Induction of multiepitopic and long-lasting immune responses against tumour antigens by immunization with peptides, DNA and recombinant adenoviruses expressing minigenes.

The development of immunization strategies to induce strong and multiepitopic T-cell responses against tumour antigens is needed for anti-tumour immunotherapy. However, a common finding after immunization with complex antigens is the preferential induction of immune responses against immunodominant epitopes. In this study, with the aim of inducing multiepitopic responses against several common tumour antigens, we have designed a minigene construct encoding four human leucocyte antigen (HLA)-A2-restricted epitopes belonging to tumour antigens CEA (CEA-691 and CEA-571), MAGE2 (MAGE2-157) and MAGE3 (MAGE3-112), as well as the universal PADRE epitope recognized by T helper lymphocytes. To optimize the activation of immune responses against these epitopes, we have used different antigen formats (short peptides encompassing individual epitopes and DNA plasmids or adenoviral constructs expressing the minigene) in single or combined immunization schedules. A single immunization with either DNA plasmid or recombinant adenovirus induced a monospecific immune response against the immunodominant epitope CEA-571, whereas immunization with the peptide pool induced responses against all epitopes. Combination of peptide priming followed by a boost with the plasmid and the recombinant adenovirus expressing the minigene induced stronger, multi-specific and long-lasting immune responses, overcoming the immunodominance imposed by the main T-cell epitope. Moreover, these combined immunization strategies were able to induce responses that were able to recognize Mel624 HLA-A2+ tumour cells expressing MAGE2. These results suggest that heterologous immunization strategies combining peptides and DNA or recombinant adenoviruses can be useful to broaden the specificity and enhance the efficacy of subunit vaccines.

Monocyte-derived dendritic cells from HCV-infected patients transduced with an adenovirus expressing NS3 are functional when stimulated with the TLR3 ligand poly(I:C).

Dendritic cells (DC) transfected with an adenovirus encoding hepatitis C virus (HCV) NS3 protein (AdNS3) induce potent antiviral immune responses when used to immunize mice. However, in HCV infected patients, controversial results have been reported regarding the functional properties of monocyte-derived DC (MoDC), a cell population commonly used in DC vaccination protocols. Thus, with the aim of future vaccination studies we decided to characterize MoDC from HCV patients transfected with AdNS3 and stimulated with the TLR3 ligand poly(I:C). Phenotypic and functional properties of these cells were compared with those from MoDC obtained from uninfected individuals. PCR analysis showed that HCV RNA was negative in MoDC from patients after the culture period. Also, phenotypic analysis of these cells showed lower expression of CD80, CD86, and CD40, but similar expression of HLA-DR molecules as compared to MoDC from uninfected individuals. Functional assays of MoDC obtained from patients and controls showed a similar ability to activate allogeneic lymphocytes or to produce IL-12 and IL-10, although lower IFN-alpha levels were produced by cells from HCV patients after poly(I:C) stimulation. Moreover, both groups of MoDC induced similar profiles of IFN-gamma and IL-5 after stimulation of allogeneic T-cells. Finally, migration assays did not reveal any difference in their ability to respond to CCL21 chemokine. In conclusion, MoDC from HCV patients are functional after transduction with AdNS3 and stimulation with poly(I:C). These findings suggest that these cells may be useful for therapeutic vaccination in chronic HCV infection.

Immunomodulation induced by synthetic peptides derived from Staphylococcus aureus protein A.

Peptides from 10 to 22 amino acids containing sequences encompassed by Staphylococcus aureus protein A were synthesized. Some of these peptides, when present in cultures of lymphomononuclear cells from healthy donors or from cancer patients (melanoma, breast carcinoma, non-Hodgkin lymphoma and renal cell carcinoma) promoted: (i) changes in the phenotype of the lymphomononuclear population, (ii) stimulation of monocytes (release of IL-1 and TNF-alpha), and (iii) an increase in cytotoxicity against K562, Daudi and HT-29 cells. Isolated monocytes responded also to those peptides with a release of IL-1 and TNF alpha and an increase of cytotoxicity against HT-29 cells. It was found that the active peptides had the following structural pattern: a length of at least 15 amino-acid residues with a proline at position 6, valine, leucine, isoleucine, glycine, alanine or lysine at position 2, and glutamic or aspartic acid at position 11. Replacement of Pro at position 6 with any other residue turned the peptide inactive. Replacement of residues at positions 2 and 11 with amino-acid residues other than those required for activity resulted in compounds with a marked decrease in the immunomodulating properties described, or lacking these properties altogether.

