Enhanced immunogenicity of a functional enzyme by T cell epitope modification
© Mucha et al; licensee BioMed Central Ltd. 2002
Received: 21 November 2001
Accepted: 25 January 2002
Published: 25 January 2002
T helper epitopes are necessary for the induction of high titers of antigen-specific IgG antibodies. We are interested in the epitope modification of intact proteins as a method to enhance their immunogenicity for the generation of recombinant protein-based vaccines.
Hartley strain guinea pig T cell epitopes were mapped for two related bacterial proteases. Two T cell epitopes were found in one of the proteases, while a comparatively reduced immunogenicity protease had no detectable T cell epitopes. A T cell epitope sequence homologous to the immunogenic protease was created in the less immunogenic protease by changing a single amino acid. Proliferative responses to the whole protein parent enzyme were two-fold higher in splenocyte cultures from variant-immunized animals. We found that the single amino acid change in the variant resulted in a protein immunogen that induced higher titers of antigen-specific IgG antibody at low doses and at early time points during the immunization protocol. The serum from parent- and variant-immunized guinea pigs cross-reacted at both the protein and the peptide level. Finally, animals primed to the variant but boosted with the parent enzyme had higher levels of antigen-specific IgG than animals immunized with the parent enzyme alone.
With a single amino acid change we have introduced a T cell epitope into a comparatively low-immunogenic enzyme and have increased its immunogenicity while retaining the enzyme's original proteolytic function. The ability to immunomodulate proteins while leaving their function intact has important implication for the development of recombinant vaccines and protein-based therapeutics.
High affinity humoral immune responses to most protein antigens require cognate interactions between antigen-specific T and B cells. Antigen-specific T cells encounter antigen presented by dendritic cells that migrate to the paracortical regions of draining lymph nodes after initial antigen contact . Only dendritic cells have the capacity to induce activation in resting peripheral T cells [2, 3]. Once activated, differentiated T helper cells contact antigen-specific B cells and provide signals for B cell differentiation via CD154-CD40 interactions, as well as by the production of cytokines [4–6]. Consistent with this general understanding of the induction of antibody responses to protein immunogens, good antibody responses to synthetic peptide-epitope constructs have been shown to depend on the presence, orientation, and multiplicity of the T cell epitope in the construct [7–11]. As a confirmation of this finding with synthetic epitope constructs, most protein immunogens studied to date contain multiple T cell epitopes, for example [12, 13].
T helper epitopes have become common components of synthetic vaccine constructs due to their ability to provide immunological help for both humoral and cell-mediated responses [11, 14–16]. T cell epitopes that can induce helper activity by binding to a wide range of HLA-DR alleles have been developed by a number of investigators using various antigen systems [17–19]. T cell epitopes that have the property of binding across a wide range of HLA-DR haplotypes are necessary for the construction of synthetic vaccines that would be useful in the human population as a whole. DNA- and peptide-based vaccines have become popular due to the comparative ease of construction, the circumvention of potential safety issues around attenuated organisms, and for their potentially enhanced immunogenicity compared to heat-killed and subunit vaccines [20–22]. DNA vaccines have the added attraction of efficiently priming both humoral and cytotoxic cell responses, a property largely lacking in subunit and attenuated organism vaccines. Priming of cytotoxic cell responses is necessary for the design of therapeutic cancer vaccines, as well as for viral vaccines. Multiple antigen peptide vaccines can also be designed to contain T and B epitope regions from numerous protein antigens derived from a complex infectious agent which could therefore confer broad protection without the potential dangers of an attenuated whole organism vaccine . However, it is of note that occasionally a well-designed peptide-based vaccine can elicit strong antibody responses to the synthetic immunogen that do not confer protection from the parasite to which they were originally designed . This may be due to either the induction of an inappropriate antibody isotype in the mouse strain used, or to an inadequate overall response.
