Killed but Metabolically Active Salmonella typhimurium: Application of a New Technology to an Old Vector
- Reprints or correspondence: Dr. Elizabeth L. Hohmann, Infectious Diseases Div., Jackson 504, Massachusetts General Hospital, 55 Fruit St., Boston, MA 02114 (ehohmann{at}partners.org).
Abstract
Previous studies have shown that attenuated salmonellae utilized as vaccine vectors engender strong immune responses; however, balancing immunogenicity with reactogenicity remains problematic. Recent work in other bacteria has shown that photochemical treatment of DNA excision repair mutants (ΔuvrAB) renders organisms “killed but metabolically active” (KBMA). Here, we extend this concept to Salmonella typhimurium. A strain of attenuated S. typhimurium previously evaluated in human volunteers was further deleted for uvrAB genes and designated CKS362. Photochemical treatment of CKS362 resulted in significant inactivation. These KBMA organisms were metabolically active as shown by radioactive methionine incorporation and lactate dehydrogenase activity. In mice inoculated intraperitoneally, KBMA CKS362 was markedly less reactogenic and stimulated a humoral immune equivalent to its live counterpart. Because the parental strain has previously been found to elicit strong immune responses to Salmonella antigens, we propose CKS362 as a prototype strain to test the immunogenicity of KBMA organisms in humans.
Rationally attenuated salmonellae have long been studied as oral vaccine vectors capable of carrying heterologous antigens and can stimulate mucosal, humoral, and cellular immune responses (reviewed in [1]). The highly attenuated vector strains studied in humans to date have not resulted in strong immune responses to the vectored antigens. Safety concerns also remain, particularly in populations with a high incidence of undiagnosed HIV-infected individuals.
Vaccines that are “killed but metabolically active” (KBMA) have been recently described as retaining the immunological properties of live organisms but having a safety profile closer to that of killed organisms. Brockstedt et al. developed Listeria monocytogenes ΔuvrAB DNA excision repair mutants that cannot replicate after photochemical treatment with UVA light and a synthetic psoralen compound known as S-59 or amotosalen [2]. Psoralens form covalent adducts with DNA in the presence of UVA light [3]. In normal bacteria, psoralen adducts would be eliminated by the ABC excinuclease nucleotide excision repair process, encoded by the UV light response genes (uvrA, uvrB, and uvrC; reviewed in [4]). These are conserved genes present in bacteria and archaea but not in eukaryotic cells. The uvrA and uvrB genes are part of the “SOS response” to DNA damage. Deletion mutants lacking any 2 of the uvr genes are unable to excise psoralen adducts and are therefore highly sensitive to psoralen and UVA light, compared with intact organisms. In a population of ΔuvrAB bacteria, a relatively small number of S-59 adducts randomly distributed about the bacterial chromosomes results in a population that cannot replicate but still expresses the entire bacterial genome. Brockstedt et al. demonstrated that these psoralen-treated, irradiated ΔuvrAB mutants of L. monocytogenes carrying foreign antigens synthesize and secrete proteins and elongate in vivo and in vitro but do not divide. When used as vaccine vectors, these “living dead” L. monocytogenes vaccine vectors induced CD4 and CD8 cell immune responses against foreign antigens and were effective vaccines in rigorous murine models of tumorigenesis and infection [2]. The authors showed that KBMA Bacillus anthracis could also be generated using this technology.
We have generated uvrAB deletion mutants of Salmonella typhimurium and demonstrate that the KBMA concept can be extended to this gram-negative organism.We show that KBMA S. typhimurium is metabolically active, more attenuated than the live organism, and strongly immunogenic in a standard mouse model of salmonellosis. A Salmonella organism that is already highly attenuated by virtue of specific defined deletions in the phoP/phoQ and aroA genes and previously evaluated in human volunteers was chosen for the initial study. Because there is ample clinical experience with live Salmonella vectors, these organisms may present an ideal opportunity to compare the immunogenicity of live and KBMA organisms in humans.
