Protein adsorption and separation on amphoteric chitosan/carboxymethylcellulose membranes

This article reported the preparation of an amphoteric natural polymeric membrane-macroporous chitosan (CS)/carboxymethylcellulose (CMC) blend membrane and the utilization of such a membrane on the membrane chromatography for bioseparation. The membranes were prepared by solution blending of CS and CMC solution, and using silica particles as porogen. Both glutaraldehyde and epichlorohydrin were used as crosslinking agent to increase its chemical stability in aqueous solution. Such a natural polymeric membrane can be served as an amphoteric membrane because of the amino group on CS and the carboxymethyl group on CMC, in which the surface charge can be changed with the environmental pH. Ovalbumin (pI = 4.6) and lysozyme (pI = 11) were selected as model proteins. These two proteins adsorption on different CS/CMC blend membranes with different initial protein concentrations at different pH values were investigated in batch systems. The results indicated that the maximum adsorption for lysozyme and ovalbumin was at pH 9.2 and 4.8 respectively, and the adsorption capacity on the membrane both increased with the increase of initial protein concentration. Though the adsorption mechanism of lysozyme and ovalbumin was found not the same, the maximum adsorption capacity of two proteins on the membranes was quite similar (about 250 mg/g). Moreover, the desorption ratio of both proteins was found to be more than 90% that implied CS/CMC blend membrane could separate proteins by adsorption-desorption process. Finally, both lysozyme and ovalbumin were successfully separated from their binary mixture only by adjusting the pH of the feed and the desorption solution.     

Drug release from a porous ion-exchange membrane in vitro.

The effect of environmental ionic strength on the rate of drug release from a cation exchange membrane was evaluated. Cationic propranolol-HCl, timolol, sotalol-HCl, atenolol and dexmedetomidine-HCl and neutral diazepam were adsorbed onto a porous poly(vinylidene fluoride) (PVDF) membrane that was grafted with bioadhesive poly(acrylic acid) chains (PAA-PVDF). Despite its porosity, the PAA-PVDF membrane acted as a cation exchange membrane. The release of adsorbed drug from the PAA-PVDF membrane was investigated by using a USP rotating basket apparatus. Adsorption of cationic drugs onto the PAA-PVDF membrane tended to increase with increasing lipophilicity of the drug. A decrease in the ionic strength of the adsorption medium increased the amount of the cationic drugs adsorbed onto the membrane, but had no effect on diazepam adsorption. The release of cationic drugs from the PAA-PVDF membrane was greatly affected by the ionic strength of both the adsorption medium and the dissolution medium, while ionic strengths did not affect diazepam release. Our results suggest that the ionic strength of both the adsorption and dissolution media substantially affects the release rate of a drug that has been adsorbed onto the ion exchange membrane, primarily via electrostatic interactions, while ionic strength has no effect on the release of a drug which has been adsorbed onto the membrane via non-electrostatic forces.     

Blood compatibility of polypropylene surfaces in relation to the crystalline-amorphous microstructure

Blood-contacting properties of polypropylene surfaces with different crystalline states at the surface layer were examined in terms of plasma protein adsorption and changes in cytoplasmic free Ca²⁺ levels in platelets. Though the wettability of polypropylene surfaces was almost constantly independent from the surface layer crystallinity and interlamellar spacing, an increase in adhesiveness was observed with decreasing surface layer crystallinity and interlamellar spacing. It is suggested that the surface properties of the sheets varied in relation to the crystalline-amorphous microstructure. Minimum magnitudes in albumin and fibrinogen adsorption were observed on the polypropylene surface with a particular surface layer crystallinity (c. 55 wt%). A decrease in interlamellar spacing resulted in enhancing albumin adsorption and diminishing fibrinogen adsorption. Transient phenomena in plasma protein adsorption were observed on their surfaces with a plasma concentration. It is considered that the polypropylene surface with a particular crystalline-amorphous microstructure reduces the denaturation of adsorbed proteins. An increase in cytoplasmic free Ca²⁺ levels in platelets was prevented at the polypropylene surface with a surface layer crystallinity of 55 wt%: the particular crystalline-amorphous microstructure of such apolar surfaces as polypropylenes acts to reduce platelet activation. Thus, it is concluded that the blood compatibility of polypropylene surfaces is greatly improved by controlling a crystalline-amorphous microstructure at the surface layer.     

