhimurium. The diagram shows the domains of PspA from S. pneumoniae Rx1 and the portions expressed in S. typhimurium 4550(pYA3193) and E. coli 6212(pYA3193). aa, amino acids.

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Structure of the recombinant plasmid vector. High-copy-number pUC replicon-based Asd+ plasmid expression-cloning vector pYA3148 (3.5 kb) that harbors the truncated S. pneumoniae pspA gene (1.5 kb) was electroporated into S. typhimurium 4550 and E. coli 6212. Restriction enzyme sites are indicated. asd, aspartate -semialdehyde dehydrogenase.

Construction, cloning, and expression of the pspA gene in E. coli and S. typhimurium. The Asd⁺ vector pYA3148 was digested with NcoI and BamHI restriction enzymes (Promega buffer C, 5 h, 37°C), while the pspA PCR product was digested first with BspHI (NEBuffer 4, 2 h 30 min, 37°C) and then separately with BamHI (Promega buffer C, 2 h, 37°C). The ligation reaction was done overnight at 16°C in the presence of T4 DNA ligase (International Biotechnologies, Rochester, N.Y.). The 5.0-kb size of the ligated product (Fig. 2) was checked by electrophoresis in a 1% TAE-agarose gel. The identity of the recombinant plasmid was confirmed by restriction digestion analysis with SacI and BamHI. The recombinant plasmid was then electroporated into E. coli 6212(pYA232) and the S. typhimurium 4550 (asd cya crp) vaccine strain. Initial selection of the recombinant clones was on L agar plates without DAP since only clones harboring the recombinant plasmid would grow on that medium. The expression of the PspA antigen was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis with the anti-PspA monoclonal antibody (MAb) Xi126 (23). S. typhimurium 4550 (pYA3193) (Table 1) was further characterized for the presence of lipopolysaccharide (LPS), growth on minimal medium supplemented with 0.5% glucose, the presence of the 90.0-kb virulence plasmid, and growth in L broth with and without DAP.

Immunization of mice and rabbits. For oral-vaccination studies, groups of 15 BALB/cJ H-2^d and CBA/N xid J H-2^k inbred 8-week-old female mice (The Jackson Laboratory, Bar Harbor, Maine) were deprived of food and water for 4.5 h and then given 30 µl of 10% (wt/vol) sodium bicarbonate (pipetted inside the mouth with a micropipettor) to neutralize stomach acidity. Approximately 30 min later, the recombinant S. typhimurium 4550(pYA3193)-PspA vaccine (1.5 × 10⁹ CFU in 30 µl of buffered saline containing 1% gelatin) was orally administered at the back of the mouth. Food and water were returned to the animals 30 to 45 min later. Two months later, a second oral dose was given according to the above procedures. Control groups of mice were orally immunized with S. typhimurium 4550(pYA3148) (host-vector controls) or given nothing (naive unimmunized controls). Blood (retroorbital puncture) and vaginal-secretion specimens (collected in a 50 µl of phosphate-buffered saline [PBS] wash) were obtained at weekly or biweekly intervals and stored at 70°C. Intestinal washes were conducted by washing the contents of the mouse large intestine into 1.0 ml of PBS and pelleting the debris by centrifugation. Supernatants were stored frozen. The responses of the common mucosal immune system were monitored by examining the vaginal washings since this method provides a means of obtaining serial secretions from each animal.

Colonization of mice with the recombinant Salmonella strain. After being given a single oral dose of S. typhimurium 4550(pYA3193) or 4550(pYA3148) (1.5 × 10⁹ CFU/mouse for both of the strains used), three mice were euthanized each on days 7 and 14 post-oral immunization. Their Peyer's patches, spleens, and livers were collected aseptically. The tissues were homogenized and plated on MacConkey agar plates with 1% maltose to examine colonization and persistence of the recombinant vaccine.

