This study demonstrated that active immunization with ψPLY and Freund's adjuvant significantly decreased the corneal damage associated with pneumococcal keratitis, whereas immunization (active or passive) against the polysaccharide capsule using the commercially available adult vaccine (PPSV23) did not. The method of active immunizations using bacterial virulence factors in the rabbit model of bacterial keratitis has been successfully used with other organisms.21–24
Active immunization with noncytolytic forms of pneumolysin has also been successfully used in other nonkeratitis models of infection, and immunization of mice with pneumolysin toxoid conferred a significant degree of protection when the mice were challenged intraperitoneally or intranasally.55,56
Before this study, the protective value of actively immunizing with PPSV23 or ψPLY had not been evaluated for the cornea.
Our results show that PPSV23 served as a poor immunogen in this model system. It required twice as many immunization boosts to reach titers considered high enough to proceed with the challenge compared with using ψPLY as an immunogen. In addition, serotype-specific antibody titers performed on serum preadsorbed with cell wall polysaccharide showed that PPSV23 immunization produced inconsistent IgG titers with anti-serotype 19A titers ranging from 0 to 12,800 and anti-serotype 3 titers ranging from 800 to 12,800. The higher titers also failed to correlate with better protection during the course of the infection (data not shown). The protective value of the PPSV23 vaccine remains highly controversial, with numerous clinical trials showing a widely varying degree of antibody production,57,58
as well as variation in the opsonic capacity and ability of these antibodies to protect against infection.59–66
It is also important to note that all clinical and observational trials regarding the protection afforded by PPSV23 immunization correspond to systemic pneumococcal infections. Our study is the first to evaluate the efficacy of PPSV23 immunization and corneal infections.
Moreover, whole-blood survival assays67
and opsonophagocytosis assays47
performed on blood and serum from PPSV23-immunized rabbits, respectively, showed that active immunization with PPSV23 failed to produce antibodies capable of opsonizing and killing either bacterial strain (data not shown). Previous studies have shown that pneumococcal polysaccharide-specific antibodies that are unable to promote opsonophagocytosis in vitro are still able to protect against pneumococcal infections in mice.68,69
Our study showed both a lack of bacterial killing in vitro and a lack of protection in vivo with PPSV23 active immunization. Antibodies capable of opsonizing serotype 3 (S. pneumoniae
strain WU2) and serogroup 19 (S. pneumoniae
strain K1443) were purchased and passively administered to rabbits immediately after infection, to confirm that the lack of protection observed after PPSV23 active immunization was not due to the rabbits' production of nonopsonizing antibodies. Our results indicate that even when the antibodies were able to opsonize and kill serotype-matched pneumococci, there was no reduction in the severity of infection (), confirming that an immunization strategy other than PPSV23 immunization is necessary to protect against pneumococcal corneal disease. The dosage of passively administered antisera was proven to protect against bacteremia caused by S. pneumoniae
WU2 in the rabbit model. Regardless, bacterial killing did not appear to be a factor in the corneal damage caused by pneumococcal keratitis, because bacterial recovery from corneas had no apparent correlation to clinical severity. This point is underscored by the observation that bacterial recovery from corneas of PPSV23-immunized rabbits mirrored the recovery from corneas of ψPLY-immunized rabbits (), yet ψPLY-immunized rabbits were more protected (). This unique observation could be due to the differences between the eye and other pneumococcal targets of the body that are vascular.
In contrast to the results obtained from PPSV23 immunizations, rabbits immunized against PLY and Freund's adjuvant showed a significant reduction in clinical scores when compared with control and PPSV23-immunized rabbits (). ψPLY and Freund's adjuvant was also highly immunogenic. Furthermore, the protective value of antibodies generated after immunization with ψPLY was confirmed by exposing HCE cells to PLY in the presence and absence of purified polyclonal anti-PLY IgG. The anti-PLY IgG isolated from the serum of immunized rabbits protected HCE cells from death caused by PLY (B, C). It is important to note that the precise concentration of PLY used for the in vitro studies varied due to different preparations of recombinant PLY. Furthermore, the use of 4 μg for the neutralization assay does not imply that it is the only dose at which anti-PLY IgG is protective. It is more likely that the ability of anti-PLY IgG to neutralize toxin is a dose-dependent phenomenon. Because previous studies suggest that corneal IgG levels are primarily due to the diffusion of serum antibodies into the cornea,70
it is feasible to predict that these serum antibodies are capable of neutralizing PLY on diffusion into the cornea during an infection in a manner similar to the neutralization of PLY in cell culture. Unfortunately, determination of IgG-specific neutralization of PLY in the cornea in this study was hindered by the production of corneal defects after intrastromal injection of IgG. It is likely that a more specific ratio of PLY to antibody, as these factors are produced and delivered into the cornea in an in vivo situation, would have shown a neutralization effect. However, determination of a specific ratio would have required the use of numerous animals for an answer to a question (whether anti-PLY IgG neutralizes PLY toxicity) that had already been determined in vitro (B, C). Furthermore, it is likely that secretory IgA molecules found within the cornea are capable of neutralizing PLY. Corneal homogenates had high titers of PLY-specific IgA (). Although we were unable to purify detectable titers for use in a neutralization assay, it is feasible that the anti-PLY IgA acts locally to neutralize the toxin's effects in a manner much like serum IgG.