Volumetric interpretation of protein adsorption: competition from mixtures and the Vroman effect

A Vroman-like exchange of different proteins adsorbing from a concentrated mixture to the same hydrophobic adsorbent surface is shown to arise naturally from the selective pressure imposed by a fixed interfacial-concentration capacity (w/v, mg/mL) for which protein molecules compete. A size (molecular weight, MW) discrimination results because fewer large proteins are required to accumulate an interfacial w/v concentration equal to smaller proteins. Hence, the surface region becomes dominated by smaller proteins on a number-or-mole basis through a purely physical process that is essentially unrelated to protein biochemistry. Under certain conditions, this size discrimination can be amplified by the natural variation in protein-adsorption avidity (quantified by partition coefficients P) because smaller proteins (MW<50 kDa) have been found to exhibit characteristically higher P than larger proteins (MW<50 kDa). The standard depletion method is implemented to measure protein-adsorption competition between two different test proteins (i and j) for the same hydrophobic octyl sepharose adsorbent particles. SDS-gel electrophoresis is used as a multiplexing, separation-and-quantification tool for this purpose. Identical results obtained using sequential and simultaneous competition of human immunoglobulin G (IgG, protein j) with human serum albumin (HSA, protein i) demonstrates that HSA was not irreversibly adsorbed to octyl sepharose over a broad range of competing solution concentrations. A clearly observed exchange of HSA for IgG or fibrinogen (Fib) shows that adsorption of different proteins (i competing with j) to the same hydrophobic surface is coupled whereas adsorption among identical proteins (i or j adsorbing from purified solution) is not coupled. Interpretive theory shows that this adsorption coupling is due to competition for the fixed surface capacity. Theory is extended to hypothetical ternary mixtures using a computational experiment that illustrates the profound impact size-discrimination has on adsorption from complex mixtures such as blood.     

Phosphorylcholine-containing polyurethanes for the control of protein adsorption and cell attachment via photoimmobilized laminin oligopeptides

In this study, we synthesized a biomaterial whose surface inhibits non-specific protein and cell attachment. The polymer was designed to mimic the external cell plasma membrane properties through the introduction of particular chemical constituents of the cell membrane: phospholipid polar headgroups. This was done by copolymerizing phosphorylcholine (PC) groups into a polyurethane polymer backbone (PCPUR). Peptides known to induce specific cell attachment were subsequently bound to the surface of this copolymer in a photoadressible manner to obtain surfaces that allowed the attachment of cells in a specific pattern. Two polymers with different phosphorylcholine concentrations were synthesized and their bulk and surface properties were characterized through differential scanning calorimetry, wettability measurements, angle-resolved X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. Protein and lipid adsorption investigation using optical waveguide light mode spectroscopy showed that the irreversible adsorption of both proteins and lipids is drastically reduced as a result of simultaneous contributions of the PC groups, molecular mobility and strong hydrophilicity of the polymers. Consequently, this leads to a marked reduction in the cellular attachment response, which further decreases with increasing PC concentration. Finally, when the polymer surface was photo-derivatized, attachment of the neural NG108-15 cell line occurred only on the areas of the PCPUR where the laminin CDPGYIGSR peptide sequence was photoimmobilized. Cell attachment was nevertheless found to be non-specific with respect to the peptide sequence used and reasons for such results are therefore discussed.     

Bluetongue virus: surface exposure of VP7.

The exposed proteins of bluetongue virus serotype 17 were determined using surface labeling and reactivity with monoclonal antibodies. Iodination of amino groups predominantly labeled VP2; however, iodination of tyrosine residues labeled both VP2 and VP5, with VP7 labeled to a significantly lesser degree. To investigate the exposure of VP7 on the intact virion further, monoclonal antibodies that reacted with this protein were used. At least two antibodies, reacting with different epitopes on VP7, bound to intact virions, as determined by adsorption of infectious particles, electron microscopic observation of antibody-bound virus, and co-sedimentation of antibody and virus. Surface iodination of viral cores was used to show that VP7 and VP3 are major exposed proteins on these particles. We conclude that a major core protein, VP7, has at least two epitopes exposed on the virus surface.     