Carcinoembryonic antigen as a target to induce anti-tumor immune responses.

Identification of relevant targets for cancer therapy is a major goal in cancer research. In this field, the identification of tumor antigens has opened the possibility of inducing specific anti-tumor immune responses. Among these antigens, carcinoembryonic antigen (CEA) is especially relevant because CEA is expressed in a wide variety of adenocarcinomas such as colon, rectum, pancreas, gastric, breast, etc. The present review focuses on different strategies to induce anti-CEA immune responses. In a first group of strategies, the antigen is administered using viral and bacterial vectors expressing CEA, dendritic cells loaded with CEA protein, or dendritic cells transfected with DNA or RNA expressing CEA. A second group of strategies is based on immunizations with antigenic peptide determinants from CEA, rather than with immunogens containing the whole protein. This has been possible due to the identification of different peptide determinants from CEA, which when presented by MHC class I molecules, are recognized by T cytotoxic lymphocytes. More recently, due to the importance of CD4(+) T cells in the induction of immune responses, T helper peptides presented by MHC class II molecules have also been identified. To overcome the poor immunogenicity of CEA-derived peptide determinants, a common feature of self-antigens, their sequence has been modified to improve binding to MHC molecules or recognition by T cell receptors. Finally, in order to enhance immunization efficacy, some of these strategies have combined the administration of immunogens and cytokines or co-stimulatory molecules. Some of the immunization protocols developed are being tested in clinical trials with promising results. Thus, CEA may prove to be a valuable target antigen for the therapy of a high number of malignancies.

Adenoviral gene transfer of interleukin 12 into tumors synergizes with adoptive T cell therapy both at the induction and effector level.

Tumors infected with a recombinant defective adenovirus expressing interleukin 12 (IL-12) undergo regression, associated with a cytotoxic T lymphocyte (CTL)-mediated antitumor immune response. In the present study we generated anti-CT26 CTLs by short-term coculture of CT26 cells and lymph node cells obtained from mice harboring subcutaneous CT26 tumors injected with an adenoviral vector expressing IL-12 (AdCMVIL-12), control adenovirus (AdCMVlacZ), or saline. Regression of small intrahepatic CT26 tumors in unrelated syngeneic animals was achieved with CTLs derived from mice whose subcutaneous tumors had been injected with AdCMVIL-12 but not with CTLs from the other two control groups. The necessary and sufficient effector cell population for adoptive transfer consisted of CD8+ T cells that showed anti-CT26 specificity partly directed against the AH1 epitope presented by H-2Ld. Interestingly, treatment of a subcutaneous tumor nodule with AdCMVIL-12, combined with intravenous adoptive T cell therapy with short-term CTL cultures, had a marked synergistic effect against large, concomitant live tumors. Expression of IL-12 in the liver in the vicinity of the hepatic tumor nodules, owing to spillover of the vector into the systemic circulation, appeared to be involved in the increased in vivo antitumor activity of injected CTLs. In addition, adoptive T cell therapy improved the outcome of tumor nodules transduced with suboptimal doses of AdCMVIL-12. Our data provide evidence of a strong synergy between gene transfer of IL-12 and adoptive T cell therapy. This synergy operates both at the induction and effector phases of the CTL response, thus providing a rationale for combined therapeutic strategies for human malignancies.

CD4-modified synthetic peptides containing phenylalanine inhibit HIV-1 infection in vitro.

Phenylalanine-containing peptides from CD4 were synthesized based on chemical similarity with active CD4(81-92)-benzylated peptides. The synthetic peptide FYIFFVEDQKEEDD blocked the binding of gp120 to CD4 and inhibited 50% human immunodeficiency virus (HIV)-induced syncytia formation at a concentration (IC50) of approximately 40-50 microM and HIV p17 expression with an IC50 of approximately 67 microM. The peptide is not toxic to cells in vitro. Moreover, acute toxicity studies carried out in Swiss mice showed the peptide to be nontoxic at a dose of 2,000 mg/kg. This phenylalanine-substituted CD4 peptide may prove to be useful in the treatment of AIDS.