Immunoassays. (i) Antibodies. Anti-PspA antibodies of the immunoglobulin G (IgG), IgM, and IgA classes in sera and vaginal secretions of BALB/c and CBA/N xid mice and anti-PspA IgG levels in rabbit sera and vaginal washings were determined by ELISA. Anti-S. typhimurium whole-cell lysate antigens and anti-S. typhimurium LPS-specific antibodies were also titrated to monitor the responses to the Salmonella strains. Purified, native, full-length PspA isolated from S. pneumoniae R36A (2) was coated onto Immulon 4 plates (Dynatech) at a concentration of 1.0 µg/well. The cloned PspA expressed by S. typhimurium in this study was derived from the pspA gene of strain Rx1, which was derived from strain R36A. Strains Rx1 and R36A are believed to express identical PspAs from identical pspA genes (4, 9, 26, 41). S. typhimurium whole-cell lysate or methylated S. typhimurium LPS (1.0 µg/well; Sigma) was coated onto Immulon 3 plates. Antigens were suspended in sodium carbonate-bicarbonate coating buffer, pH 9.6 (100 µl/well), and the coated plates were incubated at 37°C for 4 to 6 h followed by an overnight incubation at 4°C. Free binding sites were blocked with a blocking buffer (PBS [pH 7.4]-0.1% bovine serum albumin). Samples were serially diluted in the blocking buffer (dilutions were done in duplicate [100 µl/well]) and incubated overnight at 4°C. Plates were treated with goat anti-mouse IgG-biotin, goat anti-mouse IgM-biotin, goat anti-mouse IgA-biotin, or goat anti-rabbit IgG, followed by development with excess avidin-peroxidase and orthophenylenediamine. All immunoreagents were purchased from Sigma. Plates were read in an automated microtiter plate ELISA reader at 450 nm (model EL311SX; Biotek, Winooski, Vt.). The titer of each serum specimen was denoted as the log₁₀ of the reciprocal dilution of serum giving five times the absorbance of the undiluted preimmune serum.

(ii) ELISPOT. BALB/cJ mice were orally immunized once, as described earlier, with either strain 4550(pYA3193) or strain 4550(pYA3148). The numbers of antibody-secreting B cells producing anti-PspA-specific IgG, IgA, and/or IgM per 10⁶ cells of the spleen, Peyer's patches, and peripheral blood were counted. Three mice were euthanized each on days 2, 4, and 7. For these determinations, tissue samples from all three mice euthanized on the same day were pooled. The assays were done as described previously (43). Millicell-HA plates (Millipore, Mass.) coated with PspA at 2 µg/well were used in the assay. Bound anti-PspA antibodies were revealed as immunodots with Sigma Fast BCIP-NBT chromogen (Sigma).

Surface immunofluorescence. Surface immunofluorescence assays of WU2 pneumococci and S. typhimurium 4550(pYA3193) were done with sera from orally vaccinated mice. Pooled sera from mice orally immunized with the recombinant Salmonella strain, sera from mice immunized with the host Salmonella strain, and preimmune sera were used in the study. Control sera used in these studies were normal mouse sera and sera from mice immunized with the Salmonella vector (lacking PspA) only. Faint background fluorescence was observed with the control sera, but it was easily distinguished from the bright fluorescence detected with sera from mice immunized with strain 4550(pYA3193). For these studies, pneumococci were harvested, incubated with pooled immune or nonimmune sera for 2 h at 37°C, washed twice in cold PBS, and stained with goat anti-mouse IgG-fluorescein isothiocyanate (Sigma) at a 1:50 dilution for 2 h at 4°C. Surface fluorescence of pneumococcal cells was observed microscopically. S. pneumoniae WU2 stained with anti-PspA MAb Xi126 was the positive control.

Protection studies. BALB/cJ inbred mice were orally immunized twice with recombinant S. typhimurium 4550(pYA3193). Anti-PspA antibody titers were measured by ELISA prior to challenge. During the fourth week after administration of the second oral dose, mice were challenged by the intraperitoneal (i.p.) or intravenous (i.v.) route with different doses of virulent pneumococci (WU2 type 3 strain). Mice orally immunized with S. typhimurium 4550(pYA3148) and unimmunized naive mice were used as control groups. Infected mice were observed for deaths for 15 to 21 days. Virtually all deaths occurred within the first week postchallenge. Passive protection was carried out by i.p. injection of various dilutions of immune serum 1 h prior to i.v. or i.p. challenge with different doses of S. pneumoniae WU2 in 0.1 ml of Ringer solution.

