Immunoproteomic analysis of the murine antibody response to successful and failed immunization with live anti-Francisella vaccines

Francisella tularensis subspecies tularensis is one of the most virulent of bacterial pathogens for humans. Protective immunity against the pathogen can be induced in humans and some, but not all, mouse strains by vaccination with live, but not killed, vaccines. In mice, this protection is mediated predominantly by CD4+ and CD8+ T cells. This is thought to be the case too for humans. Nevertheless, it is possible that successful vaccination elicits antigen-specific antibodies that can serve as correlates of protection. To test this hypothesis we examined the repertoire of antibodies induced following successful immunization of BALB/c and CH3/HeN mice versus unsuccessful vaccination of C57BL/6 and DBA\2 mice with F. tularensis Live Vaccine Strain or following unsuccessful vaccination of BALB/c mice with highly related subspecies, F. novicida. The results showed that successful vaccination elicited antibodies to at least six proteins that were not recognized by antisera from vaccinated but unprotected mice.     

Single domain antibodies for the knockdown of cytosolic and nuclear proteins

Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, nonaggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain antibody libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant‐ and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell‐penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell‐specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb‐DNA or –protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic.     

Genetic Immunization of Wild-Type and Hepatitis C Virus Transgenic Mice Reveals a Hierarchy of Cellular Immune Response and Tolerance Induction against Hepatitis C Virus Structural Proteins

To study the effect of genetic immunization on transgenic expression of hepatitis C virus (HCV) proteins, we evaluated the immunological response of HCV transgenic mice to HCV expression plasmids. FVB/n transgenic mice expressing HCV structural proteins (core, E1, and E2) and wild-type (WT) FVB/n mice were immunized intramuscularly with plasmids expressing core (pHCVcore) or core/E1/E2 (pHCVSt). After immunization, HCV-specific humoral and cellular immune response was studied. Both WT and transgenic mice immunized with either HCV construct produced antibodies and exhibited T-cell proliferative responses against core or envelope. In WT mice immunized with pHCVSt, cytotoxic T-lymphocyte (CTL) activities were detected against E2 but not against core or E1, whereas strong CTL activities against core could be detected in WT mice immunized with pHCVcore. In pHCVSt-immunized, transgenic mice, CTL activities against the core or envelope were completely absent, but core-specific CTL activities could be detected in pHCVcore-immunized transgenic mice. A similar pattern of immune responses was also observed in other mouse strains, including a transgenic line expressing human HLA-A2.1 molecules (AAD mice). Despite the presence of a peripheral cellular immunity against HCV, no liver pathology or lymphocytic infiltrate was observed in these transgenic mice. Our study suggests a hierarchy of CTL response against the HCV structural proteins (E2 > core > E1) in vivo when the proteins are expressed as a polyprotein. The HCV transgenic mice can be induced by DNA immunization to generate anti-HCV antibodies and anticore CTLs. However, they are tolerant at the CTL level against the E2 protein despite DNA immunization.     

DNA vaccination of mice with a plasmid encoding Puumala hantavirus nucleocapsid protein mimics the B-cell response induced by virus infection

Inoculation of naked DNA has been applied for the development of prophylactic and therapeutic vaccines against different viral infections. To study the humoral immune response induced by DNA vaccination we cloned the entire nucleocapsid protein-encoding sequence of the Puumala hantavirus strain Vranica/H?lln?s into the CMV promoter-driven expression unit of the plasmid pcDNA3, generating pcDNA3-VR1. A single dose injection of 50 microg of plasmid DNA into each M. tibialis anterior of BALB/c mice induced a high-titered antibody response against the nucleocapsid protein as documented 6 and 11 weeks after immunisation. PEPSCAN analysis of a serum pool of the pcDNA3-VR1-vaccinated animals revealed antibodies reacting with epitopes covering the whole nucleocapsid protein. The epitope-specificity of the immune response induced by DNA vaccination seems to reflect the antibody response in experimentally virus-infected bank voles (the natural host of the Puumala virus) and humans. The data suggest that DNA vaccination could be used for the identification of highly immunogenic epitopes in viral proteins.     