Blood compatible aspects of DNA-modified polysulfone membrane-protein adsorption and platelet adhesion

DNA was used as a biomaterial to modify the polysulfone (PSf) membrane by blending it with PSf. The blood compatibility of the membranes was then investigated. The water contact angle decreased, and the hydrophilicity increased when a single strand DNA was blended with PSf. Because of the hydrophilic surface, the DNA-blended PSf membranes had a lower protein adsorption than the PSf membrane, but it was not significantly decreased due to the interaction between the DNA and proteins. Circular dichroism (CD) spectroscopy was used to examine the changes in the secondary structure of the proteins after adsorption onto the polymer surface and desorption from the polymer surface into the SDS solution. The conformation of the proteins adsorbed onto the PSf membrane and desorbed from the PSf membrane significantly changed, but that of the proteins for the DNA-blended PSf membranes differed only slightly from the native one. The number of platelets that adhered on the surface of the DNA-blended PSf membranes was reduced compared to that on the PSf membrane. This suggested that DNA can be regarded as a biopolymer to modify PSf, and contributes to the hydrophilic and hemocompatible wipers on the surface of the hydrophobic PSf membranes.     

Adsorption of coagulation proteins and adhesion and activation of platelets at the blood-solid interface. An experimental study of human whole blood

A procedure is presented allowing detailed studies of the adsorption of coagulation factors from whole blood on to surface. Anticoagulant (citrate or hirudin) was added to fresh venous blood. The blood was incubated in hydrophilic or hydrophobic glass tubes without contact with air. The adsorption of fibrinogen, fibronectin and factor IX was measured with an enzyme immunoassay using specific antibodies directed against these proteins. Adsorption of enzymically active kallikrein was measured using a chromogenic peptide substrate. Adhesion and activation of platelets was measured by direct examination in a scanning electron microscope and by measurement of release of beta-thromboglobulin. The results show that the adsorption of plasma proteins at the blood-solid interface is dependent on the anticoagulant used, surface energy of the test surface and incubation time. In experiments using hirudin a specific inactivator of thrombin, as anticoagulant, we found dynamic changes of the adsorbed protein film which could not be studied using citrated blood.     

VP22 herpes simplex virus protein can transduce proteins into stem cells

The type I herpes simplex virus VP22 tegument protein is abundant and well known for its ability to translocate proteins from one cell to the other. In spite of some reports questioning its ability to translocate proteins by attributing the results observed to fixation artifacts or simple attachment to the cell membrane, VP22 has been used to deliver several proteins into different cell types, triggering the expected cell response. However, the question of the ability of VP22 to enter stem cells has not been addressed. We investigated whether VP22 could be used as a tool to be applied in stem cell research and differentiation due to its capacity to internalize other proteins without altering the cell genome. We generated a VP22.eGFP construct to evaluate whether VP22 could be internalized and carry another protein with it into two different types of stem cells, namely adult human dental pulp stem cells and mouse embryonic stem cells. We generated a VP22.eGFP fusion protein and demonstrated that, in fact, it enters stem cells. Therefore, this system may be used as a tool to deliver various proteins into stem cells, allowing stem cell research, differentiation and the generation of induced pluripotent stem cells in the absence of genome alterations.     

Mechanistic aspects of blood-contacting properties of polypropylene surfaces -from the viewpoint of macromolecular entanglement and hydrophobic interaction via water molecules

Polypropylene surfaces with a particular crystalline-amorphous microstructure have been demonstrated to reduce protein adsorption and platelet activation. Such blood-contacting properties may be affected by the crystalline-amorphous microstructure of the surfaces, although wettability such as dynamic contact angles and surface free energy components were almost constant, being independent from the variation in the microstructure. In order to clarify the mechanistic aspects on their blood-contacting properties, the physicochemical properties of the surfaces were evaluated for a series of compression-molded polypropylene sheets in terms of the work of adhesion and the structure of sorbed water. The work of adhesion of the compression-molded sheets increased with decreasing surface layer crystallinity, presumably due to macromolecular entanglement with a polymeric glue used. The work of adhesion involving macromolecular entanglement may occur between proteins and the surfaces. Thus, a decrease in the surface layer crystallinity is considered to cause an increase in the protein adsorption. The structure of water sorbed into the sheets changed - it was more gaseous (isolated) at the surfaces with a higher crystallinity. This suggests that the hydrophobic interaction via water molecules increased with surface layer crystallinity, resulting in increasing protein adsorption and denaturation. Thus, it is considered that both macromolecular entanglement and hydrophobic interaction are important on the mechanistic aspects of blood-contacting properties of polypropylene surfaces. In order to confirm this hypothesis, the evaluation of the physicochemical properties and blood-contacting properties was also performed on a series of uniaxially drawn polypropylene films. A decrease in the work of adhesion and the hydrophobic interaction at the surfaces was observed with increasing draw ratio, and the protein adsorption and platelet activation were effectively prevented with increasing draw ratio. This result supports our hypothesis. Therefore, it is concluded that the excellent blood-contacting properties of polypropylene surfaces can be achieved by reducing the macromolecular entanglement and the hydrophobic interaction with proteins.     