Induction of neutralizing antibodies against human immunodeficiency virus type 1 using synthetic peptide constructs containing an immunodominant T-helper cell determinant from vpr.

Identification of immunodominant T-helper-cell determinants after natural infection is an important step in the design of immunogens for potential use in vaccination. Using cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals and a panel of peptides encompassing the sequence of the regulatory protein vpr from HIV-1, we identified the T-helper determinant QLLFIHFRIGCRHSR, which is active in 37.5% of these individuals. To gain insight on the efficacy of this peptide in helping induce neutralizing antibodies against a B-cell determinant (BD), we synthesized constructs containing B- and T-cell determinants and tested them in BALB/c mice, the highest responders to the T-cell determinant moiety among several strains tested. These immunogens induced antibodies against two chosen B-cell determinants from HIV-1IIIB gp160 (amino acids 310-322 from the V3 loop of gp120 and 736-751 from gp41) that were able to neutralize HIV-1 infection in vitro. The highest neutralization titer against HIV-1IIIB was obtained by immunization with the homopolymer of the construct containing the T-cell epitope from vpr and the B-cell epitope from the V3 loop. We believe that the immunodominant T-cell determinant from vpr is a promising epitope to consider in the design of future peptide vaccines.

Molecular basis for degenerate T-cell recognition of one peptide in the context of several DR molecules.

We report the study of one CD4+ T-cell clone that recognizes peptide HA306-320 in the context of autologous DR1101 molecules as well as of allogeneic DR1301, DR0402, DR1501, and DR1601 molecules. This degenerate T-cell recognition is mediated by a single T-cell receptor (TCR) as judged by both TCR-V beta sequencing and cold-target competition assays. Restriction analysis shows that substitutions of DR residues within the third hypervariable region result in a loss of T-cell reactivity, which is restored by additional substitutions in the first and/or second hypervariable regions. Thus, there is no correlation between antigen presentation abilities of the different allelic DR products and the degree of sequence homology between these products. DR residues whose substitution is compatible with T-cell recognition potentially interact with peptides rather than with TCRs by virtue of their location in the floor of the groove or as previously documented for residues of the alpha-helix. Furthermore, antigen presentation by allogeneic DR molecules occurs independently of their affinity for the peptide, as determined in cell surface-binding assays using biotinylated HA306-320. Altogether these data suggest that degenerate T-cell recognition mainly depends on an influence of polymorphic DR residues on the configuration adopted by the peptide in the DR groove so that the epitope is left intact.

Binding of ALA-substituted analogs of HA306-320 to DR1101, DR1301, and DR0402 molecules: correlation of DR-peptide interactions with recognition by a single TCR.

We tested the hypothesis that a cross-reactive T-cell clone could recognize HA306-320 peptide complexed to autologous HLA-DR1101, and also to allogenic HLA-DR0402 and HLA-DR1301 molecules, because of similar orientations of HA306-320 side chains in the groove of the three DR molecules. To approach peptide orientations in each HLA groove we compared the capacity of Ala-monosubstituted analogs to bind and be presented by DR1101, DR0402, and DR1301. Results indicated that the orientation of HA306-320 in DR1101 was grossly similar to the known orientation of HA307-319 in DR0101. Data suggested many similarities in peptide orientations in DR0402 and DR1301 as well. However, differences in binding were also observed. Ala substitution of Y309 had much less effect on peptide binding to DR1301 and DR0402 than to DR1101 and Ala-substitution of T314 increased affinity for DR1301 but not for DR1101 and DR0402. These alterations of peptide-DR interactions were probably communicated to the upper peptide surface. Indeed, the levels of T-cell clone reactivities against analogs mutated at positions predicted to face the TCR were lower when complexed to allogeneic DR molecules than when complexed to DR1101. Yet these epitopic alterations are likely subtle, since the decreased reactivity of the clone to allogeneic molecules could be compensated by peptide substitution at Y309, predicted to face the MHC.

Specific and general HLA-DR binding motifs: comparison of algorithms.