Amelioration of amyloid load by anti-Abeta single-chain antibody in Alzheimer mouse model

Parenteral immunization of transgenic mouse models of Alzheimer disease (AD) with synthetic amyloid beta-peptide (Abeta) prevented or reduced Abeta deposits and attenuated their memory and learning deficits. A clinical trial of immunization with synthetic Abeta, however, was halted due to brain inflammation, presumably induced by a toxic Abeta, T-cell- and/or Fc-mediated immune response. Another issue relating to such immunizations is that some AD patients may not be able to raise an adequate immune response to Abeta vaccination due to immunological tolerance or age-associated decline. Because peripheral administration of antibodies against Abeta also induced clearance of amyloid plaques in the model mice, injection of humanized Abeta antibodies has been proposed as a possible therapy for AD. By screening a human single-chain antibody (scFv) library for Abeta immunoreactivity, we have isolated a scFv that specifically reacts with oligomeric Abeta as well as amyloid plaques in the brain. The scFv inhibited Abeta amyloid fibril formation and Abeta-mediated cytotoxicity in vitro. We have tested the efficacy of the human scFv in a mouse model of AD (Tg2576 mice). Relative to control mice, injections of the scFv into the brain of Tg2576 mice reduced Abeta deposits. Because scFvs lack the Fc portion of the immunoglobulin molecule, human scFvs against Abeta may be useful to treat AD patients without eliciting brain inflammation.     

Multiepitopic HLA-A*0201-restricted immune response against hepatitis B surface antigen after DNA-based immunization

CTL together with anti-envelope Abs represent major effectors for viral clearance during hepatitis B virus (HBV) infection. The induction of strong cytotoxic and Ab responses against the envelope proteins after DNA-based immunization has been proposed as a promising therapeutic approach to mediate viral clearance in chronically infected patients. Here, we studied the CTL responses against previously described hepatitis B surface Ag (HBsAg)-HLA-A*0201-restricted epitopes after DNA-based immunization in HLA-A*0201 transgenic mice. The animal model used was Human Human D(b) (HHD) mice, which are deficient for mouse MHC class I molecules (beta(2)-microglobulin(-/-) D(b-/-)) and transgenic for a chimeric HLA-A*0201/D(b) molecule covalently bound to the human beta(2)-microglobulin (HHD(+/+)). Immunization of these mice with a DNA vector encoding the small and the middle HBV envelope proteins carrying HBsAg induced CTL responses against several epitopes in each animal. This study performed on a large number of animals described dominant epitopes with specific CTL induced in all animals and others with a weaker frequency of recognition. These results confirmed the relevance of the HHD transgenic mouse model in the assessment of vaccine constructs for human use. Moreover, genetic immunization of HLA-A2 transgenic mice generates IFN-gamma-secreting CD8(+) T lymphocytes specific for endogenously processed peptides and with recognition specificities similar to those described during self-limited infection in humans. This suggests that responses induced by DNA immunization could have the same immune potential as those developing during natural HBV infection in human patients.     

一种抗对虾白斑综合症病毒(WSSV)线性抗原表位单链抗体的筛选

对虾白斑综合症病毒(WSSV)是全世界对虾养殖业最主要的病原体之一, 虽然对该病毒的研究已较为深入, 但目前仍无有效的防治方法。本研究应用噬菌体展示技术, 构建了抗变性WSSV的单链抗体噬菌体展示文库, 分别以WSSV病毒粒子和原核表达的囊膜蛋白VP28为靶分子对该文库进行淘选。经过数轮淘选后, 得到5个能特异识别WSSV的单链抗体, 且首次获得了能特异识别WSSV线性抗原表位的单链抗体P75E8。并通过免疫胶体金电镜分析, 对5种单链抗体对应在病毒粒子上的表位进行了定位。为获取识别多种WSSV抗原的抗体提供了新的方法路线, 也为获取特异性识别线性表位的单链抗体提供了一种新的淘选技巧。     

Multivalent scFv display of phagemid repertoires for the selection of carbohydrate-specific antibodies and its application to the Thomsen-Friedenreich antigen

The Thomsen-Friedenreich disaccharide (TF) is a promising target antigen for tumor immunotherapy, since it is almost exclusively expressed in carcinoma tissues. The TF-specific antibodies generated so far are IgMs of mouse origin with limited therapeutic potential. Phage-displayed scFv repertoires are an established source for recombinant antibodies; however, we were unable to identify scFvs binding to TF when applying libraries in the standard monovalent display format of phagemid systems. Here, we report on the successful selection of TF-specific antibody fragments using a multivalent scFv phagemid library format based on shortened linkers (one amino acid residue). The libraries were constructed from mice immunized with asialoglycophorin and selected using TF displayed on two different carrier molecules in combination with the proteolytically cleavable helper phage KM13. All isolated clones encoded the same framework genes and the same complementarity-determining regions. After affinity maturation only scFv with the founder sequence were selected from secondary repertoires. This indicates a very narrow sequence window for TF-specific antibodies. Investigating other linker-length formats revealed a clear inverse correlation between linker length and binding activity both as soluble proteins and displayed on phages. The highest affinity was obtained with the tetrameric format. The selected scFv was specific for TF on various carrier molecules and tumor cells and performed well in ELISA and immunohistochemistry. We postulate that scFv phagemid library formats with short linkers (i.e. multimeric scFvs) may, in general, be advantageous in selections for the generation of scFvs against carbohydrate epitopes or other epitopes associated with low intrinsic affinity per binding site), and expect that they will be superior in applications for diagnosis or therapy.     