Amount of adsorbed albumin loss by dialysis membranes with protein adsorption

Polymethylmethacrylate (PMMA) membrane is the first synthetic polymeric hollow fiber used in dialyzers that is known to adsorb β₂-microglobulin. Polyester polymer alloy (PEPA), a blend of two polymers, i.e., polyarylate and polyethersulfone, is another dialysis membrane material with adsorption characteristics. In this study, the adsorption and permeation characteristics of BG-1.6PQ (PMMA) and FLX-15GW (PEPA) dialyzers were investigated by performing ultrafiltration experiments using chymotrypsinogen (molecular weight 25 000) and albumin (molecular weight 66 000) as test solutes. Although PMMA and PEPA had the same sieving coefficient for chymotrypsinogen at steady state, PMMA showed approximately 20% higher fractional adsorption than PEPA under the same initial concentrations. The fractional adsorption for albumin was approximately 20% in PEPA regardless of the ultrafiltration flow rate. The fractional adsorption for albumin in PMMA, however, increased as the ultrafiltration flow rate increased and reached 50%–60% after 10 h. Since PEPA has two skins, one inside and one outside the hollow fiber, proteins may have been adsorbed mainly by these two layers. However, since PMMA is a uniform membrane and since the higher the ultrafiltration flow rate, the higher the fractional adsorption found in PMMA, adsorption may be the result of the occlusion of the dense structure of the membrane. The amount of albumin loss is often clinically evaluated by measuring the amount of permeated albumin in the dialysate. However, when dialyzers with adsorption characteristics are examined, the loss by adsorption should also be taken into account.     

The interactions between antithrombin III,thrombin and surface immobilized heparin

The interactions between antithrombin III (ATIII), thrombin, and surface immobilized heparin were investigated. Carboxylated polystyrene modified with covalently immobilized albumin-heparin conjugate contain sites which can bind ATIII from buffer and plasma solutions. Approximately 65% of the ATIII molecules present at the heparinized surface either adsorbed from a buffer or plasma solution, exchanged with ATIII in buffer solution. The exchange between surface bound ATIII and ATIII in solution was repeated several times on the same heparinized surface. The number of binding sites that could bind and release ATIII was much higher when the heparinized surface was exposed to an ATIII containing buffer solution than to a plasma solution. The reduction in binding sites available for ATIII using plasma solutions as compared to buffer solutions could be explained by the competition of other plasma proteins with ATIII for the heparinized surface. It was observed that heparin binding proteins were able to compete with ATIII for binding to the immobilized heparin. Furthermore adsorption of proteins on the heparinized surface significantly reduced the availability of binding sites for ATIII. Exposure of thrombin to the heparinized surface resulted in thrombin activity at the surface. The thrombin activity on the heparinized surfaces was lower on surfaces with a higher ATIII concentration. The activity of surface bound thrombin was not affected by the presence of other plasma proteins. Enzymatically active thrombin molecules present at the heparinized surface were completely inactivated when the surface was exposed to a solution containing ATIII. The inactivation rate of surface bound thrombin by ATIII was higher than the rate of the uncatalyzed inactivation of thrombin in solution. Part of the Thrombin-Antithrombin III (TAT) complexes (10-20%) that were formed upon inactivation of thrombin remained bound to the heparinized surface. In general it was concluded that only the surface immobilized heparin molecules that can bind ATIII in a reversible way determine the anticoagulant properties of the surface. The mechanism of inactivation of a protease on a heparinized surface depends either on the catalytic effect of heparin on the inactivation rate of proteases by ATIII or on an increased uncatalytic inactivation due to increased concentrations of ATIII near the surface as compared to the concentration of ATIII in the bulk phase.     