While subunit vaccines have their inherent shortcomings, they carry the added benefit of potentially inducing complex antibody responses to multiple sites on the whole protein immunogen. Subunit vaccines are of interest in the prevention of parasite infection  and for a variety of infectious diseases . An ideal subunit vaccine for the induction of protective antibody responses would be a protein from the disease-causing organism that was minimally modified from its original structure to retain a variety of potential B cell epitopes. In addition, the modified subunit vaccine would be designed to be highly immunogenic by the addition of promiscuous T cell epitopes. To this end, we have characterized the immune response of partially outbred Hartley strain guinea pigs to two related bacterial proteases. The two bacterial enzymes studied represent important industrial enzymes used in a number of applications, including serving as an additive for laundry products, and animal feed processing. These enzymes have been well characterized as to their activity and specificity by us and by others [26, 27]. While these two proteins are approximately 60% homologous at the amino acid sequence , one of the proteases was significantly less immunogenic than the other. Reduced immunogenicity was characterized as significantly lower titers of antigen-specific IgG when animals were immunized with the same amount of the enzymes. We determined the T cell epitopes in both of the proteases using 15-mer peptides and splenocytes from immunized animals. In order to be classified as an epitope, the average proliferative response to a peptide had to reach a stimulation index of 3.0. Two regions met our criteria as epitopes in the immunogenic protease. We tested a total of 20 guinea pigs and found no epitope regions in the less immunogenic protease. A sequence comparison of the enzymes in the T cell epitope regions revealed that a single amino acid change could confer a T cell epitope onto the less-immunogenic protease. This variant was constructed, and was found to be a functional and stable protease. Immunization of Hartley strain guinea pigs showed that this single amino acid change created a T cell epitope where there was no detectable epitope before, and the presence of this epitope correlates with an improved immunogenicity in the variant. Finally, we show that the variant induces broadly cross-reactive IgG antibodies, and that priming of animals with the variant protein results in higher titers of antigen-specific antibody upon subsequent immunization with the parent enzyme.
IgG antibody titers from B. lentus subtilisin and BPN' Y217L guinea pig immunizations
T cell epitope mapping of B. lentus subtilisin and BPN' Y217L
Enzyme activity and stability
T-cell epitope mapping BPN' Y217L versus GP002
Antibody titers with BPN' Y217L and GP002
Linear B Cell Epitopes BPN' Y217L v. GP002
BPN' Y217L and GP002 Priming Immunizations
It was of interest to determine whether increased titers of IgG antibodies to BPN' Y217L could be induced by priming animals with the T cell epitope-containing variant. Four sets of four guinea pigs were immunized with 5 μg of enzyme over a six-week period in series. The animals were immunized three times, once every two weeks. This protocol was chosen to correspond with the titer data presented in Figure 6. Group A was immunized three times with BPN' Y217L, Group B was immunized first with the GP002, and then twice with BPN'Y217L. Group C was immunized the first two times with the GP002, and the third time with BPN' Y217L.
We have shown that a single conservative amino acid change in a bacterial subtilisin enzyme can create a strong T cell epitope in our guinea pig model. Immunization of guinea pigs with this modified variant results in an improved cross-reactive immune response to the unmodified parent enzyme in both antigen-specific IgG antibody production and T cell proliferative responses. The improved immunogenicity of the variant was not related to changes in the proteolytic capacity of the enzyme, because the variant exhibited 80% of the activity of the parent enzyme. The introduction of a single T cell epitope apparently allowed for the diversification of T cell responses to the protein because multiple epitopes were found in the BPN' Y217L sequence after immunization of animals with the variant (Figure 5). An increase in the complexity of the T cell response is consistent with an increase in overall antibody titer to the enzyme, as more T cell helper activity is potentially available. The antibody responses to the variant and parent enzymes were shown to be largely overlapping, indicating that the increase in the antibody titers seen were not due to the addition of a novel structure capable of being recognized by unique antibody specificities. This is shown by the last experiment where priming with the variant enzyme increased antibody titers to the parent enzyme upon subsequent immunizations.
