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Live, attenuated Salmonella has been used to express heterologous antigens for development of oral vaccines. Often, expression must be regulated because of deleterious effects on the Salmonella vector. The effect of varying expression levels on immune response parameters has not been well defined. In this study we introduced mutations in the -10 region of the trc promoter in the expression plasmid to generate series of vaccine strains with different levels of expression of a model antigen, the hemagglutin HagB from Porphyromonas gingivalis. There was no difference in growth rates of the Salmonella vaccine strains containing the wild-type, the mutant plasmids or the empty expression vector. The primary IgG response in serum in mice orally immunized with the wild type strain peaked 3-4 weeks earlier than the intermediate expression level strains, suggesting that high expression levels may favor an earlier response. While there was a trend for anti-HagB recall responses to correlate with higher expression level, the peak levels were not significantly different even for expression levels as low as 33% of wild type. A similar trend in terms of response level was seen with serum and salivary IgA. The subclass of the IgG response was predominately IgG2a regardless of expression level, consistent with a Th1 response. These data suggest that isotype distribution, immune response level and T helper cell profile is largely unaffected over a wide range of expression levels.
Live, attenuated Salmonella has been shown to be an effective vector for oral delivery of recombinant vaccine antigens, resulting in mucosal and systemic responses [1-4]. Recombinant antigen expression avoids the problem of antigen purification and stability when administered via the oral route. Synthesis of vaccine antigen can occur de novo in inductive lymphoid tissues resulting in a rich immunological response with long-term memory . As with any system involving expression of foreign proteins in bacteria, certain proteins can be toxic to the expression vector when expressed at high or even moderate levels. This can be particularly problematic with live recombinant vaccines. Vector fitness can affect growth rate, membrane integrity, and determine the ability to survive transit through the gut colonize and persist in inductive tissues. Modulation of expression level can be used to avoid toxicity but could have effects on the subsequent immune response parameters. In this study, we introduced defined mutations into the promoter of a plasmid expressing a nontoxic heterologous hemagglutinin gene of Porphyromonas gingivalis, a putative pathogen in human periodontal disease, in the cytoplasm of avirulent Salmonella enterica serovar Typhimurium and determined the effects of different levels of expression on the magnitude of the primary and recall responses in mucosal and systemic compartments. The effects on the IgG subclass distribution of the response in serum were also evaluated.
The bacterial strains and plasmids used in this study are listed in Table 1. Strains were cultured aerobically at 37°C on Luria-Bertani (LB) medium  or on agar plates, with the addition of DL-α,ε-diaminopimelic acid (50 μg/ml) for the plasmid-free Δasd strains. Cultures were maintained at -80°C as glycerol stocks.
The plasmid pCRB2922 expressing the hagB hemagglutinin of P. gingivalis under control of the trc promoter was constructed as previously described . Briefly, the hagB gene was amplified by PCR from the plasmid pAX-hagB which contains the entire open reading frame and flanking regions of hagB cloned from P. gingivalis strain 381 . A 3′ SalI site introduced into the amplified hagB gene was ligated into EcoRI-SalI digested pYA292, an expression plasmid containing the gene coding for aspartate β-semialdehyde dehydrogenase (asd) from S. enterica serovar Typhimurium . The EcoRI site was blunted by digestion with mung bean nuclease and ligated to the blunt 5’ end of the hagB gene in frame with the trc promoter. The plasmid was initially transformed into E. coli χ6097, a Δasd cloning/expression strain. The asd gene expressed by the recombinant plasmid complements the deletion in the E. coli cloning and Salmonella vaccine strains and promotes stable maintenance of the plasmid without the need for antibiotics [9, 10].