	RESULTS

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Expression and localization of the recombinant truncated PspA in S. typhimurium. The cya crp asd mutant S. typhimurium vaccine strain (4550) transformed with recombinant plasmid pYA3193 stably expressed PspA as detected by Coomassie brilliant blue staining of SDS-polyacrylamide gels and by development of Western immunoblots with anti-PspA MAb Xi126. The level of PspA expression observed in the recombinant E. coli 6212 DH5-derived construct was higher in cells grown in the presence of isopropyl--Dthiogalactopyranoside (IPTG) than in its absence, as expected due to the presence of pYA232 encoding the LacI^q repressor in 6212. Based on Coomassie blue staining, the level of expression of PspA was from 5 to 6% of the total protein in both the S. typhimurium and E. coli strains. These results were consistent with those of earlier studies of the expression of PspA in E. coli (45). Although the expected size of the cloned truncated PspA was 55 kDa, the recombinant product migrated as a series of bands ranging from 30 to about 75 kDa. This microheterogeneity was observed for recombinant PspA expressed in both S. typhimurium and E. coli, was consistent with previous studies demonstrating heterogeneity in the size of a single full-length native PspA produced by pneumococci and E. coli (25, 39, 45), and was shown previously to be due to both polymerization and degradation of PspA (39). The periplasmic fraction and the supernatant contained virtually all of the expressed PspA. The majority of the recombinant PspA was exported to the periplasmic space of S. typhimurium, with little remaining in the cytoplasm, as had previously been reported for PspA cloned in E. coli (4, 45).

Persistence, tissue distribution, and recovery of the live vaccine after oral immunization of mice. After a single oral dose of strain 4550(pYA3193) or the vector-only control strain 4550(pYA3148), the bacteria reached the Peyer's patches, spleens, and livers of mice of both strains. The numbers of CFU recovered from these tissues at 14 days were as high or higher than what was observed at 7 days (Table 2). In BALB/c mice, the PspA-producing strain showed less colonization of the spleen and liver than did the nonvaccine host strain (vector control). This difference in colonization by the host and vaccine strain was not observed in CBA/N mice. Most importantly, the vaccine strain showed very similar levels of PspA in all tissues regardless of whether the Salmonella-susceptible BALB/cJ mice or the more Salmonella-resistant CBA mice were used. The vaccine bacteria recovered on days 7 and 14 still produced PspA as detected by colony immunoblotting (data not shown).

Anti-PspA immune responses in mice and rabbits to oral vaccination with the recombinant S. typhimurium4550(pYA 3193)-PspA vaccine. The kinetics of the anti-PspA of IgG, IgM, and IgA classes of antibody in sera and vaginal secretions of mice were measured. The vaccine induced humoral IgG, IgM, and IgA anti-PspA antibody responses in the BALB/c and CBA/N xid mouse strains (Fig. 3A and C). Within a week after administration of a single oral dose, the reciprocal serum IgG anti-PspA titers had reached 1,000 and the titers of IgA and IgM had reached 100. A single oral immunization of mice with the vaccine stimulated the production, in vaginal secretions, of reciprocal IgG, IgM, and IgA anti-PspA titers of 400, 100, and 500, respectively (Fig. 3B and D). Anti-PspA immunoglobulins titers (IgG, 100; IgA, 500; and IgM, 100) were also observed in mouse intestinal washings.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Class-specific anti-PspA IgG (), IgM (), and IgA () antibody titers in sera and secretions of mice orally immunized with 1.5 × 109 CFU of S. typhimurium vaccine strain 4550(pYA3193) at weeks 0, 8, and 20 (as indicated by arrows on the horizontal axis). The titers represent the maximum end-point dilutions from the pooled sera yielding an optical density at 450 nm (OD450) five times that of undiluted preimmune serum from the vector-immunized group (OD450, 0.1). The anti-PspA titer was <1 for NMS (preimmune mice). All mice were challenged 22 weeks postimmunization with live S. pneumoniae WU2 (see Table 5). (A) Class-specific anti-PspA antibody titers in the pooled sera of 10 BALB/c mice. (B) Anti-PspA antibody titers in pooled vaginal secretions of 10 BALB/c mice. (C) Anti-PspA antibody titers in pooled sera of 10 CBA/N xid mice. (D) Anti-PspA antibody titers in pooled vaginal secretions of 10 CBA/N xid mice.