Materials and Methods
Molecular biologic analysis. The S. typhimurium strains used in this work are derived from wild-type (WT) strain SL1344 as shown in figure 1. All deletions were made using allelic replacement suicide vectors derived from pCVD442, which exploits the sacB gene of Bacillis subtilis [5]. Strain CKS362 was rederived from a ΔphoP/phoQΔaroA mutant (figure 1), to use the endogenous streptomycin resistance phenotype conferred by an intact strepA/B locus in creating the uvrA/B deletions. The genetic sequences for uvrA and uvrB were obtained from the complete sequence of S. typhimurium LT2 [6] and were used to generate primers. Deletion loci for the uvrA and uvrB genes were generated by overlap extension polymerase chain reaction (PCR); these fragments were cloned into pCVD442 to make suicide vectors for generation of in-frame deletions in the uvrA (Δ2614bp; nt 4475911–4473299; 92%) and uvrB (Δ1811bp; nt 864484–866319; 91%) loci. The strepAB gene was deleted at the conclusion of the process, so the final strain was free of antibiotic resistance genes. Clones were screened for chromosomal deletions by PCR and agarose gel electrophoresis and were subsequently confirmed by DNA sequencing.
Figure 1.
Strain derivation pathway. All plasmid deletion vectors indicated are derived from pCVD442 [5]
Bacteriological analysis. S. typhimurium cultures were grown aerobically at 37°C in Luria broth (LB; Becton Dickinson), supplemented with aromatic amino acids. Bacterial colony-forming units were enumerated by spread plating of serial dilutions on LB agar plates. KBMA and live inocula used in radioactive methionine incorporation and animal experiments were thawed from stocks frozen at –80°C in normal saline with 20% glycerol.
Photochemical treatments. Amotosalen (S-59) and the INTERCEPT UVA Illuminator System were provided and calibrated by Baxter/Cerus Corporation. The INTERCEPT illuminator device is designed for use with plastic blood component bags and delivers and records the energy dose. Overnight bacterial cultures were back-diluted (1:100) and incubated aerobically at 37°C with shaking until the OD600 reached ∼0.5 U. Varying concentrations of S-59 were added, and cultures were grown for an additional hour. Cultures were then chilled on ice and transferred to sterile 14-cm petri dishes (Nalge Nunc International) for irradiation at various energy levels (J/cm2), as per the usual process for S-59—treated blood products. Before irradiation, aliquots were removed and kept at 4°C for enumeration of live colony-forming units per milliliter. Cultures were irradiated at varying energies, and aliquots were removed for enumeration of residual live colony-forming units per milliliter. Photochemically inactivated cultures were then harvested by centrifugation (for 10 min at 5000 g), washed once with normal saline, and resuspended in 1 equivalent volume of saline. Aliquots of this saline solution were removed for plating and counting to enumerate residual live organisms, compared with input organisms, before photochemical inactivation. Subsequent dilutions to the desired concentration were made based on calculations/extrapolations from the curve of optical density at 600 nm versus colony-forming units per milliliter, generated from previous inactivation runs.
Metabolic assays. A commercially available system, the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega), was used to quantify lactate dehydrogenase activity as a measure of metabolism. This assay uses the novel tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and the electron coupling reagent phenazine ethosulfate. MTS is bioreduced by cells, and a soluble formazan product is directly quantified by absorbance at 490 nm. Serial dilutions of bacteria were incubated at 37°C in LB to which substrate was added in microtiter plates, and optical density at 490 nm was read over time.
35S-methionine incorporation. Radioactive methionine(Perkin Elmer) at a final concentration of 20 μCi/mL was added to KBMA organisms, live organisms, and heat-killed organisms, which had been suspended and previously incubated for 30 min at 37°C in M9 minimal medium containing other amino acids but lacking exogenous methionine. Incubation continued for 30 min and was followed by addition of excess “cold” methionine at 1 mmol/L and a further 15 min of incubation. Bacterial and culture supernatant proteins were harvested separately. Supernatant proteins were precipitated from medium by use of trichloroacetic acid (10%; Sigma-Aldrich). Bacteria were washed twice with sterile saline and lysed in PAGE loading buffer. Proteins were separated by PAGE, and autoradiography was used to visualize proteins in dried gels.
Macrophage studies. The J774 murine macrophage—like cell line (ATCC TIB67) was used to assess invasion and survival within mammalian cells [7]. This adherent line was chosen over more metabolically active primary macrophage cultures for ease of use and to minimize animal experimentation. Macrophages were infected for 1 h at a multiplicity of 200–300 bacteria/cell. Extracellular bacteria were then killed with gentamicin (20 μg/mL) for 30 min. Triplicate wells were lysed with sterile water to determine baseline (time 0) intracellular bacterial colony-forming units. Additional wells were allowed to incubate for another 4 or 18 h with gentamicin-containing medium, subsequently lysed, plated, and counted.