Microdomain control of block copolymers by epitaxy in mesophases

Poly(vinyl alcohol(VA)-b-4-vinylpyridine(4VP))s were prepared by mechanochemical scission of PVA in the presence of 4VP monomer under ultrasonic irradiation. After establishing the morphology of a particular specimen of these block copolymers, a solution of well-defined poly(styrene(ST)-b-4VP), prepared by anionic living polymerization, was cast on the above mentioned specimen being used as a support membrane. It is shown that the microdomain structure of poly(ST-b-4VP) may be governed by epitaxial growth of the microdomain pattern of the support membrane surface.     

Oxidation of proteins adsorbed on hemodialysis membranes and model materials

The cleaning of cellulosic hemodialysis membrane Cuprophan and model materials (glass; polystyrene and polypropylene, as such and surface-oxidized), conditioned by adsorption of blood plasma proteins (HSA, fibrinogen, IgG) was investigated in vitro. Sodium hypochlorite (NaClO) and Renalin, a product containing hydrogen peroxide and peroxyacetic acid, were used as cleaning reagents. X-ray photoelectron spectroscopy and the use of radiolabeled fibrinogen demonstrated the presence of varying amounts of a polypeptidic residue, with sulfur brought to a high oxidation stage (sulfonate-like). The trends were the same for the three proteins regarding the effectiveness of the oxidizer and the influence of the surface properties. NaClO was much more effective than Renalin to remove the adsorbed proteins. The proteins adsorbed on Cuprophan were more sensitive to the oxidizers, when compared with proteins adsorbed on other materials. This may be due to both the lower protein-surface affinity, as indicated by radiochemical measurements, and the sensitivity of the material itself to the oxidizer, as revealed by weight loss measurements. These results support the attribution of hemocompatibility improvement after regeneration of Cuprophan with Renalin to the masking of the activating surface by a residue from previously adsorbed proteins.     

Antigenic regions of poliovirus type 3/Sabin capsid proteins recognized by human sera in the peptide scanning technique.

We used the peptide scanning technique to identify regions of poliovirus type 3/Sabin capsid proteins that bind antibodies from human immune sera. Several reactive regions were seen in VP1, VP2, and VP3 while peptides resembling VP4 did not bind antibodies. Peptides derived from sequences of the previously known antigenic sites 1 and 3 were recognized to a moderate degree. Peptides imitating the four loops in the closed ends of the beta barrels or the alpha helical CD insertions of VP1, VP2 or VP3, whether exposed in the crystal structure or not, all represented major reactivity in the scans. In VP1 several additional reactive regions were found in the amino terminal quarter of the protein, which is buried in the crystal structure, and in a partially exposed region close to but separated from the carboxy terminus. In VP2 the nonexposed peak activities clustered in a bridge-like structure spanning from the outer to the inner surface of the capsid shell. Likewise, most of the novel antigenic regions of VP3 clustered in an internal location and partially composed of beta sheets with a conserved amino acid sequence. Whether any of the novel antigenic sites is capable of inducing neutralizing antibodies is not known.     

Dual radiolabeling to study protein adsorption competition in relation with hemocompatibility

Human fibrinogen (Fg) and albumin (HSA) were labeled with (3)H and (14)C, respectively. Dual counting allowed the adsorbed amount of the two proteins to be determined simultaneously. Single adsorption, adsorption of the two proteins in competition, but also exchange (substitution by molecules of the same nature) and displacement (desorption under the action of the other protein) experiments were performed on two model surfaces, glass and polystyrene (PS), as well as on pure polyvinylchloride (PVC-s) and on PVC from blood bag (PVC-b). As expected, the adsorbed amount of a single protein is higher on a hydrophobic compared to a hydrophilic surface. When the two proteins are adsorbed in competition, they are found in equal proportion on glass, while HSA is twice more abundant than Fg on PS and PVC-s and about six times more abundant on PVC-b. This trend is related to an increase of the water contact angle of the substrates. For PVC-b, the contact angle is affected by the presence of aliphatic components exposed at the extreme surface, as determined by angle-resolved X-ray photoelectron spectroscopy. In exchange and displacement experiments, the first adsorbed molecules remain dominating on PS while they can be removed from glass. Given the known importance of HSA and Fg adsorption for the fate of materials placed in contact with blood, the method described in this paper may be used as a first approach to orient the design of surfaces with improved hemocompatibility.     

