Multiple Antigen Peptide Constructs Research Articles

Differential protective impact of peptide vaccine formulae targeting the lung- and liver-stage of challenge Schistosoma mansoni infection in mice

The study aimed to elicit protective immune responses against murine schistosomiasis mansoni at the parasite lung- and liver stage. Two peptides showing amino acid sequence similarity to gut cysteine peptidases, which induce strong memory immune effectors in the liver, were combined with a peptide based on S. mansoni thioredoxin peroxidase (TPX), a prominent lung-stage schistosomula excretory-secretory product, and alum as adjuvant. Only one of the 2 cysteine peptidases-based peptides in a multiple antigenic peptide construct (MAP-3 and MAP-4) appeared to adjuvant protective immune responses induced by the TPX peptide in a MAP form. Production of TPX MAP-specific IgG1 serum antibodies, and increase in lung interleukin-1 (IL-1), uric acid, and reactive oxygen species (ROS) content were associated with significant (P < 0.05) 50 % reduction in recovery of lung-stage larvae. Increase in lung triglycerides and cholesterol levels appeared to provide the surviving worms with nutrients necessary for a stout double lipid bilayer barrier at the parasite-host interface. Surviving worms-released products elicited memory responses to the MAP-3 immunogen, including production of specific IgG1 antibodies and increase in liver IL-33 and ROS. Reduction in challenge worm burden recorded 45 days post infection did not exceed 48 % associated with no differences in parasite egg counts in the host liver and small intestine compared to unimmunized adjuvant control mice. Alum adjuvant assisted the second peptide, MAP-4, in production of IgG1, IgG2a, IgG2b and IgA specific antibodies and increase in liver ROS, but with no protective potential, raising doubt about the necessity of adjuvant addition. Accordingly, different vaccine formulas containing TPX MAP and 1, 2 or 3 cysteine peptidases-derived peptides with or without alum were used to immunize parallel groups of mice. Compared to unimmunized control mice, significant (P < 0.05 to < 0.005) 22 to 54 % reduction in worm burden was recorded in the different groups associated with insignificant changes in parasite egg output. The results together indicated that a schistosomiasis vaccine able to entirely prevent disease and halt its transmission still remains elusive.

Protective immune potential of multiple antigenic peptide (MAP) constructs comprising peptides that are shared by several cysteine peptidases against Schistosoma mansoni infection in mice

In vaccine trials, Schistosoma mansoni cathepsin B1 (SmCB1), helminth cathepsins of the L family (e.g., SmCL3), and papain consistently induce highly significant reductions in challenge worm burden and egg viability, but generated no additive protective effects when used in combination. The protective capacity of the cysteine peptidases is associated with modest (SmCB1) and poor (cathepsins L) production of cytokines and antibodies, essentially of the type 2 axis, and is only marginally reduced upon use of proteolytically inactive enzymes. In this work, peptides shared by SmCB1, cathepsins of the L family, papain and other allergens were selected, synthesized as tetrabranched multiple antigen peptide constructs (MAP-1 and MAP-2), and used in two independent experiments to immunize outbred mice, in parallel with papain. The two peptides elicited significant (P < 0.05) reduction in challenge worm burden when compared to unimmunized mice, albeit lower than that achieved by papain. Protection was associated with modest serum type 2 cytokines and antibody levels in MAP-, and papain-immunized mice. Immunization with papain also elicited a reduction in parasite egg load, viability, and granuloma numbers in liver and intestine. MAP-1 and MAP-2 immunogens displayed some opposite effects- MAP-1 leading to higher egg numbers with poor vitality, whereas MAP-2 immunization yielded fewer eggs. Cysteine peptidase thus appear to carry peptides that elicit opposing outcomes, highlighting the difficulty of reaching fully fledged protection, unless a vaccine is based on carefully selected peptides and combined with an effective adjuvant.

Multiple Antigenic Peptide-Based Vaccines Targeting Ixodes ricinus Neuropeptides Induce a Specific Antibody Response but Do Not Impact Tick Infestation.