Murine studies. Female BALB/c mice (Taconic Farms) 8–12 weeks old were used, and experiments were reviewed and approved by the IACUC at Massachusetts General Hospital. In experiments designed to evaluate virulence, groups of 5 animals were tested per dose. Animals received inocula in saline (300 μL intraperitoneally [ip] or 50 μL by oral feeding with a micropipette tip). Animals were observed several times daily when ill, and those deemed moribund and certain to die were killed, and the use of WT controls was minimized. The LD50 doses were determined by the method of Reed and Munch [8]. Animals were killed by CO2 inhalation; blood samples were obtained at the time of killing by cardiac puncture.
ELISAs. ELISAs were developed in the laboratory to detect serum IgG directed against Salmonella-specific antigens and were similar to those described for prior clinical studies [7, 9]. The blocking solution was PBS plus 0.05% Tween-20 and 5% dried milk. Maxisorp microtiter plates (96 well; Nalge Nunc International) were coated with 10 μg/mL antigen in 40 mmol/L sodium carbonate buffer (pH 10.0) overnight at 4°C with shaking. S. typhimurium lipopolysaccharide (LPS) was purchased from Sigma-Aldrich. Sheared flagella were isolated and purified from S. typhimurium as described elsewhere [7] and contained 3.2 ng/μg endotoxin (0.32%) as measured by the limulus lysate assay (Associates of Cape Cod). Salmonella-secreted proteins were isolated from spent culture medium by precipitation, as described for listerial-secreted proteins [9, 10]. Serum samples were diluted 1:20 in blocking solution and serially 2-fold across the microtiter plate. Secondary antibody was affinity-purified phosphatase-labeled goat anti—mouse IgG (1:2000 in blocking solution; Kirkegaard and Perry Laboratories). Plates were developed with p-nitrophenyl phosphate (2 mg/mL) in 1 mol/L TRIS (pH 8.0). Plates were read by the optical density at 405 nm by use of the Vmax Molecular Devices Kinetic Microplate Reader and SoftMax Pro software. The end-point dilution titer was empirically defined as the highest dilution at which OD405 was ⩾0.15 U. A 4-fold or greater increase in endpoint dilution titer over saline-injected control animals was considered to be seroconversion.
Results
Bacteriological analysis and virulence assessment. Derivation of S. typhimurium CKS362 (ΔuvrAB) from the WT SL1344 background strain and its relationship to CKS257 (evaluated in humans) is shown in figure 1. Strain CKS362 was evaluated in vitro under ambient light without photochemical treatment to determine whether deletion of the uvrAB genes resulted in any obvious additional independent attenuation or growth defect. There were no obvious alterations in colony morphology or growth curves in LB between strain CKS362 with uvrAB deletions and its sister strain, CKS257, lacking these deletions, although the uvrAB deletion mutants grew to slightly lower final optical density (∼0.2 OD600 U). As shown in figure 2 and elsewhere [9], the attenuating deletions in both strains confer a defect in intracellular survival within J774 macrophages, compared with WT organisms. Murine studies were performed to evaluate virulence, using organisms grown to stationary phase (16 h).We have previously used ip inoculation of female BALB/c mice as a measure of virulence before performing clinical studies of S. typhimurium vaccine strains. The LD50 for strain CKS362 (ΔuvrAB) without photochemical treatment was 1.4 × 108 cfu, essentially identical to CKS257 with these loci intact, at 1.3 × 108 cfu. The LD50 for WT strain SL1344 concurrently studied was <20 organisms. In a second experiment, we compared CKS362 (ΔuvrAB) grown with psoralen but not irradiated and CKS362 (ΔuvrAB) grown with psoralen and irradiated (KBMA), and the respective LD50 determinations were 5.7 × 107 and 7.3 × 108 cfu. There was ∼1 live organism/780,000 particles in the KBMA inoculum used. In this second experiment, bacteria were harvested for photochemical treatment/inoculation or live inoculation while in mid-log-phase growth, whereas the first experiment comparing live CKS257 and CKS362 utilized bacteria grown to stationary phase. Because growth phase and many environmental signals regulate Salmonella invasiveness and virulence [11], the LD50 values cannot be compared across experiments that use log- and stationary-phase organisms.
Figure 2.