Serum antibody responses and corneal antibody responses were different after infection with S. pneumoniae
, with serum being high in titers of PLY-specific IgG and relatively moderate in titers of IgM antibody isotypes, whereas corneal homogenates had high titers of PLY-specific IgA and relatively moderate titers of IgG. The lack of serum IgA response after a pneumococcal corneal infection is consistent with what has been seen in P. aeruginosa
and S. aureus
Studies of serum and corneal antibody responses after P. aeruginosa
keratitis in two mouse strains suggested that corneal IgA production is a local response, while corneal IgG levels may have primarily diffused from the serum with lower levels of local production.70
The lack of serum IgA antibodies in ψPLY-immunized rabbits before infection supports the idea that IgA antibodies found in the cornea after infection were made locally and did not diffuse from the serum.
It is interesting to note that bacterial CFUs did not appear to play a role in the clinical severity of the corneal infection in the groups in this study. For the active immunization experiments, the log10
CFUs recovered from ψPLY-immunized rabbits were higher for WU2-infected corneas but significantly lower for K1443-infected corneas when compared with the corresponding controls (). PLY, which is released extracellularly, has recently been shown to also localize to the cell wall of pneumococci.73
It is possible that antibodies were able to target PLY within the cell wall of strain K1443 but not WU2 because the polysaccharide capsule of K1443, although present, appears considerably less mucoid than that of WU2 as colonies on blood agar (Norcross EW, unpublished observation, 2008), and any cell-wall–associated PLY present in K1443 would be more exposed to the antibodies in the extracellular milieu. This antibody targeting of PLY within the cell wall may have resulted in a reduction of recovered CFUs from the corneas infected with S. pneumoniae
K1443 by targeting the bacteria for killing within the macrophage. Alternatively, biochemical differences in capsule composition other than sheer mass, or differences in other components altogether, could account for the differences in bacterial recovery. Differences in opsonization have been observed previously for types 6B and 19F,74
but have not been specifically reported for type 3 versus type 19A. Although higher CFUs were obtained from WU2-infected corneas after immunization with ψPLY, it is important to note that antibody to PLY has not been linked to an increase in bacterial counts for other types of infection. In fact, immunization with PLY has shown a reduction in recovered bacteria as well as an increase in survival after pneumonia and sepsis.55,56,75–82
It is also important to note that we did not test extraocular tissues for the presence of bacteria to examine whether bacteria in the cornea could have spread to other sites. Although rabbits immunized with ψPLY, PPSV23, or PBS did not exhibit any symptoms of pneumococcal spread such as lethargy, disorientation, or decreased appetite after intracorneal infection with S. pneumoniae
, it is impossible to confirm that the spread of bacteria did not occur. Previous research has shown that, although treatments capable of suppressing the host immune system (neutropenia, MyD88 deficiency) may prevent pathology in ocular tissues, such treatments could also allow for bacterial spread to the brain and spleen.83
Although it is possible that immunization with PLY toxoid could prevent disease within the cornea yet allow for extraocular spread of bacteria, the current literature does not provide information on PLY and possible immune suppression in the ocular setting. There are experimental findings showing that what occurs in the cornea is opposite to what occurs in other tissues. For instance, cystic fibrosis transmembrane conductance regulator (CFTR) in lung tissue increases the bacterial clearance of P. aeruginosa
, whereas CFTR-mediated uptake in the cornea leads to pathogenesis.84
Although it is possible that immunization with ψPLY has negative impacts on bacterial spread into other organ tissues in our rabbit model, it seems unlikely, as immunization with PLY toxoid has not been shown to increase bacterial counts in other systems but rather leads to increased protection and survival.55,56,75,81
It is unclear why immunization with either ψPLY or PPSV23 led to significantly higher bacteria recovered from the cornea than in the mock-immunized rabbits after WU2 infection, but significantly lower bacteria after K1443 infection (). The presence of more bacteria is not a desirable effect for an immunization. Therefore, caution should be observed in extrapolating the findings to the clinical setting.
