Abstract

BackgroundEconomical and effective vaccines against Streptococcus pneumoniae (pneumococcus) are needed for implementation in poorer countries where the disease burden is highest. Here, we evaluated Lactococcus lactis intracellularly producing the pneumococcal surface protein A (PspA) as a mucosal vaccine in conferring protection against pneumococcal disease

MethodsMice were intranasally (inl) immunized with the lactococcal vaccine. Control groups were also immunized with similar amounts of recombinant PspA administered inl or subcutaneously with alum. PspA-specific antibodies in serum samples and lung lavage fluids were measured before challenge in intraperitoneal sepsis and inl respiratory-infection models of pneumococcal disease

ResultsThe lactococcal vaccine afforded better protection against respiratory challenge with pneumococcus than did vaccination with purified antigen given inl or by injection with alum. This finding was associated with a shift toward a Th1-mediated immune response characterized by reduced antibody titers to the PspA antigen. In the sepsis model, the lactococcal vaccine afforded resistance to disease on a par with that obtained with the injected vaccine, demonstrating its efficacy against different forms of pneumococcal disease

ConclusionGiven the safety profile of L. lactis there is considerable potential to develop a pneumococcal vaccine for use in humans and to broaden this approach to combat other major pathogens

Streptococcus pneumoniae (pneumococcus) is a leading cause of invasive disease and respiratory infections worldwide [1]. In developing countries, acute lower respiratory infections due to pneumococcus cause at least 1 million deaths annually in children <5 years of age [2]. In the United States, it is estimated to cause 3000 cases of meningitis, 50,000 cases of bacteremia, 125,000 cases of pneumonia, and 6 million cases of otitis media annually [3]. Furthermore, treatment of pneumococcal disease is being compromised by the worldwide emergence of multidrug-resistant strains [4]

Vaccines are one of the most cost-effective methods to prevent infectious disease, but, for vaccines against pneumococcus, there is still room for improvement. The currently licensed pneumococcal polysaccharide vaccine, comprising 23 of the 90 known capsular polysaccharide serotypes, is relatively cost-effective for high-risk groups, such as the elderly and immunocompromised patients with a sufficiently high CD4+ T cell count, but is poorly immunogenic in children <2 years of age and only 60% effective in adults [5, 6]. Moreover, cost and poor protective efficacy against mucosal pneumococcal infections have prevented their implementation in large-scale immunization programs, particularly in poorer countries [2, 7]. Recently developed polysaccharide-protein conjugate vaccines are immunogenic and effective in children [8, 9]. The afforded protection is, however, restricted to only 7, 9, or 11 clinically relevant serotypes, and the emergence of disease caused by nonvaccine serotypes is of serious concern [1]. As a result, there is considerable interest in using conserved surface proteins as vaccine antigens to provide broad protection in all age groups; a number of leading candidates have been shown to elicit protection in mice [7, 8]

There is growing interest in the use of lactic acid bacteria (LAB) as mucosal vaccine vehicles because of their long and safe association with humans and their food [10]. Such vaccines would be easier to administer via oral, intranasal (inl), or urogenital routes; would be economical to produce; and would be inherently safer than an attenuated pathogen. Furthermore, mucosal immunization is considered better for optimal secretory IgA responses at mucosal surfaces [11]. Here, we report the development of a mucosal vaccine against pneumococcus with recombinant Lactococcus lactis as a vaccine-delivery vehicle. Several heterologous antigens have been expressed in L. lactis [12–14], and, in some cases, mucosal immunization was shown to elicit antibody and cellular responses [15–17]. Oral immunization and protective immunity were first demonstrated in mice challenged with tetanus toxin [18]. More recently, protection from infection was demonstrated by mucosal vaccination with recombinant L. lactis strains expressing vaccine antigens from Streptococcus pyogenes [19], the swine pathogen Erysipelothrix rhusiopathiae [20], and Helicobacter felis [21]. Partial protection against challenge with an HIV Env-expressing vaccinia virus was also achieved in mice administered a lactococcal vaccine with cholera toxin as an adjuvant [22]