Using panels of peptides well characterized for their ability to bind to HLA DR1, DRB1*1101, or DRB1*0401 molecules, algorithms were deduced to predict binding to these molecules. These algorithms consist of blocks of 8 amino acids containing an amino acid anchor (Tyr, Phe, Trp, Leu, Ile, or Val) at position i and different amino acid combinations at positions i+2 to i+7 depending on the class II molecule. The sensitivity (% of correctly predicted binder peptides) and specificity (% of correctly predicted non-binder peptides) of these algorithms, were tested against different independent panels of peptides and compared to other algorithms reported in the literature. Similarly, using a panel of 232 peptides able to bind to one or more HLA molecules as well as 43 non-binder peptides, we deduced a general motif for the prediction of binding to HLA-DR molecules. The sensitivity and specificity of this general motif was dependent on the threshold score used for the predictions. For a score of 0.1, the sensitivity and specificity were 84.7% and 69.8%, respectively. This motif was validated against several panels of binder and non-binder peptides reported in the literature, as well as against 35, 15-mer peptides from hepatitis C virus core protein, that were synthesized and tested in a binding assay against a panel of 19 HLA-DR molecules. The sensitivities and specificities against these panels of peptides were similar to those attained against the panels used to deduce the algorithm. These results show that comparison of binder and non-binder peptides, as well as correcting for the relative abundance of amino acids in proteins, is a useful approach to deduce performing algorithms to predict binding to HLA molecules.

Fine analysis of immunoreactivity of V3 peptides: antibodies specific for V3 domain of laboratory HIV type 1 strains recognize multiple V3 sequences synthesized from field HIV type 1 isolates.

Production of cross-reactive antibodies recognizing the V3 loop--that is, the principal neutralizing determinant (PND)--of various HIV-1 isolates is an important challenge in the development of passive immunotherapy or vaccinations against AIDS. We have produced two types of antibodies to the V3 domain of HIV-1: (1) antibodies against the HIV-1 MN laboratory strain generated in rabbits and (2) antibodies targeted to the HIV-1 LAI laboratory strain induced in chimpanzees. These antibodies were shown to be specific for HIV-1 subtype B. The cross-reactivity of these antibodies has been evaluated against a large panel of peptides representing different parts of the V3 loop. Seventy-five peptides, referred to as clinical peptides, were synthesized according to HIV-1 sequences recovered from PMBCs of 27 patients followed in three Parisian hospitals. Thirteen V3 peptides derived from 4 HIV-1 laboratory strains (MN, LAI, SF2, and RF) were also included in the study. The results show that both the amino-terminal and central parts of the V3 loop are immunogenic. The rabbit antibodies against the amino-terminal end of the PND proved to be highly cross-reactive against the clinical peptides. The anti-gp160 antibodies induced in one chimpanzee recognized a significant proportion of the panel of V3 clinical sequences. These antibodies cross-reacted mainly with the apex of the V3 loop. These data give some additional indications on the immunogenicity of the V3 loop and further demonstrate that extensive cross-reactivity of anti-V3 antibodies can be obtained on field HIV-1 isolates despite the high variability of the V3 loop amino acid sequence.

B cell epitopes of HIV type 1 p24 capsid protein: a reassessment.

The objective of the present study was to identify p24 antigenic domains recognized during natural human immunodeficiency virus type 1 (HIV-1) infection, the determination of the major epitopes of p24 having significant applications for both the improvement of diagnostic approaches and the development of vaccines. Reactivity of 20 HIV-1-infected patients and 8 HIV-1-negative patients was analyzed using an enzyme-linked immunosorbent assay (ELISA) developed with 45 overlapping synthetic pentadecapeptides, spanning amino acids 133 to 363 of HIV-1 p55gag precursor. Two peptides covering aa 178-192 and 288-302 of p55 were recognized by 40 and 45% of HIV-1 antibody-positive human samples, respectively. A peptide covering aa 272-322 of p55 was synthesized and recognized by most human sera in indirect ELISA. However, inhibition assays indicated that this sequence does not contain all of the immunodominant domains of p24 since it was not sufficient to block binding of human sera to whole p24. A three-dimensional model of p24 derived from the Mengovirus VP2 suggests that the two distant sequences recognized by human sera containing antibodies to HIV-1 could possibly be a part of a conformational epitope built up by two loops corresponding to aa 183-186 and 289-292.

Further insights on the inhibition of HIV type 1 infection in vitro by CD4-modified synthetic peptides containing phenylalanine.