Adsorption of low molecular weight proteins to hemodialysis membranes: experimental results and simulations.

Adsorption of alpha-lactalbumin and Cytochrome C on different hollow-fiber hemodialysis modules, whose main membrane constituent was polyacrylonitrile (PAN), polymethylmethacrylate (PMMA) or polysulfone (PS), were compared under different pH and flow rate conditions. These proteins were chosen as models of small scarce proteins like beta2-microglobulin from which the patient's blood should be epurated. Influence of pH suggests the importance of electrostatic interactions on a charged membrane since adsorption greatly decreases at pH 10.9 for Cytochrome C (pI = 10.6) and at pH 7.4 for alpha-lactalbumin (pI = 5). The difference in adsorbance between different membranes is most probably due to their different microstructures. However, the chemical nature of the support plays a non-negligible role since PMMA membranes have smaller initial adsorption rates than PAN membranes. This is correlated with the density of membrane surface charge showing the importance of electrostatic interactions. The influence of the wall shear rate on adsorption kinetics is analyzed through numerical simulations, in terms of transport limitation in the liquid phase and interfacial reaction.     

Desorption and exchange properties of adsorbed albumin on apatite. Influence of long-term interfacial residence times

A technique using 125I-labeled proteins was employed to study static adsorption properties and slow exchange and desorption processes of human albumin in contact with synthetic hydroxyapatite beads. With the aid of a thermostated "minicolumn," the adsorption isotherm was obtained during a so-called multiadsorption process, and could be described by a Langmuir adsorption model (K = 1.10 x 10(10) cm3.mol-1). All kinetic desorption and exchange experiments could be fitted by a simple exponential function of time. No influence of long-term interfacial residence times on characteristic relaxation times or percentage of desorbed or exchanged proteins could be detected in the present system. On the other hand, as compared to the low surface coverage domain, the small percentage of desorbable and high percentage of exchangeable molecules in the adsorption plateau domain was attributed to a bimolecular exchange process.     

Characteristics of Streptolysin O Action

A study of the lysis of rabbit erythrocytes by streptolysin O (SO) revealed at least two steps in the hemolytic process. The initial interaction between SO and erythrocytes is the adsorption of the toxin molecule to the cell surface. Adsorption occurred at 4 C and was independent of ionic strength and pH; these results suggest that hydrophobic interactions between SO and the cell may be important in this process. Cholesterol was shown to prevent the adsorption of toxin to the cell, and it is proposed that cholesterol in the red cell membrane may be the site of toxin adsorption. The concept of a lipid attachment site is supported by the findings that proteolytic enzymes and sulfhydryl inhibitors known to affect external erythrocyte proteins did not affect SO hemolysis. Although the number of toxin molecules that will adsorb to a cell is limited, more than one toxin molecule was required for hemolysis. The step(s) following adsorption was dependent on temperature, ionic strength, and pH. Thus, it is evident that this step(s) is readily separable from adsorption, suggesting that an ionic interaction occurs between toxin and an erythrocyte membrane molecule. The step(s) following adsorption was also inhibited by divalent cations. Since N-ethyl maleimide will also inhibit lysis after toxin adsorption, it is possible that divalent cations may prevent SO hemolysis by reacting with free sulfhydryl groups on the toxin molecule.     

T cell epitopes in coxsackievirus B4 structural proteins concentrate in regions conserved between enteroviruses

The present study aimed to characterize systematically the target epitopes of T cell responses in CBV4 structural proteins. These were studied by synthesizing 86 overlapping 20-aa-long peptides covering the known sequence of CBV4 structural proteins and analyzing the proliferation responses of 18 CBV4-specific T cell lines against these peptides. Recognized peptides differed depending on the HLA-DR genotype of the T cell donor. They were concentrated to the VP4 and VP2 regions as six of seven common peptide epitopes located in this region, whereas there was only one in the VP3 region and none in the VP1 region. Peptides from conserved areas were recognized more often (on average, 15% of them stimulated each T cell line) than those derived from variable areas (3%) (P < 0.0001, Fisher's exact test). Some conserved peptides inducing T cell responsiveness in most subjects were identified, a knowledge which can be useful in the development of new synthetic vaccines.