The pneumococcal antigen pneumococcal surface protein A (PspA) used here has been shown to protect mice from fatal bacteremia, with its N-terminal α-helical domain being the target of protective antibodies [7, 8, 23]. Humans naturally infected or colonized with pneumococcus develop anti-PspA antibodies in both serum and mucosal secretions, with antibody to the α-helical domain of PspA also implicated in preventing pneumococcal carriage [24]. Although present on all pneumococci, PspA demonstrates antigenic strain variability, particularly in the α-helical domain. However, protein-sequence comparisons of this domain in >24 alleles indicated that PspA forms 3 protein families, with >95% of isolates belonging to families 1 and 2 [25]. Moreover, a recent phase 1 clinical trial using a single recombinant PspA (rPspA) showed that elicited antibodies were broadly cross-reactive against different PspA variants [26] and protected mice against different pneumococcal strains expressing heterologous PspA antigens [27]. On the basis of these findings, a vaccine containing only 2 or 3 PspA antigens—1 from each major family—may elicit protection against most strains infecting humans

Many pneumococcal strains (notably serotypes 2–6) are virulent in mice, causing fatal sepsis when administered intravenously or intraperitoneally (ip) [7]. Administration of pneumococcal strains inl to anesthetized mice or directly into the lungs has also been shown to cause acute pulmonary inflammation followed by a generalized sepsis [28]. Here, we compared the efficacy of the lactococcal PspA vaccine to a conventional injected vaccine (i.e., PspA plus alum) in ip sepsis and inl respiratory-infection models, the latter to better mimic the natural route of infection

Materials and Methods

Bacterial strains and growth conditionsEncapsulated S. pneumoniae TIGR4 (serotype 4), a highly virulent clinical isolate [29], was grown at 37°C on blood agar supplemented with 5% defibrinated horse blood (Oxoid) or in Todd Hewitt broth containing 0.5% yeast extract (THY) supplemented with 10% fetal bovine serum (FBS). Escherichia coli strains DH5α and BL21λ(DE3) (Novagen)—used for cloning and production of recombinant protein, respectively—were grown aerobically at 37°C in Luria-Bertani medium containing 34 μg/mL kanamycin to select for plasmids pTRCHis-TOPO and pET28b (Invitrogen). L. lactis strains MG1363 [30] and F17847(ΔnisA) [31] were propagated in M17 medium containing 0.5% glucose (GM17; Oxoid) at 30°C. Plasmids were electroporated into L. lactis [32], and transformants carrying pTnis plasmids [33] were selected on GM17 plates containing 5 μg/mL erythromycin

Purification of rPspAThe 1254-bp DNA fragment encoding the α-helical domain of PspA (pspATIGR4; aa 1–418) was amplified from genomic DNA by polymerase chain reaction (PCR) with primers LLPSPAF1 (5′-ccatggaagaatctccacaagttgtcg-3′) and LLPSPAR1 (5′-ttgcggccgcagtttcttcttcatctccatcagggc-3′). The amplicon was cloned into pTrcHis-TOPO, excised with NcoI and NotI, and then recloned into pET28b in-frame with a carboxyl-terminal His6 tag. This construct (pET28b-pspA) was transformed into E. coli BL21λ(DE3), and His6-tagged rPspA product was purified using Ni2+ affinity chromatography columns (Qiagen). Eluted rPspA was analyzed by SDS-PAGE, and selected fractions were pooled, dialyzed against PBS/10% glycerol solution (pH 7.0), and quantified using the Bradford assay [34]. Protein purity was confirmed by matrix-assisted laser desorption/ionization-time of flight (TOF) mass spectrometry of tryptic peptides using an Ultraflex TOF mass spectrometer (Bruker Daltonics)

Cloning and expression of PspA in L. lactisThe pTnis plasmid, a derivative of the broad gram-positive host range vector pTREX1 [33, 35], was used to express PspA in L. lactis The α-helical domain of pspATIGR4 was amplified by PCR with primers LLPSPAF2 (5′-acatgcatgcaagaatctccacaagttgtcg-3′) and LLPSPAR2 (5′-gaagatctttattcttcttcatctccatcagggc-3′), cut with SphI-BglII and cloned into pTnis downstream of a nisin-inducible promoter [36]. The cloned pspATIGR4 DNA was identical to that expressed in E. coli except for 1 bp resulting in a Glu→Gln replacement at amino acid position 2 of rPspA. This construct (pTnis-pspA) was transformed into L. lactis F17847, and expression was induced using nisin (Sigma) [36]. To optimize PspA expression, cultures were grown at 30°C to an OD600 of ∼0.5 before inducing with nisin (0–50 ng/mL) for 2.5 h. Protein was extracted [35], and expression of PspA was confirmed by Western blot analysis using polyclonal anti-PspA antibodies generated in New Zealand White rabbits [37]