Bacterial persistence within adherent murine J774 macrophage—like cells. CKS362 (ΔuvrAB ) is less persistent than wild-type Salmonella typhimurium and is comparable to its sister strain, CKS257 (uvrAB intact). Macrophages were infected with wild-type (●), CKS257 (Δ), or CKS362 (◯) at a multiplicity of ∼300:1 for 30 min at 37°C. Cells were rinsed free of excess bacteria, and medium containing gentamicin was added for 30 min. Macrophages were then lysed in sterile H2O, and intracellular bacteria were enumerated by serial plating (time 0). Identical triplicate cultures were lysed and enumerated at 4 h and 18 h. Error bars represent SE values.
S. typhimurium CKS362 (ΔuvrAB) cultures grown in the presence of S-59 and treated with varying doses of psoralen S-59 and UV radiation. Viable bacterial colony-forming units were determined and compared with nonirradiated bacteria. S-59 alone at escalating doses was found to be nontoxic to bacterial cells with intact or absent uvrAB loci (data not shown). The UVA illuminator has 7 fixed energy doses, and the dose range was studied. Bacterial inactivation was positively correlated with psoralen dose and radiation dose. CKS257 organisms lacking the uvrAB deletions were markedly less susceptible to photochemical treatment than CKS362 with uvrAB deletions (figure 3). In several experiments, at various radiation doses, a psoralen concentration of 500 ng/mL (1.5 μmol/L) was found to produce maximal results; larger doses resulted in no additional effect (figure 3). Superior inactivation was accomplished by irradiating bacteria in log-phase growth in LB after 1 h of exposure to S-59. Washing bacteria and suspending them in normal saline rather than LB before irradiation and utilizing a longer exposure of bacteria to S-59 (i.e., adding psoralen for the entire growth period after back dilution) were not advantageous.
Figure 3.
Greater sensitivity to photochemical inactivation in CKS362 (ΔuvrAB) than in its sister strain, CKS257 (uvrAB intact). Mid-log-phase cultures were treated with varying concentrations of psoralen S-59 and grown for 1 h. Bacteria were then illuminated with 7.2 J/cm2 UVA light, and viability was assessed by serial dilution and plating for colony-forming units. Points represent mean values of duplicate plates counted at the most appropriate dilution.
Metabolic assessment. KBMA Salmonella CKS362 were evaluated using a commercial assay that measures the lactate dehydrogenase enzyme activity. Assays were performed by resuspending KBMA Salmonella in LB in 96-well plates and measuring optical density at 490 nm over 2–4 h. Control wells included medium alone and an identical inoculum of live bacteria (both metabolically active and replicating and therefore much more metabolically active). Figure 4B shows the metabolic activity of KBMA organisms over time, compared with that of live replicating organisms. KBMA bacteria clearly reduced MTS, compared with negative control wells, but less so than live replicating organisms. We demonstrated that MTS reduction continued at 4 h (data not shown) but have not yet more carefully analyzed the rate or duration of metabolism. Similar results were seen in an assay measuring glucose fermentation in which medium containing 2% glucose and phenol red was inoculated, and the development of a yellow color was monitored as an indicator of acidic fermentation products (data not shown). Heat-killed bacteria result in no activity in either assay. A radiation dose of ⩾7.2 J/cm2 seemed to offer the best balance of inactivation and metabolic activity as measured by bacterial killing and the MTS assay, respectively (figure 4). Incorporation of radioactive 35S-methionine and polyacrylamide gel electrophoresis of labeled proteins was also used to evaluate bacterial metabolism. As shown in figure 5, KBMA CKS362 synthesize and also secrete protein in a radiation dose—dependent manner. Heat-killed bacteria and “no bacteria” control wells show no 35S-methionine incorporation (even with prolonged exposures; data not shown).
Figure 4.
Metabolic activity in CKS362 (ΔuvrAB; ◯) after photochemical treatment, despite markedly reduced viability, compared with parent strain CKS257 (uvrAB intact; □). Mid-log-phase cultures were treated with psoralen S-59 (500 ng/mL) and grown for 1 h. Cultures were illuminated at the indicated energy levels, and viability (colony-forming units) was determined by serial dilution and plating (mean of duplicate plates). Panel A shows viability as a function of energy. No viable bacteria could be detected in cultures receiving ⩾7.2 J/cm2. Panel B shows reduction in MTS substrate by bacterial lactate dehydrogenase over time, as measured by an increase in optical density at 490 nm.
Figure 5.
Synthesis of cell-associated and secreted protein by photochemically inactivated CKS362 (ΔuvrAB ) and control strains, as measured by 35S-labeled methionine incorporation. Whole cell lysate and trichloroacetic acid—precipitated supernatant proteins were fractionated by SDS-PAGE and visualized by autoradiography (exposure times: secreted protein, 17 h; cell lysate, 6 h). Heat-killed bacteria and control lanes show no incorporation. KBMA, killed but metabolically active.