Synthetic peptide vaccines were designed to target the neuropeptides innervating Ixodes ricinus salivary glands and hindgut and they were tested for their capacity to afford protective immunity against nymphs or larvae and Anaplasma phagocytophilum-infected nymph infestation, in mice and sheep, respectively. In both models, the assembly of SIFamide (SIFa) or myoinhibitory peptide (MIP) neuropeptides into multiple antigenic peptide constructs (MAPs) elicited a robust IgG antibody response following immunization. Nevertheless, no observable detrimental impact on nymphs was evidenced in mice, and, unfortunately, the number of engorged nymphs on sheep was insufficient for firm conclusions to be drawn, including for bacterial transmission. Regarding larvae, while vaccination of the sheep did not globally diminish tick feeding success or development, analyses of animals at the individual level revealed a negative correlation between anti-SIFa and MIP antibody levels and larva-to-nymph molting success for both antigens. Our results provide a proof of principle and precedent for the use of MAPs for the induction of immunity against tick peptide molecules. Although the present study did not provide the expected level of protection, it inaugurates a new strategy for protection against ticks based on the immunological targeting of key components of their nervous system.

Dissecting the Immune Response Elicited by WbALT-2, ALT MAP in Clinical Populations and Mouse Model: A Prophylactic Measure Against Lymphatic Filariasis.

Abundant Larval Transcript (ALT) is one of the major groups of immune-dominant proteins produced by filarial worms during their larval stage. The major B-cell and T-cell epitopic domains of the ALT-2 antigen were mapped to develop a multiple antigenic peptide (MAP) prophylactic antigen against lymphatic filariasis. ALT MAP was constructed by solid phase peptide synthesis. The reactivity of whole ALT protein and ALT MAP against clinical sera described a high reactivity of endemic normal sera against ALT MAP compared to WbALT-2 protein. The antibody isotype pattern revealed elevated levels of IgG1 and IgG2 against ALT MAP, followed by IgG3 and IgG4. In this study we also analyzed the immune response pattern elicited by ALT MAP, ALT in mice models, which revealed similar pattern of humoral response, while low T cell proliferation in ALT MAP groups. The low proliferation could be attributed to T/B epitope arrangement on the construct, MHC restriction, and incomplete signal delivery by T cell receptor. The immunodominant epitopes in ALT MAP were found to play a crucial role in inducing high antigen specific proliferation. This revealed the significance of ALT MAP in stimulating innate immunity in offering protective immune response probably through the activation of complement cascade along with stimulation of cellular response. An improved understanding, including the construction of ALT MAP and parasite challenge study in jirds to determine the worm clearance would give a better insight in the characterization ALT MAP construct as a prophylactic vaccine candidate.

Induction of Protective Immune Responses Against Schistosomiasis haematobium in Hamsters and Mice Using Cysteine Peptidase-Based Vaccine.

One of the major lessons we learned from the radiation-attenuated cercariae vaccine studies is that protective immunity against schistosomiasis is dependent on the induction of T helper (Th)1-/Th2-related immune responses. Since most schistosome larval and adult-worm-derived molecules used for vaccination uniformly induce a polarized Th1 response, it was essential to include a type 2 immune response-inducing molecule, such as cysteine peptidases, in the vaccine formula. Here, we demonstrate that a single subcutaneous injection of Syrian hamsters with 200 μg active papain, 1 h before percutaneous exposure to 150 cercariae of Schistosoma haematobium, led to highly significant (P < 0.005) reduction of >50% in worm burden and worm egg counts in intestine. Immunization of hamsters with 20 μg recombinant glyceraldehyde 3-phosphate dehydrogenase (rSG3PDH) and 20 μg 2-cys peroxiredoxin-derived peptide in a multiple antigen peptide construct (PRX MAP) together with papain (20 μg/hamster), as adjuvant led to considerable (64%) protection against challenge S. haematobium infection, similar to the levels reported with irradiated cercariae. Cysteine peptidases-based vaccination was also effective in protecting outbred mice against a percutaneous challenge infection with S. haematobium cercariae. In two experiments, a mixture of Schistosoma mansoni cathepsin B1 (SmCB1) and Fasciola hepatica cathepsin L1 (FhCL1) led to highly significant (P < 0.005) reduction of 70% in challenge S. haematobium worm burden and 60% reduction in liver egg counts. Mice vaccinated with SmCB1/FhCL1/rSG3PDH mixture and challenged with S. haematobium cercariae 3 weeks after the second immunization displayed highly significant (P < 0.005) reduction of 72% in challenge worm burden and no eggs in liver of 8–10 mice/group, as compared to unimmunized mice, associated with production of a mixture of type 1- and type 2-related cytokines and antibody responses.