Preparation of bacterial cells for immunizationOvernight cultures of L. lactis F17847 strains carrying pTnis-pspA (referred to hereafter as “LL-PspA”) or pTnis vector (referred to hereafter as “LL-control”) were diluted 1:100 in GM17 supplemented with 5 μg/mL erythromycin and grown at 30°C to an OD600 of ∼0.5. Expression of rPspA was induced by adding 10 ng/mL nisin and incubating for a further 2.5 h to late-exponential phase (OD600 >2). Harvested cells were washed twice by centrifugation (3220 g at 4°C) in sterile, ice-cold PBS before being suspended in vaccine buffer (0.2 mol/L sodium bicarbonate, 5% casein hydrolysate, and 0.5% wt/vol glucose) at 5×1010 cfu/mL. The L. lactis vaccine strain contained between 0.25 and 0.5 μg of PspA per 1×109 cells as determined by immunoblotting using purified E. coli-derived rPspA as a standard. For chemical inactivation, bacterial suspensions were pretreated with mitomycin C [33]

ImmunizationsOn days 0, 21, and 42, lightly anesthetized (1.5% vol/vol halothane) CBA/ca mice (7–9 weeks old; n=10 or 20; Harlan) were inl immunized with 1×109 cfu (in 20 μL of vaccine buffer) of the LL-PspA or LL-control strains. Separate groups were immunized with 1 μg of rPspA given inl (in 20 μL of vaccine buffer) or subcutaneously (sc) mixed with alum (1:1; 100 μL; Pierce Biotech). All vaccination experiments included a naive or sham-vaccinated (inl immunized with 20 μL of vaccine buffer) control group. Tail blood samples were collected from each mouse on days 14, 35, and 56 after primary immunization. Lung lavage fluids were obtained 7 days after the final boost, as described elsewhere [16]. Samples were stored at −20°C until analysis

ELISA detection of antigen-specific antibodies in serum and lung lavage fluidsIgG, IgG1, and IgG2a antibody titers were determined by ELISA [33] using plates coated with rPspA (50 ng/well). For serum samples, end-point titers were calculated as the dilution resulting in the same OD405 as a 3×1:50 dilution of pooled preimmune serum. For lung lavage fluids, end-point titers were calculated as the dilution producing the same OD405 as 3 times the background level. Lung lavage fluids were also tested for PspA-specific and total IgA, as described elsewhere [38], by use of microplates coated with either rPspA (50 ng/well) or anti-mouse IgA (α-chain specific; Sigma) (250 ng/well). Concentrations of PspA-specific and total IgA using a standard curve of known amounts of mouse myeloma IgAk (Sigma) were included on all ELISA plates

Pneumococcal challenge experimentsMice were challenged 3 weeks after the final immunization. In the sepsis model, mice were ip challenged with 2×105 cfu of standard inoculum (500 μL; 1000 LD50; calculated 7 days after challenge). In the respiratory-infection model, lightly anesthetized mice were inl challenged with 1×106–2×106 cfu of standard inoculum (50 μL; 10–100 LD90). Challenged mice were monitored every 2–3 h for 7 days for symptoms of infection; moribund mice were humanely killed, and the time at which they were killed was recorded. To estimate the challenge dose, groups of 8–10 nonimmune mice were challenged with serial dilutions of standard inoculum

To prepare the standard inoculum, S. pneumoniae TIGR4 cultures, resuspended and serially diluted in PBS, were administered ip to mice. After challenge, mice showing symptoms were exsanguinated, and the blood containing pneumococci was used to inoculate serum broth (THY containing 20% heat-inactivated FBS). Cultures were grown to late exponential phase and used to prepare a standard inoculum that was aliquoted, snap-frozen, and stored at −80°C. For respiratory-challenge experiments, the TIGR4 strain was inl repassaged to produce a standard inoculum with enhanced virulence via this route. When required, aliquots of standard inoculum were thawed slowly and diluted in PBS before use. All protocols were performed in accordance with UK Home Office and institutional regulations

Statistical analysisThe results are expressed as mean±SE values. Statistical significance was evaluated by the Mann-Whitney U test. P<.05 was considered to be significant