View this table: [in this window] [in a new window]   TABLE 3.   Reciprocal titers of antibody to LPS and S. typhimurium lysate in sera and vaginal secretions of BALB/c mice orally immunized with PspA-expressing S. typhimurium  4550(pYA3193)a

Surface fluorescence of S. pneumoniae WU2. Polyclonal immune sera (pooled from 10 mice) collected after oral immunizations with S. typhimurium 4550(pYA3193) reacted with the native PspA expressed on the surface of the virulent WU2 human isolate of S. pneumoniae as revealed by an immunofluorescence assay test, demonstrating that sera from vaccinated mice could recognize native PspA (data not shown).

Evaluation of protective immunity. BALB/cJ mice were vaccinated with either the recombinant Salmonella strain 4550(pYA3193) or the host strain 4550(pYA3148), lacking PspA expression, or were left unimmunized. After two oral immunizations, the mice were challenged i.p. with 3 × 10³ CFU of S. pneumoniae WU2 (Table 4). In unimmunized BALB/cJ mice, the 50% lethal dose (LD₅₀) of S. pneumoniae WU2 was <10² CFU by this route. When mice immunized with the PspA-expressing vaccine strain were challenged, 66% survived, compared to 30% of the mice immunized with the non-PspA-expressing host strain. This challenge dose killed 100% of unimmunized control mice, indicating that the host strain by itself had elicited some level of nonspecific host immunity. The time to death/survival ratio of mice immunized with the PspA vector was significantly (P = 0.009) greater than that of nonimmunized mice and significantly (P = 0.004) less than that of mice immunized with the PspA⁺ Salmonella strain.

View this table: [in this window] [in a new window]   TABLE 5.   Passive protection of mice from fatal pneumococcal infection with anti-PspA serum from mice orally immunized with S. typhimurium 4550(pYA3193)a

	DISCUSSION

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These studies have demonstrated that oral immunization with an attenuated live Salmonella strain expressing PspA can be used to elicit protective humoral immunity to an encapsulated bacterium, S. pneumoniae. In these studies, protection against pneumococcal sepsis was measured. However, since the vaccine also induced mucosal immune responses, it was anticipated that immunization by this route might also induce protection against normal acquisition of pneumococci and carriage of that organism in the upper respiratory tract (42). Intraperitoneal immunization of mice with recombinant bacillus Calmette-Guerin (rBCG) expressing PspA induced a protective humoral response against pneumococcal challenge, but mucosal immune responses against PspA delivered by rBCG have not been reported (19). This is the first report of oral immunogenicity resulting from administration of a cya crp-based recombinant Salmonella strain to rabbits.

The Salmonella vaccine was attenuated by deletion of the genes encoding adenylate cyclase and cyclic AMP receptor protein. This approach can render wild-type Salmonella strains completely avirulent but still immunogenic (12). Since Salmonella strains with cya crp mutations do not possess antibiotic resistance genes, they are appropriate for vaccines intended for use in humans or animals. The ability of strain 4550(pYA 3193) to produce PspA at immunogenic concentrations was probably an important element of its ability to elicit high-level mucosal and serum antibody responses to PspA.