Mouse experiments. BALB/c mice were inoculated ip with live organisms, KBMA organisms, heat-killed organisms, or saline. Animals were observed daily after inoculation and rated for illness on a scale of 0 (totally well) to 8 (expired), as described in the figure 6 legend. Animals receiving heat-killed organisms exhibited minimal evidence of systemic reaction on day 1 after inoculation only. Animals receiving KBMA organisms showed signs of moderate illness for ∼4 days, whereas animals receiving a 10-fold lower dose of live organisms were ill for ∼8 days and reached a peak severity of illness that was greater than that of any other group. A separate group of animals was also inoculated concurrently with a live dose of 54 cfu, comparable to the remaining residual live organisms present in the KBMA (7.2 J/cm2) inoculum. These animals showed no signs of illness (figure 6A). These findings were mirrored in spleen weights (figure 6B). Animals were killed 18 days after inoculation, and serum IgG directed against S. typhimurium LPS, flagella, and Salmonella-secreted proteins was measured to quantify systemic immune responses to S. typhimurium (figure 7). As expected, even large doses of heat-killed bacteria resulted in small immune responses. For all 3 antigens, animals receiving KBMA CK362 generated a geometric mean antibody titer that was 1.5–6.5-fold greater than that induced by live CKS362. Results were radiation dose—dependent, with bacteria that received a maximal UVA dose of 10.8 J/cm2 stimulating a less vigorous humoral immune response than those that received a dose of 7.2 J/cm2. Small residual numbers of live bacteria (26 cfu) were left in the KBMA inoculum irradiated at 7.2 J/cm2; there were no residual live bacteria in the parallel inoculum irradiated with 10.8 J/cm2. To be certain that the humoral immune responses were not attributable to proliferation of this small number of residual live organisms, a control group of animals that received a dose of 54 live bacteria was included. As shown in figure 7, no humoral immune responses were detected in animals that received 54 cfu of live bacteria. In a separate experiment, identical numbers of KBMA and live organisms were inoculated (106 cfu), and immune responses to LPS were comparable to those shown in figure 7, which were the same for KBMA and live organisms, but KBMA organisms resulted in 5-fold greater titers to flagella and secreted proteins.
Figure 6.
Health status and splenic weight of BALB/c mice (3–4/group) injected intraperitoneally with live or killed but metabolically active (KBMA) CKS362 (ΔuvrAB). Mice received saline, live organisms at 2 × 107 or 54 cfu, KBMA (7.2 J/cm2), KBMA (10.8 J/cm2), or heat-killed organisms (all at an equivalent dose of 4 × 107 organisms). Mice were graded on a scale of 1 to 8 for signs of illness as follows: 1, fully healthy; 2, trace illness; 3, mild illness—reduced activity or less responsive; 4, mild-moderate illness—reduced activity, hair on end, less alert; 5, moderate illness—reduced activity, hair on end, eyes open, movement when touched; 6, moderate-severe illness—reduced activity, hair on end, crouched, eyes closed, some movement when provoked, may survive; 7, moribund, not moving, killed; 8, expired. Mean illness grading over time (A) and mean splenic weight at killing on day 18 (B), which generally mirrors systemic illness, is plotted. All mice were fully healthy after day 12. Error bars represent SDs.
Figure 7.
Strong systemic humoral response to Salmonella antigens in mice immunized with killed but metabolically active (KBMA) CKS362 (ΔuvrAB). BALB/c mice were immunized as described in the figure 6 legend and were killed 18 days after immunization, and blood was collected. Serum IgG directed against S. typhimurium lipopolysaccharide (LPS), flagella, and secreted proteins was detected by ELISA and are reported as endpoint titers. Nos. within bars for the KBMA groups represent the no. of residual live organisms present in the KBMA inocula. Results are the logarithm of the geometric mean titer (GMT) for each group.
Like its sister strain, CKS257, S. typhimurium CKS362 secretes an HIV Gag antigen, as a fusion protein of the Salmonella SopE protein, via the type III secretion system [9]. Similar to results in clinical studies of CKS257, serum antibodies to p24 antigen were not detected in serum from any of the groups (data not shown). Oral inoculation of mice with 1 or 2 doses (day 0 and day 22) of either live or KBMA S. typhimurium CKS362 or CKS257 resulted in little to no measurable systemic Salmonella immunity in mice (data not shown). This is in direct opposition to human studies, in which CKS257 was shown to produce strong mucosal and humoral response to S. typhimurium LPS and flagella in humans that received single live oral doses of 107–1010 cfu [9].