Results

Expression of PspATIGR4 in L. lactisand E. coliTo obtain antigen for antibody production and vaccination, the α-helical domain of PspATIGR4 was overexpressed in E. coli as a C-terminal His-tagged fusion protein. This migrated as 3 predominant polypeptides of 55, 50, and 42 kDa rather than the expected 48.4-kDa protein (figure 1). This heterogeneity has been observed previously with rPspA expressed in E. coli and S. typhimurium [39, 40] as well as with native PspA [41]. In the LL-PspA strain, PspA was produced in a nisin dose-dependent manner with 2 major polypeptides of 55 and 42 kDa (figure 1). Western blot analysis using antiserum to rPspA purified from E. coli readily detected rPspA but showed no reaction with proteins from L. lactis and E. coli carrying empty vector (figure 1)

Figure 1

Intracellular expression of pneumococcal surface protein A (PspA) in Lactococcus lactis (LL) Shown is a Western blot analysis of total cell extracts (equivalent to 1×107 cfu) prepared from LL-PspA induced for 2.5 h with nisin (0–50 ng/mL). Total cell extract prepared from LL-control (10 ng/mL nisin) and 10 ng of purified recombinant PspA (rPspA) were also included. Molecular weights of protein standards (kilodaltons) are indicated

Figure 1

Intracellular expression of pneumococcal surface protein A (PspA) in Lactococcus lactis (LL) Shown is a Western blot analysis of total cell extracts (equivalent to 1×107 cfu) prepared from LL-PspA induced for 2.5 h with nisin (0–50 ng/mL). Total cell extract prepared from LL-control (10 ng/mL nisin) and 10 ng of purified recombinant PspA (rPspA) were also included. Molecular weights of protein standards (kilodaltons) are indicated

Enhancement of Th1-type serum antibody responses by lactococcal vaccine deliveryGroups of mice were immunized inl with the lactococcal vaccine, vector control strain, rPspA, and sham vaccine or sc with rPspA/alum (see Materials and Methods). At all time points, mice administered the injected vaccine (rPspA/alum) exhibited the highest PspA-specific serum IgG titers (figure 2A). A dose of 1×109 cfu of the lactococcal vaccine was sufficient to elicit measurable IgG, although end-point titers (day 56) were significantly lower than those elicited by the injected vaccine (P=.0113). Antibody responses to the lactococcal vaccine were also lower than responses to the inl rPspA vaccine, but the difference was not significant. Antibody responses to the vector control and sham vaccines were below background levels

Figure 2

Analysis of pneumococcal surface protein A (PspA)-specific IgG, IgG1, and IgG2a antibodies in serum. A Serum IgG titers on days 14, 35, and 56, measured by ELISA in individual mice (n=10) immunized intranasally (inl) with either Lactococcus lactis (LL)-PspA (black squares), LL-control (white squares) a sham vaccine (white triangles) and purified recombinant PspA (rPspA) (gray triangles) or rPspA administered subcutaneously (sc) with alum (stars) (see Materials and Methods). B Anti-PspA IgG1 (gray bars) and IgG2a (black bars) end-point titers, measured in individual mice on day 56 after primary immunization. Vaccine groups are as indicated. Data are the mean±SE results

Figure 2

Analysis of pneumococcal surface protein A (PspA)-specific IgG, IgG1, and IgG2a antibodies in serum. A Serum IgG titers on days 14, 35, and 56, measured by ELISA in individual mice (n=10) immunized intranasally (inl) with either Lactococcus lactis (LL)-PspA (black squares), LL-control (white squares) a sham vaccine (white triangles) and purified recombinant PspA (rPspA) (gray triangles) or rPspA administered subcutaneously (sc) with alum (stars) (see Materials and Methods). B Anti-PspA IgG1 (gray bars) and IgG2a (black bars) end-point titers, measured in individual mice on day 56 after primary immunization. Vaccine groups are as indicated. Data are the mean±SE results

Levels of PspA-specific serum IgG1 and IgG2a subclasses were measured to determine the effect that L. lactis and vaccination route have on T helper subset responses (figure 2B). Although the injected rPspA/alum vaccine elicited a dominant PspA-specific IgG1 response (mean IgG1:IgG2a ratio, 59.5±10.46) in serum, the inl lactococcal vaccine elicited similar IgG1 and IgG2a titers (mean IgG1:IgG2a ratio, 3.49±0.72), suggesting the preferential induction of Th1-type responses. The inl rPspA vaccine elicited intermediate IgG1:IgG2a ratios (mean, 10.71±4.55)