The vaccine strain was designed so that the fragment of PspA produced would contain the PspA signal peptide; the entire -helical region, which makes up the N-terminal half of PspA; the central proline-rich region; and a portion of the first five repeats of the C-terminal choline binding domain of PspA. The PspA -helical region contains the known protection-eliciting epitopes of PspA (22, 40). By including the proline-rich region and a portion of the repeat region in the construct, we hoped to optimize the conformational stability of the -helical portion of the molecule. Since PspA, as well as the truncated fragment of it cloned here, has a leader sequence but lacks a membrane attachment site, it was anticipated that the cloned molecule would be secreted into the periplasmic space. This was observed, but there was a considerable (but smaller) amount of PspA that appeared in the supernatant fluid. Whether this represents secretion across the outer membrane or lysis and release of periplasmic proteins will have to be determined in future studies.

The use of recombinant live Salmonella vaccines for mucosal immunization may have several advantages over immunization with isolated antigens. With mucosal immunization with isolated antigens such as PspA, adjuvants must be used to obtain significant mucosal responses (42, 43). One advantage of using live S. typhimurium to produce the vaccine antigen in vivo is that the presence of the live Salmonella cells alleviates the need for any additional adjuvant. Another advantage is that the immunizing protein need not be produced in vitro, isolated, purified, and characterized. Finally, the ability of S. typhimurium to colonize gut tissue following oral administration should permit elicitation of strong mucosal as well as humoral immune responses.

The present study demonstrated that the recombinant Salmonella strain was well tolerated by both rabbits and mice. The S. typhimurium 4550-PspA-based recombinant vaccine persisted in the spleen, liver, and gut lymphatic system. The elicitation of common mucosal immunity was apparent from the detection of anti-PspA antibodies in vaginal washings following oral immunization. The observation that the anti-LPS titers induced by strains 4550(pYA3148) and 4550(pYA3193) were comparable indicated that the expression of PspA by S. typhimurium 4550(pYA3193) did not interfere with the immunogenic potential of the bacteria. Oral immunization combined with another route of administration (37), such as intranasal or systemic, might stimulate even better combined mucosal and humoral immune responses. It is likely that the combination of mucosal and systemic immunity to PspA will be more protective against natural infections than systemic immunity alone.

Mice orally immunized with PspA-expressing S. typhimurium were more resistant to pneumococcal infection than mice immunized with the Salmonella host (nonvaccine) strain. It was also observed, however, that compared to mice given no immunization, those immunized with the host strain were somewhat resistant to infection with pneumococci and exhibited a significant delay in time to death. This partial resistance elicited by the vector alone did not appear to be able to be transferred with serum and may have been the result of a nonspecific host immune response to immunization caused by the live Salmonella cells. These results are very reminiscent of our previous data showing that pneumococcal infection itself elicits a host immune response that can play a major role in extending the lives of mice infected with pneumococci (2, 3).

It is important to note that the host strain exhibited a greater capacity to colonize the livers and spleens of BALB/cJ mice (and presumably elicited more nonspecific host immunity) than did the PspA-producing strain. Thus, the contribution of the anti-PspA immunity to immunization-enhanced resistance in BALB/cJ mice may have been even greater than was apparent from these studies. The efficacy of the anti-PspA immunity was further documented by passive transfer studies, in which it was apparent that as little as 0.1 ml of a 1/10 dilution of serum from the immunized animals could provide statistically significant protection from a fatal pneumococcal infection. The fact that the oral vaccine elicited specific and nonspecific protection even 4 weeks postboost argues for the overall efficacy of live Salmonella oral vaccines.

This is the first report of an avirulent cya crp-based recombinant oral Salmonella vaccine that has been employed in mouse protection studies by using a clinical human isolate of mouse-virulent S. pneumoniae WU2. Recombinant Salmonella strains may be a valuable vaccine vehicle for inducing primary protection against a wide range of pathogens which gain entry via mucosal surfaces. In addition, this vehicle has the potential to be an inexpensive delivery system for polyvalent vaccines. This demonstration that a mucosal attenuated Salmonella vaccine can elicit protection against systemic infection with pneumococci may encourage subsequent studies evaluating and identifying Salmonella attenuation systems that would be safe for immunization of young children. As a group, young children, especially those in developing countries, who may be malnourished or infected with other agents, may provide the most demanding environment for establishing the correct balance between attenuation and virulence of live bacterial and viral vector vaccines.