Discussion
These results demonstrate that the KBMA concept can be applied to S. typhimurium. Radiation of pathogens to make vaccines has been tried with varying success for several organisms, including Malaria [12], Brucella [13], Burkholderia [14], Pasturella [15], and Listeria [16] species, and typically involves high doses of γ or UV radiation. The addition of psoralen in the presence of uvrAB mutations significantly lowers the dose of radiation required to prevent replication and may allow more complete and efficient expression of the bacterial transcriptional repertoire. For unknown reasons, complete photochemical inactivation of S. typhimurium, as measured by colony-forming units, required approximately a 10-fold higher concentration of psoralen (∼1.5 μmol/L) than in previous studies with the gram-positive organisms L. monocytogenes and B. anthracis [2]. We have not comprehensively studied the longevity of metabolic activity in KBMA Salmonella organisms nor demonstrated what degree of metabolism is essential for immune responses. Additional experiments with lethally radiated bacteria and fixed, inactivated bacteria or bacterial “ghosts” that are not metabolically active will be done in future to address this.
The uvrAB mutations did not independently alter virulence in animals, but photochemical treatment of the uvrAB mutants markedly reduced illness in inoculated animals. KBMA CKS362 organisms engendered briefer clinical reactions midway between heat-killed and live organisms. As is true of the live strains CKS257 and CKS362, KBMA organisms at very high ip doses were still able to cause death, likely a result of LPS overload. This death is very unlikely due to residual live organisms: the small, “subimmunogenic” numbers of live residual organisms remaining in some experimental inocula (<100 organisms) do not cause death or illness, and animals tolerate even large numbers of the highly attenuated live strain also (up to 107 cfu).
It was surprising and encouraging that mice inoculated with KBMA organisms were less sick but had systemic immune responses to Salmonella antigens that were at least as good as those engendered by live organisms. This could be in part due to the absence of prolonged systemic illness or the lack of other deleterious effects of live salmonellae on innate immunological function [17]. We did not evaluate mucosal or cellular immune responses or challenge animals with WT organisms in these early experiments, but these experiments are planned. Neither the live nor KBMA organisms resulted in systemic immunity when administered orally to mice, a finding that contradicts data in human volunteers who showed systemic seroconversion and strong mucosal immune responses to Salmonella antigens to CKS257 [9]. Because the 2 strains are highly similar in terms of growth, persistence in mouse macrophages, and virulence in the mouse ip model, CKS362 seems a good strain to test whether KBMA organisms can stimulate immune responses to Salmonella antigens in humans. The phoP/phoQ deletion eliminates a major virulence regulatory locus in salmonellae, and the aroA deletion renders the organism auxotrophic for aromatic amino acids not available in mammalian tissues. Both of these loci have been independently demonstrated to reduce virulence in animals and humans in several prior studies. Psoralens have various clinical applications already. Photochemical treatment with S-59 is used clinically to eliminate replicating leukocytes and infectious agents in human blood products (primarily platelets). Photochemical treatments are also used to control the exuberant dermal replication in psoriasis and in cancer therapy (photodynamic therapy). On the basis of this murine and prior human data [7, 9], we think humans would respond immunologically to KBMA Salmonella administered orally. Because the strain under study here is already highly attenuated, superior results may be seen with less attenuated organisms, for example, single aroA or phoP/phoQ mutants. Perhaps even photochemically treated WT organisms or PhoPconstitutive mutant Salmonella could be utilized, the latter being highly immunogenic in animal models [18, 19] but unsuitable as live human vaccines because of reversion. Study of these strains could also allow analysis of environmentally regulated antigen expression in KBMA organisms, a very promising approach in animal models [20, 21]. Additionally, the inactivation afforded by photochemical treatment might allow exploration of intranasal administration of Salmonella vectors, given that this route is of special interest for generation of genital mucosal immunity.
Acknowledgments
We thank Thomas Dubensky and John Hearst of Cerus Corporation for helpful discussions.
Footnotes
-
Potential conflicts of interest: none reported.
-
Financial support: National Institutes of Health (grant R01 AI67103 to E.L.H.).
- Received September 15, 2006.
- Accepted November 18, 2006.
- © 2007 by the Infectious Diseases Society of America
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