Phenylalanine-containing peptides from CD4 were synthesized on the basis of chemical similarity with active CD4(81-92)-benzylated peptides. Systematic replacement of amino acids of these peptides bearing the benzyl group by phenylalanine, afforded several peptides that were able to block the binding of gp120 to CD4 and to inhibit HIV-induced syncytium formation. These experiments showed that substitution of residues 81 and 85 by phenylalanine was the most important for activity. Following optimization of the length of phenylalanine-substituted peptides it was found that FYICFVED and FYICFVEDE were the most active. Their IC50 for the inhibition of syncytium formation was around 1.2-1.6 microM. This activity is at least 30 times higher than that of the parent peptide FYIFFVEDQKEEDD previously reported (Lasarte et al., J Acquir Immune Defic Syndr 1994;7:129-134). Binding competition experiments with two different anti-peptide antisera recognizing the V3 region of gp120 and FYICFVEDE, show that the active peptides bind to V3 or to a sterically near region of V3. None of the active peptides was toxic to cells in vitro. The enhanced activity and simplicity of these new phenylalanine-substituted CD4 peptides might be a good starting point for the development of mimotopes of potential use for the treatment of AIDS.

An insight into a combination of ELISA strategies to diagnose small ruminant lentivirus infections.

A single broadly reactive standard ELISA is commonly applied to control small ruminant lentivirus (SRLV) spread, but type specific ELISA strategies are gaining interest in areas with highly prevalent and heterogeneous SRLV infections. Short (15-residue) synthetic peptides (n=60) were designed in this study using deduced amino acid sequence profiles of SRLV circulating in sheep from North Central Spain and SRLV described previously. The corresponding ELISAs and two standard ELISAs were employed to analyze sera from sheep flocks either controlled or infected with different SRLV genotypes. Two outbreaks, showing SRLV-induced arthritis (genotype B2) and encephalitis (genotype A), were represented among the infected flocks. The ELISA results revealed that none of the assays detected all the infected animals in the global population analyzed, the assay performance varying according to the genetic type of the strain circulating in the area and the test antigen. Five of the six highly reactive (57-62%) single peptide ELISAs were further assessed, revealing that the ELISA based on peptide 98M (type A ENV-SU5, consensus from the neurological outbreak) detected positives in the majority of the type-A specific sera tested (Se: 86%; Sp: 98%) and not in the arthritic type B outbreak. ENV-TM ELISAs based on peptides 126M1 (Se: 82%; Sp: 95%) and 126M2 0,65 0.77 (Se: 68%; Sp: 88%) detected preferentially caprine arthritis encephalitis (CAEV, type B) and visna/maedi (VMV, type A) virus infections respectively, which may help to perform a preliminary CAEV vs. VMV-like typing of the flock. The use of particular peptide ELISAs and standard tests individually or combined may be useful in the different areas under study, to determine disease progression, diagnose/type infection and prevent its spread.

Characterization of T-cell responses against immunodominant epitopes from hepatitis C virus E2 and NS4a proteins.

Successful clearance of hepatitis C virus (HCV) infection has been associated with strong cellular immune responses against viral antigens. However, although the magnitude of these responses is clearly important for viral eradication, more studies are needed to unravel the fine specificity of the protective anti-HCV immunity in infected patients. This was the aim of the present study. Overlapping peptides spanning the sequence of HCV E2 and NS4a proteins were used to stimulate T cells from patients with chronic hepatitis C divided into three groups: naïve patients, patients who exhibited sustained response to interferon (IFN)-alpha therapy and patients who failed to respond to the treatment. Interleukin-2 production by stimulated cells was measured in each case. Patients with sustained response to therapy had stronger responses to E2 peptides than nonresponders, whereas naïve patients demonstrated intermediate reactivity. In the case of NS4a, responses against peptides where similar in all groups of patients. Analysis of the peptides recognized by T cells showed that responses were broad and heterogeneous, and some immunodominant epitopes, preferentially recognized by patients exhibiting sustained response to treatment, were found. These results confirm the role of cellular immune responses in viral clearance, and stress the importance of immunodominant regions within HCV antigens. These viral sequences may represent valuable immunogens for preparation of therapeutic or prophylactic vaccines.

The purification of myosin long subfragment 2 by DEAE-Sepharose CL-6B ion-exchange chromatography.