It was of interest that the unmodified protease induces antigen-specific IgG in the absence of detectable immunodominant T cell epitopes. BPN' Y217L may possess weak T cell epitopes that are below our limit of detection in our cell culture assay. Our criterion for an epitope was an average stimulation index of 3.0. In addition, it is notable that both of the immunogens tested display considerable proteolytic activity. The proteolytic activity of the parent enzyme could be responsible for inducing T cell independent B cell differentiation via a number of different mechanisms [33–35]. Finally, we have seen than both chemically inactivated and an inactive variant of the parent protease used in these experiments are incapable of inducing IgG1 in a mouse model (data not shown). The proteolytic activity of these molecules also acts as an adjuvant for specific IgG production .
The ability to induce improved immune responses by the subtle changes described here could have a number of potentially vital applications. The use of a T cell epitope-modified whole protein immunogen confers a number of advantages. The first advantage is the priming of a diverse immune response to the protein that allows the generation of both a complex and robust T helper responses, and secondly the concomitant affinity maturation of the antibody response to naturally occurring antibody epitopes. This is especially relevant to the design of subunit vaccines for malaria and schistosomiasis where high titers of high affinity antibodies are usually the best defence against invading organisms [11, 16, 24]. Creating T cell epitopes in expressed proteins has been demonstrated here for Hartley strain guinea pigs, and for various inbred strains of mice [37, 38]. Interestingly, the introduction of T cell epitopes from a hen-egg lysozyme or ovalbumin into mouse TNF-alpha also resulted in an increased cross-reactive antibody response in mice . However, the protein construct was no longer cytotoxic for L929 cells, suggesting that the modified protein was not in the correct tertiary configuration. Incorrect tertiary configuration could lead to the production of specific antibodies that do not cross-react with the native molecule. T cell responses to the variants were not well characterized. In this paper, and in the report from Tsujihata  T cell epitopes were created within the molecule of interest, without affecting the activity of the enzyme. We created a naturally occurring T cell epitope by comparison to a related protein. In addition, we found that the creation of a T cell epitope in BPN' Y217L resulted in a complex pattern of T cell response to the enzyme. The challenge of utilizing this technique for the creation of subunit vaccines for the human population will be to create T cell epitopes within a protein of interest that will not have deleterious effects on the activity or the stability of the protein of interest, and that would be effective across a number of HLA-DR haplotypes .
The improved antibody response measured in this paper was increased titers of guinea pig polyclonal IgG. We did not measure potential differences in guinea pig IgG isotypes. Changing the interaction of peptide, MHC class II molecules, T cell receptors and other antigen-presenting cell specific ligand interactions to foster stronger, higher affinity T cell signalling has been shown in mice to skew T cell differentiation from Th2 to Th1-like patterns [40–50]. Differences in Th-differentiation patterns have downstream effects on the type of immune response mounted, and in particular on the isotypes of antibody expressed. This has been well documented for mice and humans, but less is known about Th differentiation in Hartley strain guinea pigs.
In our system, we cannot distinguish if the amino acid change we have created has its effects on binding to the guinea pig MHC, interaction with the T cell receptor, or more efficient processing of the epitope from the protein precursor . Any or all of these mechanisms could contribute to the enrichment for peptide-specific proliferative responses seen in vitro.
The ability to up regulate antibody and T cell responses to whole protein immunogens by the modification of T cell epitopes suggests that the converse would also be possible. Down regulation of T cell responses to functionally intact enzymes would have obvious implications for both existing and potential protein therapeutics.
The immunogenicity of an enzyme can be increased, while maintaining the majority of its stability and proteolytic activity, by incorporating a single amino acid change. The variant enzyme was found to have a major T cell epitope where none existed in the parent. In addition to the increased cellular response, humoral responses were increased at low doses, and as well for high doses early in the immunization schedule. Enhancing the T cell response to an enzyme, in addition to the humoral response, has applications in the construction of recombinant vaccines as well as in protein-based therapeutics.
Materials and Methods
GP002 was created by site-directed mutagenesis of the sequence of BPN' Y217L and expressed in B. subtilisn. Enzymes were purified from fermentation broths utilizing the BioCAD 700E system (Perceptive Biosystems, Foster City, CA) using POROS HS/M resin (Perceptive Biosystems, Foster City, CA) on porous polystyrene beads, and was stored as stock solutions at 20 mg/ml in 20 mM MES, pH 5.5 with 1 mM CaCL2 at -20°C.