Vaccination and resistance to pneumococcal sepsisTo determine the efficacy of the lactococcal vaccine, immunized mice were challenged ip with S. pneumoniae TIGR4 (1000 LD50). In comparison to a naive group, only mice immunized inl with the lactococcal vaccine (P=.0036; no survivors) or sc with rPspA/alum (P=.0376; 10% survival) were significantly resistant to disease at this high challenge dose. Their corresponding efficacies were also significant, compared with those of mice immunized inl with rPspA or the vector control strain (P<.05). Both vaccines performed equally well in the sepsis model; they produced equivalent mean survival times that were not significantly different (figure 3)

Figure 3

Protection against pneumococcal sepsis conferred by mucosal vaccination with recombinant Lactococcus lactis (LL)-pneumococcal surface protein A (PspA). Separate groups of mice were immunized on days 0, 21, and 42 and challenged intraperitoneally 3 weeks later with ∼1000 LD50 of Streptococcus pneumoniae TIGR4. Vaccine groups are as indicated. All vaccines were administered intranasally (inl) unless otherwise indicated. Results are the mean±SE survival time (n=10). Each group of mice immunized with either the LL-PspA (inl) or recombinant PspA (rPspA) (subcutaneous [sc]) vaccines differed from the naive controls at P=.0036 and P=.00376, respectively, as calculated by the Mann-Whitney 2-sample&amp;rank test. *P<.05, compared with inl rPspA and vector control vaccine groups

Figure 3

Protection against pneumococcal sepsis conferred by mucosal vaccination with recombinant Lactococcus lactis (LL)-pneumococcal surface protein A (PspA). Separate groups of mice were immunized on days 0, 21, and 42 and challenged intraperitoneally 3 weeks later with ∼1000 LD50 of Streptococcus pneumoniae TIGR4. Vaccine groups are as indicated. All vaccines were administered intranasally (inl) unless otherwise indicated. Results are the mean±SE survival time (n=10). Each group of mice immunized with either the LL-PspA (inl) or recombinant PspA (rPspA) (subcutaneous [sc]) vaccines differed from the naive controls at P=.0036 and P=.00376, respectively, as calculated by the Mann-Whitney 2-sample&amp;rank test. *P<.05, compared with inl rPspA and vector control vaccine groups

Vaccination and protection from respiratory pneumococcal challengeImmunized mice were inl challenged with S. pneumoniae TIGR4 (10 LD90). Groups administered the inl rPspA or sc rPspA/alum vaccines were significantly protected, with 15% and 20% of mice surviving challenge, respectively (figure 4A and 4B). However, the most striking protection was obtained with the lactococcal vaccine, with 40% of mice surviving and a mean survival time significantly higher than that of the control groups. This was also significantly better than the protection afforded by either of the rPspA vaccines. Although not statistically significant, 20% of mice immunized with the vector control strain survived (figure 4B), suggesting that L. lactis alone may also contribute to nonspecific host immunity. No direct correlation between titers of serum antibody to PspA and survival was established. In groups receiving the lactococcal vaccine, surviving mice had higher mean titers of antibody (IgG, IgG1, and IgG2a) to PspA, although these were not significantly different (P>.05). This trend was not apparent in groups that received rPspA vaccines

Figure 4

Protection against respiratory pneumococcal challenge conferred by mucosal vaccination with Lactococcus lactis (LL)-pneumococcal surface protein A (PspA). Three weeks after the final immunization, CBA/ca mice were intranasally (inl) challenged with ∼10 LD90 of Streptococcus pneumoniae TIGR4. No. of survivors was recorded hourly, and percentage of survival was determined at 7 days after challenge. A Mean survival times for each vaccine group as indicated. All vaccines were administered inl unless otherwise indicated. Results are the mean±SE survival time (n=20). Statistical differences were calculated. B Survival time and no. of survivors in groups immunized inl with LL-PspA (gray squares), LL-control (white squares) recombinant PspA (rPspA) (white spheres) or sham vaccine (gray triangles) or subcutaneously (sc) with rPspA/alum (black spheres)

Figure 4

Protection against respiratory pneumococcal challenge conferred by mucosal vaccination with Lactococcus lactis (LL)-pneumococcal surface protein A (PspA). Three weeks after the final immunization, CBA/ca mice were intranasally (inl) challenged with ∼10 LD90 of Streptococcus pneumoniae TIGR4. No. of survivors was recorded hourly, and percentage of survival was determined at 7 days after challenge. A Mean survival times for each vaccine group as indicated. All vaccines were administered inl unless otherwise indicated. Results are the mean±SE survival time (n=20). Statistical differences were calculated. B Survival time and no. of survivors in groups immunized inl with LL-PspA (gray squares), LL-control (white squares) recombinant PspA (rPspA) (white spheres) or sham vaccine (gray triangles) or subcutaneously (sc) with rPspA/alum (black spheres)