A new method for the purification of myosin long subfragment 2 is presented. This method is based on the fractionation, by DEAE-Sepharose CL-6B ion-exchange chromatography, of either chymotryptic hydrolysates of heavy meromyosin or tryptic hydrolysates of myosin total rod. Although emphasis is given to the purification of long subfragment 2, the method could be easily adapted to the purification of short subfragment 2.

Evidence for periodicity in the amino-acid sequence of myosin.

We have characterized CNBr digests of rabbit myosin heavy chain, total rod, light meromyosin and mouse heavy chain by combined isoelectric focusing and dodecylsulfate gel electrophoresis. Conditions for digest were chosen so as to have less than complete cleavage at most or all of the methionine residues. Examination of the gels in the dodecylsulfate dimension shows a remarkable periodicity in the molecular weights of fragments produced in all these digests. Nearly all lie in a monotonic sequence with an interval of approximately 3800 between them. One interpretation of this finding is that there is a high degree of periodicity in the position of methionines of, at the very least, the light meromyosin region. The impression of periodicity is further re-inforced by a regular distribution of fragments in the isoelectric focusing dimension of the gels as well as in the dodecylsulfate dimension. The heterogeneity in the isoelectric focusing dimension could, unlike that in the dodecylsulfate dimension, be artefactual (e.g. as a consequence of de-amidation and or cyanylation) or as a result of protein heterogeneity due to gene duplication. However, it is also possible that it too reveals a periodicity, this time in the distribution of charged residues in the amino-acid sequence.

Engineering of immunogenic peptides by co-linear synthesis of determinants recognized by B and T cells.

The potential of synthetic peptides as vaccines is restricted by their frequent lack of immunogenicity. As with haptens, coupling to a carrier protein is usually required to provide T cell help to anti-peptide antibody-producing B cells. In spite of their short length, a few natural or synthetic peptides are immunogenic: they all include both a determinant recognized by B cells and a proven or putative determinant recognized by T cells. We speculated that it should be possible to induce immunogenicity in peptide haptens by the inclusion of a well characterized determinant recognized by T cells. We thus synthesized two peptides, corresponding to different regions of the major protein VP6 of bovine rotavirus, co-linearly linked to a peptide of influenza virus hemagglutinin which had been shown to induce T helper cells in BALB/c mice. Both peptides induced anti-rotavirus antibodies and were more immunogenic than the corresponding bovine serum albumin-conjugated peptides.

Enhancement of peptide immunogenicity by linear polymerization.

The effect of linear homopolymerization on the immunogenicity of synthetic peptides was studied using either haptenic peptides (representing amino acid sequences 103-115 and 133-147 of bovine rotavirus major protein) or immunogenic peptides TD-103-115 and TD-133-147 which were constructed by co-linear synthesis of the former peptides and an amino acid sequence representing a determinant recognized by T helper cells (TD). It was found that the two haptenic peptides were rendered immunogenic by linear homopolymerization. Moreover, homopolymerization also enhanced the immunogenicity of TD-103-115 but not that of TD-133-147. In the three cases where polymerization enhanced immunogenicity, a reinforced amphipathic pattern was predicted in the neighborhood of the junction of the monomers. The possibility that polymerization might have generated a new T cell determinant is discussed.

An immunodominant cytotoxic T cell epitope on the VP7 rotavirus protein overlaps the H2 signal peptide.

C57BL/6 (H-2b) mice were primed with the bovine RF strain of rotavirus to study the induction of CD8+ cytotoxic T lymphocytes (CTLs). These rotavirus-specific CTLs were detected only after in vitro restimulation with the virus. Using a recombinant vaccinia virus we identified the RF VP7 protein as a major target of these CTLs. The response against this protein was obtained also after in vitro restimulation with simian SA11 and human WA strains of rotavirus. Using published Db and Kb allele-specific motifs to predict possible CTL epitopes in the RF VP7 protein, we synthesized and tested 18 predicted peptides of VP7. Only one peptide was able to sensitize target cells at a concentration below 5 x 10(-7) M. This CTL epitope was also induced by immunization with the RF VP7 expressed with a baculovirus vector, and was shown to be immunodominant by its capacity to inhibit, in an unlabelled target assay, the bulk response against cells infected with recombinant vaccinia virus expressing VP7. This CTL epitope overlaps the H2 signal peptide of the protein.