Mutations were introduced into -10 region (TATAAT) of the trc promoter to regulate expression of hagB as previously described . Briefly, pCRB2922 was amplified using a primer pair, one of which contained random nucleotide substitutions in the -10 region (TANNNT). Clones of E. coli χ6097 generated from the mutated library were screened for variable expression using colony immunoblotting with an IgG fraction of rabbit anti-HagB IgG raised against histidine-tagged recombinant HagB purified by nickel-nitriloacetic acid agarose (Qiagen Inc., Valencia, CA) . The IgG fraction was obtained using immobilized Protein A chromatography (Sigma-Aldrich, St. Louis, MO). The promoter regions of candidate mutant plasmids were sequenced to determine the mutations and then a restriction fragment containing the promoter and hagB reading frame was cloned into a similarly restricted copy of pYA292 to minimize chances that any mutations occurred elsewhere in the plasmid. Plasmids from selected clones were electroporated into the vaccine strain S. enterica serovar Typhimurium χ4072.
Growth curves were obtained using the Bioscreen C Microbiology Reader (Oy Growth Curves AB Ltd., Helsinki, Finland). Overnight LB static cultures of Salmonella vaccine strains containing the empty expression vector (pYA292) or mutated expression plasmids were diluted 1:40 in LB and grown with shaking to OD= 0.5. The cultures were diluted 1:100 in fresh LB, and the growth of all strains was monitored simultaneously at 30 min intervals at 600nm at 37°C with continuous shaking.
Cell lysates of vaccine strains were prepared and assayed for expression level as previously described . Briefly, strains were grown in LB medium to an optical density of 0.8 at 600nm. The cells were collected by centrifugation of a 1.5ml portion at 13,000 × g for 5 min at 4°C. The pellet was washed 2x using 7mM sodium phosphate buffer (pH 7.4) containing 272mM sucrose and 1mM MgCl2. The pellet was resuspended in 1x SDS-PAGE sample buffer (0.0625M Tris-HCl, pH 6.8, containing 2% (w/v) sodium dodecyl sulfate (SDS) and 10% (v/v) glycerol) and boiled for 10 minutes to solubilize proteins.
To quantitate HagB, ELISA plates were coated with purified polyclonal rabbit anti-HagB IgG. After blocking with 0.1% bovine serum albumin, serial dilutions of purified HagB standard , or bacterial lysates were applied to the plates. Biotinylated anti-HagB IgG was applied and the plates were incubated overnight at 4°C. After washing, the plates were incubated for 30 min with avidin peroxidase (1 μg/ml), washed, and developed with 0.04% o-phenylenediamine dihydrochloride in 0.1 M phosphate buffer, pH 5.0, containing 0.012% H2O2. The absorbance at 490nm was recorded using a BioRad model 550 microplate reader (BioRad, Hercules, CA). Samples were compared to standard curves generated employing 4-parameter logistic regression using Microplate Manager III software (BioRad).
The total protein concentration of each lysate was determined using the Micro BCA™ Protein Assay Reagent Kit (Pierce, Rockford, IL). The specific HagB concentration in each lysate was normalized to total protein concentration to allow comparison of the expression level of each strain as ng HagB/μg total protein in lysate. The expression level of the wild-type promoter strain pCRB2922 (143.3 ng HagB/μg protein) was taken as 100%.
Female BALB/c VAF/Plus mice, 6-8 wks of age (Charles River, Wilmington, MA) were housed in filter-top cages in the Infectious Disease Isolation Unit at the University of Florida Animal Resource Center and given sterilized food and water ad libitum. The bedding was changed to reduce coprophagy and the food supply was removed 4 hours prior to immunization to minimize the amount of material in the stomach. Vaccine strains were grown as a static cultures in LB broth overnight at 37°C, diluted 1/20 in fresh LB broth and grown at 37°C with aeration to an OD600 of 0.8. Cultures were centrifuged and resuspended in LB broth to a density of 1010 CFU/ml based on calibrated curves of OD600 vs cell number. Mice were given 0.1ml of 0.1M NaHCO3 by gastric intubation 10 min prior to the vaccine to neutralize gastric acid. Groups of seven mice were immunized by gastric intubation with 109 cells on days 1, 3, and 5 of week 0. The food supply was returned following immunization. Boosting was carried out in the same manner at week 14. Serum and saliva were collected and assayed as previously described [11, 12].