Inactivated lactococcal vaccine immunogenicity and protection against respiratory challengeTo determine whether an inactivated lactococcal vaccine would be as effective as a live one, respiratory-challenge experiments included mice that were immunized with mitomycin C-inactivated LL-PspA harvested from the same culture as the live vaccine. A combined vaccine group composed of the live LL-control (1×109 cfu) mixed with 1 μg of rPspA was included to address the need for a recombinant vaccine. In these experiments, a higher respiratory-challenge dose (100 LD90) was used to accentuate any differences in the efficacies of the live, combined, and inactivated vaccines. With this high challenge dose, mice immunized sc or inl with the purified rPspA vaccines or the combined vaccine were not significantly protected, compared with the sham group (figure 5A). However, both the live and inactivated lactococcal vaccines afforded protection, with the efficacy of the live vaccine being significantly better than that of all other vaccines (P<.05). Protection conferred by the inactivated vaccine was significant, compared with that conferred by the sc rPspA/alum, and the inl combined, vector control, and sham vaccines

Figure 5

Inactivated lactococcal vaccine immunogenicity and protection against respiratory pneumococcal challenge. Three weeks after the final immunization, CBA/ca mice were intranasally (inl) challenged with ∼100 LD90 of Streptococcus pneumoniae TIGR4. No. of survivors and mean survival time were determined at 7 days after challenge. A Mean survival times for each vaccine group as indicated. All vaccines were administered inl unless otherwise indicated. The live vaccine (*) provided the best protection, with a mean survival time that was significantly higher than that obtained with the inactivated vaccine (P=.0468), combination vaccine (P=.0006), inl recombinant pneumococcal surface protein A (rPspA) vaccine (P=.0005), subcutaneous (sc) rPspA/alum vaccine (P=.0000), or vector (P=.0003) and sham (P=.0000) controls. Results are the mean±SE survival time (n=20). B Mean IgG1:IgG2a ratios. Anti-PspA IgG1 and IgG2a end-point titers were measured in individual mice on day 56 after primary immunization (before respiratory challenge), and mean IgG1:IgG2a ratios determined. Vaccine groups are as indicated. Results are the mean±SE IgG1:IgG2a ratio (n=20). Statistical significance was evaluated by the Mann-Whitney U test, and P<.05 was considered to be significant. LL, Lactococcus lactis

Figure 5

Inactivated lactococcal vaccine immunogenicity and protection against respiratory pneumococcal challenge. Three weeks after the final immunization, CBA/ca mice were intranasally (inl) challenged with ∼100 LD90 of Streptococcus pneumoniae TIGR4. No. of survivors and mean survival time were determined at 7 days after challenge. A Mean survival times for each vaccine group as indicated. All vaccines were administered inl unless otherwise indicated. The live vaccine (*) provided the best protection, with a mean survival time that was significantly higher than that obtained with the inactivated vaccine (P=.0468), combination vaccine (P=.0006), inl recombinant pneumococcal surface protein A (rPspA) vaccine (P=.0005), subcutaneous (sc) rPspA/alum vaccine (P=.0000), or vector (P=.0003) and sham (P=.0000) controls. Results are the mean±SE survival time (n=20). B Mean IgG1:IgG2a ratios. Anti-PspA IgG1 and IgG2a end-point titers were measured in individual mice on day 56 after primary immunization (before respiratory challenge), and mean IgG1:IgG2a ratios determined. Vaccine groups are as indicated. Results are the mean±SE IgG1:IgG2a ratio (n=20). Statistical significance was evaluated by the Mann-Whitney U test, and P<.05 was considered to be significant. LL, Lactococcus lactis

PspA-specific IgG1 and IgG2a serum end-point titers (day 56) were also measured. Compared with the live vaccine, the inactivated vaccine elicited a significantly higher mean end-point IgG titer (29,252±5608 vs. 64,081±10,655; P=.0173) and IgG1:IgG2a ratio (figure 5B), indicating a shift toward a Th2 response. Although the combined vaccine induced end-point titers and IgG ratios that were similar to the live recombinant vaccine, it did not elicit significant protection, indicating that the lower efficacy was not simply due to a lack of an antibody response (figure 5B). The most significant changes in IgG1:IgG2a ratios were those observed with rPspA administered sc or inl (P=.0000), the latter producing lower IgG1:IgG2a ratios, which indicate more effective induction of Th1 and Th2 responses via this route