Incorporation of Disulfide Containing Protein Modules into Multivalent Antigenic Conjugates: Generation of Antibodies against the Thrombin-Sensitive Region of Murine Protein S

Antigenic peptide conjugates can be used as vaccines and for the production of antibodies for clinical and research use. A method is presented here for the construction of conjugates incorporating oxidatively folded protein domains in their native conformation. This method was used to prepare multiple antigenic peptide constructs of the thrombin-sensitive loop region of murine anticoagulant protein S.

Cell-mediated immune response to epitopic MAP (multiple antigen peptide) construct of LcrV antigen of Yersinia pestis in murine model

Yersinia pestis is the causative agent of plague. Cellular immunity seems to play an important role in defense against this disease. The subunit vaccine based on V (Lcr V) antigen has been proved to be immunogenic in animals and in humans. The multiple antigen peptide (MAP), incorporating all the relevant B and T cell epitopes is highly immunogenic in mice through intranasal route of immunization in PLGA particles containing CpG-ODN as an immunoadjuvant inducing humoral and mucosal immune response. In the present study, cell-mediated immune response using same MAP was studied in murine model. Primary and memory T cell responses were studied in outbred and inbred mice immunized intranasally with MAP in the presence of two immunoadjuvants (Murabutide and CpG-ODN). All the three compartments (Spleen, Lamina propria and Peyer’s patches) of the lymphoid system showed increased lymphoproliferative response. Highest lymphoproliferative response was observed especially with CpG-ODN. Cytokine profile in the culture supernatant showed highest Th1 and Th17 levels. FACS analysis showed expansion of both CD4+ and CD8+ T cells producing gamma-interferon, perforin and granzyme-B with major contribution from CD4+ T cells.

Enhanced humoral and mucosal immune responses after intranasal immunization with chimeric multiple antigen peptide of LcrV antigen epitopes of Yersinia pestis coupled to palmitate in mice

Yersinia pestis is the causative agent of the most deadly disease plague. F1 and V antigens are the major vaccine candidates. Six protective epitopes of V antigen of varying length (15-25aa) were assembled on a lysine backbone as multiple antigen peptide (MAP) using standard Fmoc chemistry. Palmitate was coupled at amino terminus end. Amino acid analysis, SDS-PAGE, immunoblot and immunoreactivity proved the authenticity of MAP. MAP was immunized intranasally encapsulated in PLGA (polylactide-co-glycolide) microspheres and with/without/adjuvants murabutide and CpG ODN 1826 (CpG), in three strains of mice. Humoral and mucosal immune responses were studied till day 120 and memory response was checked after immunization with native V antigen on day 120. Epitope specific serum and mucosal washes IgG, IgA, IgG subclasses and specific activity were measured by indirect ELISA and sandwich ELISA, respectively. IgG and IgA peak antibody titers of all the MAP construct formulations in sera were ranging from 71,944 to 360,578 and 4493 to 28,644, respectively. MAP with CpG showed significantly high (p<0.0001) antibody titers ranging from 101,690 to 360,578 for IgG and 28,644 for IgA. Mucosal peak IgG and IgA titers were ranging from 1425 to 8072 and 1425 to 7183, respectively in intestinal washes and 799-4528 and 566-4027, respectively in lung washes. MAP with CpG showed significantly high (p<0.001) SIgA titers of 8000 in lung and 16,000 in intestinal washes. IgG isotyping revealed IgG2a/IgG1 ratio>1 with CpG. Serum and mucosal antipeptide IgG and IgA specific activities correlated well with antibody titers. All the constituent peptides contributed towards immune response. Structural analysis of MAP revealed little or no interaction between the peptides. Present study showed MAP to be highly immunogenic with high and long lasting antibody titers in serum and mucosal washes with good recall response with/without CpG as an adjuvant which can be used for vaccine development for plague.