Differences in immune responses between groups were determined by one-way analysis of variance on log-transformed data using Tukey’s post test for mutiple comparisons. Analysis was performed using InStat software (GraphPad Software, San Diego, CA).
The library of promoter mutations in χ6097 was screened by colony immunoblotting using anti-HagB. Clones with variable levels of expression could be detected by observing differences in staining intensity (Fig. 1). Quantitative expression levels were determined in the Salmonella vaccine strain using pCRB2922 (143.3 ng HagB/μg protein), which possesses the wild-type trc promoter, was taken as 100%. Three reduced expression mutants, pCRB65 (75.8%), pCRB1232 (33.2%), and pCRB148 (1.7%), were chosen for immunization studies. There was no difference in growth rates of the Salmonella vaccine strains containing the wild-type, the mutant plasmids or the empty expression vector pYA292 (Fig. 2).
To examine the effects of the cytoplasmic expression level of HagB on the magnitude and kinetics of the immune response, mice were orally immunized at week 0, then boosted at week 14 with the wild-type and expression level mutant vaccine strains. Immune responses were assessed in serum and saliva. In both serum and secretions, there was a general correspondence between expression level and peak responses (Fig. 3). Primary serum IgG responses to the wild-type strain peaked at around 5 weeks while the peak primary response to pCRB65 (75.8%) and pCRB1232 (33.2%) appeared to be delayed by 3-4 weeks (Fig 3a). Peak recall responses occurred around week 19, with the wild-type strain exhibiting a more rapid rise and reaching the highest level. Differences between peak recall responses at week 19 were not statistically significant except for pCRB148 (1.7%), which was significantly lower than the other vaccine strains (p<0.001), reaching around 800 ng/ml during weeks 17-21 (ca. 1% of wild-type).
Peak primary serum IgA responses occurred somewhat later (weeks 7-9) than IgG responses (Fig. 3b). This delay in the response is consistent with other cytoplasmic strains we have examined . Peak recall serum IgA responses occurred between weeks 17-21, with the wild-type reaching the highest levels. Differences in peak recall responses were not statistically significant except for the pCRB148 (1.7%), which was significantly lower than the other vaccine strains (p<0.001). For both serum IgG and IgA, there was little difference in response between the intermediate pCRB65 (75.8%) and pCRB1232 (33.2%) expression level mutants.
Primary salivary IgA responses followed kinetics similar to serum IgA (Fig. 3c). Peak responses were similar among the wild-type and two intermediate expression level strains.
The subclass distribution of IgG anti-HagB antibodies was determined during the early primary response (week 3), peak primary (week 5), pre-boost (week 13), and peak recall responses (week 19). Regardless of expression level, IgG2a responses to HagB predominated during the course of the primary and recall response (Fig 4 a-c), which is characteristic of a Th1 type response [13, 14].
The majority of pathogens initially colonize and/or invade via mucosal surfaces. As a consequence, much effort has been directed at designing vaccines to stimulate mucosal immunity [15, 16]. Because of compartmentalization of the immune system, parenteral immunization routes are ineffective at inducing mucosal responses. Stimulating a mucosal response requires delivering the vaccine antigen to a mucosal inductive site such as the Peyer’s patches in the gut via the oral route. Oral mucosal vaccine approaches must preserve the integrity of the antigen during transit through the gut, and deliver the antigen in such a way that enhances its immunogenicity. For purified component vaccines, a variety of methods have been used including physical encapsulation, and conjugation to mucosal adjuvant molecules . Live, attenuated whole-cell vaccines, such as the oral cholera vaccines have been shown to be capable of inducing effective responses .
For recombinant antigens, live, attenuated viral or microbial vectors have been used for delivery. Salmonella has the advantage of having a tropism for the mucosal inductive tissues of the Peyer’s patches, allowing in situ expression of antigen while enhancing its immunogenicity. Attenuated Salmonella has been used to express over 50 different foreign antigens of various origins [1-4].