Respiratory antibody responses elicited by lactococcal vaccinationGroups of mice were immunized, and, 1 week after the final boost, lung lavage fluids were collected to measure PspA-specific antibody titers (figure 6A and 6B). Compared with those of the vector control group, significantly elevated levels of PspA-specific IgG1, IgG2a, and IgA were detected in mice inl immunized with the live/inactivated lactococcal and rPspA vaccines (P<.0001). No PspA-specific antibodies were detected in lung lavage fluids from sham-vaccinated mice. As with the serum antibody response, the inactivated lactococcal vaccine elicited higher IgG1 and IgG2 and significantly higher IgA levels (P=.02) than did the live vaccine, reflecting a slightly elevated Th2 response. Surprisingly, mice inl immunized with purified rPspA produced mean titers of PspA-specific IgG1, IgG2a, and IgA in lung lavage fluids that were strikingly higher than those of the other vaccine groups (P<.001). This contrasted with the sc rPspA/alum vaccine, which elicited significant levels of IgG1 but very low levels of IgG2a (P=.6015), with only 1 of 5 mice producing a detectable IgA response (P=.2087), confirming previous studies showing injected vaccines to be poor inducers of mucosal immunity. As in serum, the inl rPspA vaccine elicited much lower IgG1:IgG2a ratios than did the sc vaccine. These results confirm reports showing the nasal route to be the most efficient route for inducing specific IgA responses in the airways while eliciting a more Th1-like antibody subclass pattern [42]. Responses to rPspA were substantially reduced when it was inl administered in combination with live lactococci, indicating that immune modulation by L. lactis causes a shift toward a Th1 immune response. Of the respiratory antibody responses to mucosally administered vaccines, those to PspA were lowest for the combined vaccine group

Figure 6

Respiratory antibody responses to pneumococcal surface protein A (PspA). One week after the final boost, mice were killed, and lung lavage fluids were assayed by ELISA for anti-PspA antibody levels. A Anti-PspA IgG1 (gray bars) and IgG2a (black bars) end-point titers in individual mice on day 49 after primary immunization. Vaccine groups are as indicated. B Anti-PspA-specific IgA responses. Vaccine groups are as indicated. Data are the mean±SE results (n=5). *P<.05, compared with the sham and vector control groups (Mann-Whitney U test). LL, Lactococcus lactis; MitC, mitomycin C; rPspA, recombinant pneumococcal surface protein A; sc, subcutaneous

Figure 6

Respiratory antibody responses to pneumococcal surface protein A (PspA). One week after the final boost, mice were killed, and lung lavage fluids were assayed by ELISA for anti-PspA antibody levels. A Anti-PspA IgG1 (gray bars) and IgG2a (black bars) end-point titers in individual mice on day 49 after primary immunization. Vaccine groups are as indicated. B Anti-PspA-specific IgA responses. Vaccine groups are as indicated. Data are the mean±SE results (n=5). *P<.05, compared with the sham and vector control groups (Mann-Whitney U test). LL, Lactococcus lactis; MitC, mitomycin C; rPspA, recombinant pneumococcal surface protein A; sc, subcutaneous

Discussion

This investigation set out to ascertain whether mucosal immunization with a recombinant L. lactis vaccine expressing a pneumococcal protein immunogen would be an effective alternative approach to an injected protein vaccine. The α-helical domain of PspATIGR4 was intracellularly expressed in L. lactis because it is identical to PspAEF3296, shown previously to provide cross-protective immunity against pneumococcal strains of different capsular types and PspA backgrounds [43, 44]. When tested as an inl vaccine by use of the ip route of infectious challenge, the lactococcal vaccine significantly increased resistance against pneumoccal sepsis, and the increase was similar to that obtained with an injected vaccine (rPspA/alum). Because pneumonia normally precedes invasive disease, an efficacy study was also performed using the inl route of infectious challenge. Here, the lactococcal vaccine repeatedly elicited protection against respiratory pneumococcal challenge that was significantly better than that obtained by inl vaccination with rPspA or sc with rPspA/alum