Increased survival time in mice vaccinated with a branched lysine multiple antigenic peptide containing B- and T-cell epitopes from T. gondii antigens

To develop a multiple antigenic peptide (MAP) vaccine against toxoplasmosis, tri-epitope MAP constructs were made in dimeric fashion. The constructs included one B-cell and two T-cell epitopes derived from Toxoplasma gondii antigens (SAG1, GRA4 and GRA1) situated in tandem through the GGG spacer sequence, with the latter positioned adjacent to a polylysine core. Immunization of BALB/c and Kunming mice with the MAP construct in Freund's adjuvant induced not only humoral immunes response but cellular responses. These responses were accompanied by significant levels of splenocyte proliferation and interferon gamma (IFN-γ) in vitro. After lethal challenge, vaccinated mice had increased survival time in comparison to unvaccinated controls. Our data demonstrate that a MAP construct could trigger strong humoral and cellular responses against T. gondii, and that this MAP is a vaccine candidate worth further development.

Absence of amplification role of the protein KLH on antibody response generated by a MAP Staphyloccocus aureus enterotoxin A (SEA) peptide comparing with the corresponding monomeric peptide

Staphuloccocus aureus enterotoxin A (SEA) is a toxin involved in numerous food poisoning cases. To develop a detection test specific for this toxin, different approaches have been envisaged to obtain the highest antibody titer sera against a peptide sequence from this protein. The present work compares the properties of antibodies raised against the peptide epitope by classical and multiple antigen peptide (MAP) system approaches. Different immunization protocols were used: BALB/c mice were immunized either with the peptide or the MAP alone in Freund's adjuvant, co-immunized with the keyhole limpet hemocyanin (KLH) protein or, with the peptide, conjugated to this carrier KLH protein.The extent of reaction of the antibodies to the MAP construct with the parent protein was found to be significantly less than the antibodies raised against the monomeric peptide co-immunized with or conjugated to a carrier protein but more that the antibodies raised against the peptide alone. Inversely, co-immunization of the MAP with the KLH was not able to raise the immune response as it was observed with the monomeric peptide. The results suggest that, for the epitope chosen here, MAP constructs were not the most effective option to induce sera with high levels of antibodies that react with the native protein.

Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase is a lung-stage schistosomula surface membrane antigen

We have previously reported that Schistosoma mansoni larvae emerging from host lung at pH 7.5-7.8 and then fixed with diluted formaldehyde (HCHO) readily bind radiation-attenuated cercariae (RA) vaccine serum antibodies, as assessed by indirect membrane immunofluorescence (IF). Here we show that S. mansoni schistosomula emerging from lung pieces under 5% CO2 (pH < or = 7.3) readily bind RA vaccine serum antibodies, provided they have been incubated for 12 h at pH 7.5-7.8 in foetal calf serum-free RPMI medium, and fixed with diluted HCHO. Ex vivo larvae exposed during incubation to GW4869, a specific inhibitor of tegument-bound, neutral sphingomyelinase (nSMase) displayed significantly diminished binding of RA vaccine serum antibodies, thus suggesting that nSMase activity at pH > or = 7.5 leads to exposure of lung-stage larvae surface membrane antigens to specific antibody detection. More importantly, ex vivo larvae readily bound antibodies directed to dipeptidic multiple antigen peptide constructs, based on S. mansoni-specific sequences in S. mansoni glyceraldehyde 3-phosphate dehydrogenase (SG3PDH). Lung-stage schistosomula IF reactivity was diminished following antiserum absorption with recombinant SG3PDH. The data together indicate that intact ex vivo, as well as, 5-day-old in vitro-grown larvae express SG3PDH on their surface membrane. The findings are discussed in relation to the importance of surface membrane proteins as candidate vaccine antigens.