Although the precise reason for the variable bacterial clearance is unknown, a lack of correlation between reduced clinical scores and bacterial clearance from the cornea has been seen previously. After immunization with S. aureus
α-toxin toxoid, there was no difference in bacterial killing between the immunized and mock-immunized rabbits, even though the SLE scores were lower for the immunized group.24
In addition, when rabbits infected with S. pneumoniae
WU2 were passively immunized with anti-PLY sera, there was no difference in recovered CFUs, although there was a decrease in the clinical scores of the passively immunized group.30
It should be noted that Freund's complete adjuvant was used in the initial immunizations for all the groups regardless of immunogen. Freund's complete adjuvant contains multiple mycobacterial cell components and has been reported to elicit a variety of responses in host animal species ranging from the characteristic tubercle skin lesions to changes in delayed type hypersensitivity (for review, see Ref. 85
). Moreover, it is possible that the components in this adjuvant cause production of antibodies that are cross-reactive with pneumococcal components, which would indicate that the effectiveness of active immunization reported herein is not all due to anti-PLY antibodies. However, the Western blot of anti-PLY serum produced by one of the rabbits in this study showed high specificity for PLY and no reactivity with other bacterial components in pneumococcal extracts (). Freund's adjuvant was chosen for the present study based on its known ability to generate high titers, but cannot be used in the human situation. Alternate modes of immunization would have to be explored for possible use of PLY as an immunogen in humans.
PLY has been shown to play a significant role in pneumococcal keratitis. Studies showed that rabbits infected with a PLY-deficient S. pneumoniae
strain had reduced pathology when compared with those infected with the wild-type strain.37,41
Much of the damage associated with pneumococcal keratitis is due to the inflammatory response caused by PLY.41
Antibodies directed against PLY could serve to bind to and inactivate the toxin, thereby preventing the release of chemokines and the invasion of PMNs and other immune cells. Histology of WU2-infected eyes supports this idea, in that the ψPLY-immunized eyes showed reduced infiltration of immune cells when compared to mock- or PPSV23-immunized eyes (). The histologic evidence is supported by the relative amounts of myeloperoxidase in the corneal homogenates such that within each infection group, rabbits immunized with ψPLY had reduced myeloperoxidase in their corneas compared with rabbits immunized with PPSV23 or PBS indicating that immunization with ψPLY results in lower quantities of neutrophils infiltrating the cornea.
It is important that immunizing against S. pneumoniae
provide protection against a broad range of pneumococcal serotypes. The currently available 23- and 13-valent pneumococcal vaccines were designed to target the most prevalent disease-causing capsule types of S. pneumoniae
in the United States. Although the vaccines are successful against those serotypes, they have limited effectiveness in preventing infections overall due to the development of serotype replacement in which capsule types that are circulating in the community but are not included in the vaccine replace vaccine serotypes as the strains causing most disease.86
To successfully immunize against S. pneumoniae
using PLY as the immunogen, it is necessary that PLY immunization protect against a broad range of serotypes. The ply
gene has limited variability across all pneumococcal strains regardless of serotype and thus is a promising immunogen.87
Previous research showed that actively immunizing mice with PLY toxoid provided protection against at least nine pneumococcal serotypes in intraperitoneal and intranasal infection models,56
and the use of protein-based immunizations, including those based on PLY-based vaccines, has been shown to be effective in other models of pneumococcal infections.27,88,89
In the present study, two different pneumococcal strains were used—one that has been characterized and one that is an uncharacterized clinical keratitis strain—to determine whether immunization could protect against more than one strain and type. Although it is beyond the scope of this study to test additional pneumococcal serotypes, it is likely that the generalized protection provided by immunization with PLY as reported in mouse intraperitoneal and intranasal infections can be extrapolated to corneal infections. Since the protection observed in this study was significant but not complete, the use of additional components such as conserved pneumococcal proteins other than PLY or specific peptides that target pneumococcal virulence factors might improve the immunization method described herein. To date, there have been no reports of any other pneumococcal virulence factors for keratitis, and PLY cannot be the only factor in pneumococcal keratitis, since disease still occurs in rabbit corneas infected with a PLY-negative strain, albeit significantly less severe than in those infected with a wild-type strain.32
Determination of these factors will aid in refining treatment strategies for pneumococcal keratitis.
In summary, ψPLY served as an effective immunogen, and actively immunizing rabbits with ψPLY and Freund's adjuvant provided protection against corneal damage after challenge with S. pneumoniae, whereas active immunization with PPVS23 and Freund's adjuvant and passive immunization with serotype-specific anticapsular antisera did not. Furthermore, PPSV23 acted as a poor immunogen in our model system. In addition, anti-PLY IgG effectively neutralized the toxin in cell culture cytotoxicity assays. This study illustrates the importance of PLY in ocular infections and provides evidence that adding a protein-based immunogen to the current vaccination regimen may ameliorate pneumococcal ocular disease.