Enzyme activity and stability measurements
Proteolytic activity was measured by hydrolysis of the substrate succinyl-AAPF-p-nitroanilide (suc-AAPF-pNA). Serial dilutions of the proteases were made in 100 mM Tris buffer, pH 8.6, containing 10 mM CaCl2. Enzyme activity was measured at 25°C in 100 mM Tris/HCl, pH 8.6 containing 1.6 mM suc-AAPF-pNA (Bachem Bioscience, Torrance, CA), 0.0005% Tween-80 and 1% DMSO by monitoring the absorbance change at 410 nm using a spectrophotometer. Each dilution of enzyme was tested in triplicate. Three dilutions were tested for each enzyme. Activity was then determined by linear regression, and is reported as OD units/mg enzyme.
Comparative thermostability was determined for BPN' Y217L and GP002 by incubating the enzymes (0.5 ppm) at 37°C in 50 mM TES buffer, pH 7.3 containing 0.0005% Tween-80. Aliquots of the enzyme solutions were removed over time, diluted and assayed for activity using 1 mM suc-AAPF-pNA substrate in 100 mM Tris pH 8.6 containing 0.0005% Tween-80. The half-lives of the enzymes were then calculated using a linear regression.
1. BPN' Y217L, B. lentus subtilisin, and GP002 immunizations
Guinea pig immunizations were outsourced to EL Labs in Santa Cruz, CA. Female Hartley outbred guinea pigs weighing 200–600 grams were used in all studies. There were three or four dosages of the appropriate enzyme and for each dosage there were four animals immunized. Each group of four animals was immunized every two weeks, immediately after the removal of a serum sample. There were a total of six immunizations. The first immunization was in Complete Freund's Adjuvant. Incomplete Freund's Adjuvant was used for all subsequent immunizations. Each immunogen was provided as a stock solution at 20 mg/ml in 20 mM sodium acetate, 1 mM calcium chloride, and was diluted to 1,5,10, or 20 μg/ml in cold DPBS to a total volume of 1 ml. This was emulsified at a 1:1 ratio with CFA or IFA. Each guinea pig was immunized subcutaneously with a total of 500 μl of emulsion.
2. Priming study with BPN' Y217L and GP002
Guinea pig immunizations were out-sourced to EL Labs in Santa Cruz, CA. The following immunization protocol was followed: four groups of four Hartley guinea pigs were distinguished as groups A through D. All animals received 5 μg of immunogen. The first immunization was performed in CFA, then IFA for all subsequent immunizations. Each animal received a total of 500 μl of emulsion. All immunizations were performed subcutaneously using the following schedule: First immunization – Group A received BPN' Y217L, groups B-D GP002. Week two – Groups A and B received BPN' Y217L, Groups B and C received GP002. Week 4 – Groups A through C received BPN' Y217L, Group D received GP002. Serum samples were drawn at week 6.
3. T-cell epitope mapping for GP002 and BPN' Y217L
Guinea pig immunizations were outsourced to Strategic BioSolutions, Ramona, CA. Groups of 10 female Hartley strain guinea pigs were immunized every two weeks with 5 ug per dose of BPN' Y217L or GP002 in Complete Freund's adjuvant, then Incomplete Freund's as described above. Two weeks following the third immunization, animals were sacrificed and their spleens and serum samples collected.