In the present study, expression of HagB at any level had no effect on the growth rate of the vaccine strains. However, the metabolic burden resulting from overexpression of heterologous gene products, or even low levels of expression of certain more toxic genes, can compromise the live vaccine vector [17-19]. Reductions in growth rate, interference with membrane stability or altering expression of important vector genes may reduce its ability to colonize in the gut and persist long enough in mucosal inductive tissues to induce an effective immune response.
We have used attenuating promoter mutations to allow successful expression HagB on the surface of S. enterica serovar Typhimurium using the Lpp-OmpA fusion system, which is toxic at moderate expression levels . Using in vivo-inducible promoters can avoid toxicity during vaccine growth, although the actual expression level in vivo is difficult to predict, and the problems associated with toxicity would resurface following in vivo induction. It may be desirable to use promoter attenuation in combination with in vivo-inducible promoters for particularly toxic vaccine antigens.
How expression levels affect immune response parameters has not been well defined. In the present study, we examined the effect of different constitutive expression levels on immune responses using a model antigen that we have previously shown to be immunogenic and nontoxic at high expression levels [5, 11, 12, 20]. The primary IgG response in serum to the wild type strain peaked 3-4 weeks earlier than the intermediate expression level strains (Fig 3a), suggesting that high expression levels may favor an earlier response. While there was a trend for anti-HagB recall responses to correlate with higher expression level, the peak levels were not significantly different even for expression levels as low as 33% of wild type. A similar trend in terms of response level was seen with serum IgA, however the peak primary response occurred later than the peak IgG response regardless of expression level. Likewise, primary mucosal IgA responses in saliva follow a similar pattern to serum IgA reponses and peak later than the IgG response.
IgG subclass distribution is influenced by several factors including the cytokine environment during presentation, type of antigen presenting cell and costimulatory signals, as well as the nature and dose of antigen . While antigen dose can affect T helper cell polarization, there is controversy over whether high or low doses of antigen favor Th1 or Th2 responses. Over the range of expression levels we studied, the predominant subclass of anti-HagB was IgG2a which is characteristic of a Th1 response [13, 14]. The predominance of IgG2a antibodies agrees with our previous studies with HagB . Most recombinant antigens expressed in oral, avirulent Salmonella vaccines induce a predominately Th1 response, as does the vector itself [12, 22, 23]. An exception is cholera toxin B subunit, which induces a Th2 response [24, 25]. Some temporal changes in Th bias have been seen in during immune responses to oral Salmonella vaccines [26, 27].
Normally, mucosal IgA production is strongly Th2-dependent. In particular IL-5, IL-6, and IL-10 are major cytokines that enhance IgA responses. While IL-4 is necessary for mucosal IgA responses to soluble proteins, it is not necessary for mucosal IgA responses to passenger antigens delivered by live Salmonella [23, 28]. In the presence of a strong Th1 response induced by live, recombinant Salmonella vaccines, IL-6, IL-10 from alternative sources can compensate for the lack of the Th2 cytokines IL-4 and IL-5 [15, 23, 28].
The present study suggests that there is a wide latitude in expression level, and that the highest levels of expression are not necessarily required for an elevated immune response to heterologous recombinant antigens expressed in live attenuated Salmonella vectors. Furthermore, expression level does not significantly affect isotype distribution and IgG subclass profile, an indication that the T helper cell profile is unaffected. With toxic heterologous recombinant antigens, promoter attenuation can be of value in increasing the in vivo fitness of the vaccine vector. Even with in vivo-inducible promoters, limiting maximum expression level by introducing promoter mutations could be of additional benefit with potentially toxic antigens.
We thank Roy Curtiss III (Arizona State University) for providing vaccine strains and expression vectors. We also thank Kimi Bloom and Sheila R. Gillespie for their excellent technical assistance. This work was supported by Grants DE-10963 and DE-07200.
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