Immunogenic and Protective Potential of Mutans Streptococcal Glucosyltransferase Peptide Constructs Selected by Major Histocompatibility Complex Class II Allele Binding

Mutans streptococcal glucosyltransferases (GTF) have been demonstrated to be effective components of dental caries vaccines. We had previously selected peptide subunits of GTF for vaccine development based on putative functional significance and conservation of GTF primary structure among enzyme isoforms. In this study, 20 20-mer linear GTF peptides were synthesized, 17 identified on the basis of the highest potential major histocompatibility complex (MHC) class II-binding activity using computer-generated algorithms (Epimatrix and ProPred) and 3 with previously demonstrated functional significance. The immunoreactivities of these peptides were explored with rodent systems. Sera from GTF-immunized rats, assessed for binding to linear peptides by enzyme-linked immunosorbent assay, demonstrated immunoglobulin G antibody reactivity with peptides 6 and 11 and a T-cell proliferation response to peptides 6, 9, 11, and 16. Multiple antigenic peptide (MAP) constructs were synthesized from promising linear sequences. Rats that were immunized with MAP 7, 11, or 16, respectively, responded well to the immunizing MAP. Most importantly, a robust immune response (antibody and T-cell proliferation) was observed to native GTF following MAP 11 (amino acids 847 to 866; VVINNDKFVSWGITDFEM) immunization. This response inhibited GTF enzyme function. Two dental caries pathogenesis experiments were performed wherein rats were immunized with MAP constructs 11, 16, and/or 11 plus 16, followed by infection with cariogenic Streptococcus sobrinus. In both experiments cariogenic bacterial recoveries were reduced relative to total streptococci in the MAP 11- and MAP 11 plus 16-immunized groups, and the extent of dental caries was also significantly reduced in these groups. Thus, we have identified a peptide with projected avid MHC-binding activity that elicited immunoreactivity with native GTF and demonstrated protection against dental caries infection after immunization, implying that this peptide may be important in a subunit dental caries vaccine.

Immunological and Protective Effects of Diepitopic Subunit Dental Caries Vaccines

As a prelude to development of broader-spectrum vaccines for dental caries, we explored the immune potential of constructs combining epitopes from mutans streptococcal glucosyltransferases (GTF) and glucan binding protein B (GbpB). Two diepitopic peptide constructs were synthesized in a multiple antigenic peptide (MAP) format. Both constructs contained SYI, a 20-mer GbpB peptide that included a sequence having major histocompatibility complex class II binding characteristics. One diepitopic construct (SYI-CAT) also contained a 22-mer sequence from the catalytic domain of GTF. Another diepitopic construct (SYI-GLU) contained a 22-mer sequence from the glucan binding domain of GTF. To assess the ability of each construct to induce antibody reactive with GbpB and GTF native proteins, rats were injected subcutaneously with SYI-CAT, SYI-GLU, or the constituent monoepitopic constructs. Only the SYI-CAT construct induced significant levels of serum immunoglobulin G (IgG) and IgA antibody to both pathogenesis-associated proteins. Also, immunization with SYI-CAT significantly (P < 0.001) enhanced the antibody response to the CAT peptide. Experiments then compared experimental dental caries after immunization with SYI-CAT, SYI, or CAT MAP constructs, followed by infection with Streptococcus mutans strain SJr. Dental caries were lower in each peptide-immunized group than in the sham-injected group. The level of protection after SYI-CAT immunization was similar to that after immunization with constituent MAP constructs. In another experiment, rats were infected with Streptococcus sobrinus strain 6715 under an identical protocol. Significant protection was observed on buccal surfaces in both SYI-CAT and CAT construct-immunized, but not in the SYI construct-immunized, groups. Thus, addition of the GbpB-derived SYI peptide to the GTF-derived CAT peptide construct not only enhanced the immunological response to CAT and GTF epitopes, but also extended the protective effect of the construct to include both S. mutans and S. sobrinus.

Immunological response of female macaques to the PH-20 sperm protein following injection of recombinant proteins or synthesized peptides