ELISA protocols (whole protein IgG)
Medium binding EIA plates (Costar, Cambridge, MA) were coated with 10 μg/ml of acid-denatured enzyme in DPBS. Enzyme was coated onto the plates at 4°C overnight. Plates were then washed three times with PBS/0.25% Tween-20 (Sigma, St. Louis, MO). Plates were blocked with 1% BSA (Rockland, Gilbertsville, PA)/PBS at room temperature for 30 minutes. Ten-fold serial dilution of sera in 1% BSA/PBS were performed. The plates were covered and incubated at room temperature for two hours. Secondary biotin anti-IgG antibody (guinea pig IgG Fc specific, Jackson ImmunoResearch, West Grove, PA) was diluted 1:10,000 in 1%BSA/PBS. 100 μl of this dilution were placed per well on the plates. The plates were again covered and incubated at room temperature for 1 hour. Plates were washed (as above). Avidin-HRP (Jackson ImmunoResearch) was diluted 1:1000 in 1%BSA/PBS and 100 μl per well were added to the plates. Plates were covered and incubated at room temperature for 30 minutes. The plates were washed (as above). The ELISAs were developed with ABTS in citrate buffer with 0.03% H2O2, at room temperature for 40 minutes and then read at OD = 405 nm. Titers were found by using a log-based slope equation where the O.D. = 0.5.
1. Proliferation assays: 15-mer peptides offset by 3 amino acids describing the proteins of interest were obtained as a Pep Set from Mimotopes (San Diego, CA). Pep Sets were constructed for Bacillus lentus subtilisin (B. lentus subtilisin) and Bacillus amyloliqufaciens subtilisin (subtilisin BPN' Y217L). Peptides were resuspended in DMSO at 1 mM.
2. Linear epitopes: Biotinylated 15-mer peptides from obtained from Mimotopes (San Diego, CA). The 15-mer peptides were offset by 5 amino acids, and described the sequence of BPN' Y217L. The biotin moiety at the N-terminus was separated from the peptide sequence of interest by a 4 amino acid spacer. Biotinylated peptides were resuspended at 1 mM in DMSO.
T-cell epitope mapping of enzymes B. lentus subtilisin, BPN' Y217L and GP002
T cell epitope mapping was performed using splenocytes from immunized Hartley strain guinea pigs. Splenocytes from immunized guinea pigs were resuspended in AIM-V (Life Technologies Gaithersberg, MD) containing 100 units/ml penicillin, 100 ug/ml streptomycin, 1 mM glutamine, and either 10% autologous serum or 10% FCS (Sigma, St. Louis, MO). Using round-bottom 96 well plates (Costar, Cambridge, MA), either 5 × 105 or 1 × 106 cells/well in 200 uL of media. 5 uM of peptide were placed in each well. Each peptide was tested minimally in duplicate. DMSO was present at 0.5%. Control cultures contained 0.5% DMSO in the absence of peptide. The cultures were incubated at 37°C for 5 days. On day 5, cells were pulsed with tritiated thymidine at a concentration of 0.5 μCi/well. On day 6, cells were harvested and counted. A proliferative response was counted as positive if the average counts for a particular peptide were three times higher than the control levels.
Linear B-cell epitope mapping of enzymes BPN' Y217L and GP002
Biotinylated peptides were resuspended in 1 ml of DMSO (1 mM). A stock plate was made by diluting 10 μl of each peptide into 200 μl of PBS/ 0.025% Tween-20 (Sigma, St. Louis, MO). Streptavidin-coated plates (Pierce, Rockford, IL) were blocked with 1%BSA/PBS for 30 minutes at room temperature. Approximately 5 μM of biotinylated peptide was coated onto each well. Each peptide was tested minimally in duplicate. Plates were incubated for one hour at room temperature and then washed three times. Guinea pig serum was diluted 1:1000 in PBS/Tween-20. 100 μl of dilute sera was placed into each well and plates were incubated at room temperature for one hour and then washed three times. The secondary antibody, anti-guinea pig IgG-HRPO) (Jackson ImmunoResearch, West Grove, PA) was diluted 1:1000 in 1%BSA/PBS and 100 μl of dilute conjugate was added to each well. Plates were incubated at room temperature for 1 hour and then washed three times in PBS/Tween-20. Plates were then washed twice in PBS only. ELISAs were developed using ABTS in citrate buffer, 0.03% H2O2. Plates were then incubated at room temperature for up to 45 minutes and then read at 405 nm. OD readings for each peptide were compared to background readings with no peptide. Any peptide giving readings at least twice the background was considered a positive.
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