The superior efficacy of the lactococcal vaccine was surprising because purified antigen administered by either route elicited higher levels of anti-PspA immunoglobulins in serum and the lungs. In contrast to the rPspA vaccines (which predominantly elicited IgG1 and IgA), the lactococcal vaccine elicited almost equivalent anti-PspA IgG1:IgG2 titers, indicative of a shift toward a Th1 response. When inactivated, the lactococcal vaccine still afforded significant protection against respiratory challenge, even when compared with that afforded by the injected vaccine. Compared with its live counterpart, however, the inactivated vaccine showed reduced efficacy against respiratory challenge despite eliciting significantly higher PspA-specific IgG titers and IgG1:IgG2a ratios in serum and significantly higher IgA titers in the lungs. This supports the notion that the Th1 component of the immune response is important in enhancing protection against pneumococcal infections, particularly at mucosal surfaces

A correlation between increased protection and lower PspA-specific IgG1:IgG2a ratios has been reported in a study in which a PspA-based DNA vaccine was used [45]. Similarly, coadministering PspA with interleukin-12 was shown to enhance protection in mice [46]. Although the important role that antibody and complement play in mediating efficient clearance of S. pneumoniae by pulmonary macrophages and recruited neutrophils is well documented [47–49], it is apparent that T cells are also critical. Human pneumococcal infections are characterized by lymphopenia due to the disappearance of activated T lymphocytes with a type 1 cytokine profile that reappear with clinical improvement [50]. More recently, experiments performed in antibody-deficient MuMT−/− mice and using live pneumococcal-based or killed nonencapsulated whole cell vaccines demonstrated that long-term immunity to pneumococcal colonization could be acquired in the absence of antibody via a CD4+ T cell-dependent mechanism [51]. The innate immune response or T cell responses to L. lactis itself may also have an impact on the chemotaxis and activation of neutrophils and macrophages seen with S. pneumoniae [52]. This may explain why L. lactis alone elicited similar (although not significant) protection against respiratory infection to the sc administered PspA. Studies have shown that ingestion of dietary LAB can reduce nasal colonization by pathogenic bacteria including S. pneumoniae [53] or can reduce the incidence and severity of respiratory diseases in young children [54, 55]. These protective effects may be related to the stimulation of neutrophil activity as shown with oral intake of L. lactis in humans [56] or by increasing the number of activated alveolar macrophages and lymphocyte populations in the tracheal lamina propria in mice after inl pretreatment with Lactobacillus fermentum [57]. The L. lactis strain used in this study is a known inducer of Th1 cytokine responses [15] and may also elicit Toll-like receptor 2-dependent production of tumor necrosis factor (TNF)-α in macrophages, as do other LAB [58]. Such mechanisms could be particularly relevant to this pulmonary disease model because reduced expression of TNF-α in the lungs of CBA/ca mice during the early stages of infection with pneumococcus may decrease recruitment of neutrophils and increase susceptibility to pneumococcal pneumonia [59]. Surprisingly, coadministering (inl) rPspA with L. lactis did not improve protective efficacy despite inducing antibody responses that were similar to those induced by the live recombinant vaccine. We conclude, therefore, that, as a recombinant vaccine, L. lactis is better able to stimulate PspA-specific cell-mediated and humoral responses that are together more effective at eliciting protection against pneumococcal infection

This is, to our knowledge, the first demonstration of protection against respiratory and invasive pneumococcal disease induced by mucosal administration of a recombinant lactococcal vaccine. Moreover, the lactococcal vaccine was better than an injected vaccine in protecting against respiratory challenge and caused the induction of antibody responses that were dependent on the use of L. lactis In addition to clarifying the cellular basis of the elicited immunity, future work will improve this vaccine prototype by optimizing antigen production and/or coexpressing immunoregulatory cytokines while ensuring biological containment [60]. A mucosal recombinant L. lactis-based vaccine producing 1 or more conserved antigens would be broadly protective and amenable to large-scale vaccination programs in developed and developing countries. Because it may also reduce carriage rates shown to be important in pneumococcal disease, a lactococcal vaccine could have enormous potential in any future strategies aimed at combating this important pathogen

Acknowledgments

We thank Peter Mayne for assistance and advice with the purification of recombinant pneumococcal surface protein A and Francis Mulholland for the matrix-assisted laser desorption/ionization-time of flight mass spectrometry

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Potential conflicts of interest: none reported
Financial support: European Framework 5 Programme (grant QLK3-2000-00340 to J.M.W.)
Present affiliation: Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, and Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek, Zeist, The Netherlands