Because of its location on the sperm surface and its multiple functions during fertilization, the PH-20 protein is a potential target for contraceptive vaccines. Cynomolgus macaques were immunized using four different adjuvants together with synthesized peptides or recombinant proteins representing selected regions of macaque PH-20. The synthesized peptide (amino acids 387–412, designated Peptide 4) was used as a linear molecule in a 1:1 ratio with a peptide sequence of tetanus toxoid, as well as a multiple antigenic peptide (MAP) matrix held together by scaffolding lysine residues. In the MAP construct, the ratio of Peptide 4 to tetanus peptide was 4:1. To circumvent the poor production of recombinant PH-20 in bacterial cells, two truncated forms of the molecule were expressed in Escherichia coli, G18 (encoding amino acids 143–510) and E10 (encoding amino acids 291–510). The adjuvants were Montanide ISA 51, Titermax Gold, Syntex adjuvant formulation (SAF), and QS-21. All of the antigen/adjuvant combinations produced significant immune responses as measured by ELISA. The circulating antibodies from immunized animals recognized macaque sperm surface PH-20 on Western blots and were shown by indirect immunofluorescence to bind to the surface of macaque sperm. Montanide and Titermax were associated with higher titers of anti-PH-20 antibodies than QS-21 and SAF adjuvants. Immunization with Titermax, however, resulted in sterile abscesses in 4 of 8 animals injected. We conclude that antigens derived from synthesized peptides and recombinant proteins representing selected regions of the PH-20 molecule can be used as vaccine components in combination with the adjuvant Montanide to elicit a significant sperm-directed antibody response in immunized macaques.

Double dimer peptide constructs are immunogenic and protective against Plasmodium falciparum in the experimental Aotus monkey model.

Multiple antigen peptide constructs (MAPs) have been used to obtain defined multimeric peptide molecules useful in the development of possible synthetic malaria vaccines. In this context, a method was developed, named double dimer constructs (DDCs), involving the direct synthesis of a dimeric peptide with a C-terminal cysteine. A tetrameric molecule was then obtained by oxidation of sulfhydryl groups. Dimer synthesis was optimized using a Fmoc/tBu strategy, dimers were purified by HPLC, oxidized with DMSO and characterized by HPLC and MALDI-TOF-MS. The tetramers or DDCs obtained by this method were used as immunogens in the search for a possible malaria vaccine. It was found that they were immunogenic in the experimental Aotus monkey model, and were able to induce protective immunity when challenged experimentally with a highly infective Plasmodium falciparum malaria strain.

Assessment of synthetic peptides for hepatitis A diagnosis using biosensor technology

In the present work we demonstrate the application of a commercial biosensor instrument (BIACORE 1000, Biacore AB, Uppsala) for the detection of antibodies against the hepatitis A virus (HAV) in human serum samples using linear and branched synthetic peptides related to the VP3 capsid protein of HAV. We also studied the conformation of the synthetic peptides by circular dichroism (CD) in order to analyse the changes in secondary structure of the constructs that could influence their recognition by antibodies. Linear and dimeric VP3(110–121) multiple antigen peptides (MAP) were the most sensitive and appropriate for serological studies of serum from HAV infected patients using BIACORE. Immobilization of tetrameric MAPs via amine groups apparently failed to preserve the active conformation of the peptide epitope since it led to lower antibody binding compared to linear and dimeric peptides. The CD analysis showed that the tetrameric MAP constructs tend to adopt a β-sheet structure due to intermolecular aggregation, which limits epitope accessibility. Our results demonstrate the value of biospecific interaction analysis technology using synthetic peptides for the diagnosis of acute hepatitis A.

Efficient protection against Mycobacterium tuberculosis by vaccination with a single subdominant epitope from the ESAT-6 antigen.

We have investigated the vaccine potential of two peptides derived from the 6-kDa early secretory antigenic target (ESAT)-6 antigen in the mouse model of tuberculosis. The peptides were both strongly immunogenic in B6CBAF1 (H-2b,k) mice and primed recall responses of the same intensity after immunization. However, both tuberculosis infection and immunization with ESAT-6 resulted in responses focused towards ESAT-61-20. Multiple antigen peptide constructs as well as free peptides were emulsified with dimethyl dioctadecylammonium bromide/monophosphoryl lipid A/IL-2 and tested as experimental vaccines in an i.v. and aerosol model of tuberculosis in mice. The peptide were highly immunogenic and induced cellular responses of the same magnitude. However, only vaccines based on the subdominant ESAT-651-70 epitope promoted significant levels of protective immunity and the level of protection was equivalent to that achieved with ESAT-6 and BCG. These findings demonstrate the potential of peptide-based vaccines against tuberculosis and indicate that there is not direct correlation between the hierarchy of response to naturally processed peptides and their ability to induce protective immunity against Mycobacterium tuberculosis.

The immunogenicity of various peptide antigens inducing cross-reacting antibodies to a cell surface protein antigen of Streptococcus mutans.

A cell surface protein antigen (PAc) of Streptococcus mutans may be involved in the binding of bacteria to the tooth surface, and has long been focused upon as a candidate for a preventive vaccine of dental caries. Previously the peptide PAc (365-377) was shown to raise an antibody in B10.D2 mice which inhibited the binding of salivary components to the PAc molecule. Using this peptide as a unit peptide, two constructs based on multiple antigenic peptides, and several types of tandem repeats of two or three copies were synthesized to estimate the immunogenicity of these peptides. Increase in the immunogenicity was observed for all constructs with the use of an adjuvant compared to the unit peptide alone. However, the tandem repeat constructs generally induced antibody production in the absence of adjuvant, while the multiple antigenic peptide constructs did not induce antibody production under the same condition. Although such a phenomenon may be restricted to this particular peptide sequence, these results may influence the strategy for the design of peptide vaccines.

Interaction of a Multiple Antigenic Peptide of Hepatitis A Virus with Monolayers and Bilayers of Acidic, Basic, and Zwitterionic Phospholipids

The interaction of the synthetic multiple antigenic peptide construct MAP4-VP1(11-25) with lipid membranes was studied by a combination of spectroscopic and biophysical techniques. MAP4-VP1 showed an important surface activity when spread in an air–water interface, indicative of an amphipatic conformation. Since this peptide has a net anionic charge, we studied the degree of interaction with model membranes of zwitterionic dipalmitoylphosphatidylcholine (DPPC), anionic phosphatidylinositol (PI and DPPC/PI 9:1), or cationic stearylamine (SA and DPPC/SA 9:1). MAP4-VP1 was readily soluble in aqueous buffer, yet spontaneously inserted from an aqueous subphase into cationic and zwitterionic monolayers up to limiting pressures of 20–30 mN/m. The induced surface pressure increases were in the order cationic > zwitterionic > anionic monolayers. This indicates that the interaction is mainly driven by electrostatic forces, although there is an important hydrophobic component, responsible for the penetration in monolayers of neutral DPPC. Interaction of MAP4-VP1 with unilamellar vesicles of DPPC, DPPC/SA (9:1), and DPPC/PI (9:1) labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) or with 1-anilino-8-naphthalene sulfonic acid (ANS) was determined by changes in fluorescence polarization as a function of temperature. Results showed that the presence of the positively charged SA in the membrane strongly enhances the incorporation of MAP4-VP1 and that the peptide increases the fluidity of the bilayer and decreases the temperature of the phase transition.

Direct synthesis and characterisation of multi‐dendritic peptides for use as immunogens

The direct synthesis and subsequent rapid characterisation of multiple antigen peptides (MAPs) for use as immunogens has presented difficulties, partly because of the formation of incomplete or truncated peptide sequences during the synthetic procedure. Therefore, many researchers have resorted to ligation procedures for the synthesis of MAP constructs. This article describes a method to improve the yield of MAP constructs by direct synthesis methods, as well as a general procedure that enables easier characterisation of the synthetic products. In particular, during the synthesis of MAP constructs, a capping procedure was introduced after each amino acid coupling step, thus improving significantly the yield of the desired multi-dendritic peptidic immunogens. Through the use of this capping procedure, problems arising from the incomplete amino acid residue coupling at the point of synthesis were minimised, and any deletion peptides which formed could be eliminated more readily during the subsequent purification procedures. In addition, previous difficulties in purification and characterisation of MAP construct by, e.g. electrospray mass spectroscopy (ES-MS), often led to the multi-dendritic peptidic immunogens being used without full characterisation after dialysis and recovery of the product(s). This article describes an enzymatic (tryptic) digestion method with the MAP construct, followed by characterisation of the enzymatic digest by reversed phase high-performance liquid chromatography-ES-MS. With this method, fragments of the MAP construct cleaved at specific amino acid residue sites (e.g. lysine or arginine) within the sequence of the parent peptide can be readily determined and the kinetics of the digestion easily followed. This enzymatic digestion procedure thus provides a facile approach to confirm that all of the multi-dendritic arms of the purified MAP construct have been equivalently elongated during the peptide synthesis and that consequently the purified construct structure contains the correct peptide sequence.