Therapeutic antibodies elicited by immunization against TNF-alpha.

Tumor necrosis factor-alpha (TNF-alpha) is critically involved in the pathogenesis of several chronic inflammatory diseases. Monoclonal antibodies against TNF-alpha are currently used for the treatment of rheumatoid arthritis and Crohn's disease. This report describes a simple and effective method for active immunization against self TNF-alpha. This vaccination approach leads to a T-cell-dependent polyclonal and sustainable anti-TNF-alpha autoantibody response that declines upon discontinuation of booster injections. The autoantibodies are elicited by injecting modified recombinant TNF-alpha molecules containing foreign immunodominant T-helper epitopes. In mice immunized with such molecules, the symptoms of experimental cachexia and type II collagen-induced arthritis are ameliorated. These results suggest that vaccination against TNF-alpha may be a useful approach for the treatment of rheumatoid arthritis and other chronic inflammatory diseases.     

Epitope mapping of the human biliary amphipathic, anionic polypeptide: similarity with a calcium-binding protein isolated from gallstones and bile, and immunologic cross-reactivity with apolipoprotein A-I.

Biliary amphipathic anionic polypeptide (APF) the major protein of the pigment-lipoprotein complex in bile, and calcium-binding protein (CBP) from gallstones are both small (less than 10 kDa), highly acidic, amphipathic proteins present in bile and closely associated also with pigmented areas in human gallstones. Polyclonal antibodies against APF have shown cross-reactivity with plasma high density lipoproteins (HDL). This study examines the hypothesis that APF and CBP might be closely related or even identical, and might also share common epitopes with the larger apoA-I (23 kDa). To assess this, immunoreactivity of the three delipidated, highly purified proteins was determined against a panel of 12 monoclonal antibodies (MAbs) prepared against APF and a panel of 4 MAbs against apoA-I. APF was isolated from bile by zonal ultracentrifugation. CBP was isolated from proteins precipitated from bile by CaCl2, as well as from the calcium bilirubinate shells of cholesterol gallstones, by extraction successively with methyl-t-butyl ether, methanol, and Na2EDTA, followed by Sephadex G-25 chromatography and two-stage preparative SDS-PAGE. ApoA-I was prepared by two types of chromatography: Sephacryl S200 chromatography and heparin-chromatographic immunoaffinity. Specific polyclonal antibodies to APF and apoA-I were prepared from immunized rabbits. MAbs to APF and apoA-I were prepared by immunization of mice, using standard hybridoma technique. Western blotting of APF and CBP in 15% SDS-PAGE yielded one band with an apparent molecular weight of 6.5 kDa, which, along with apoA-I, was immunostained by polyclonal antibodies to APF and apoA-I. Using 12 MAbs against APF with three types of ELISA (direct antigen binding, competitive antigen displacement, and epitope competition between antibodies), it was shown that APF and delipidated apoA-I shared six epitopes, three of which were detected also on the surface of intact HDL particles. Six other epitopes were present in APF but not apoA-I, four of which were exposed on the surface of HDL. Four MAbs against apoA-I reacted with APF and CBP. Amino acid analyses of APF and CBP were similar with 20-23% acidic and 7-11% basic amino acids and low contents of cysteine, methionine, and tyrosine; both differed from apoA-I in containing isoleucine and cysteine. Using ELISA and one MAb (no. 32) against APF, this polypeptide was detected in human plasma HDL, the pigment-lipoprotein complex in the bile of humans, dogs, and rats, and in both pigment and cholesterol gallstones.(ABSTRACT TRUNCATED AT 400 WORDS)     

The antibody repertoire to proteins of Mycobacterium leprae. Genetic influences at the antigen and epitope level.

The effects of both H-2 and non-H-2 genes on the antibody response to the proteins of Mycobacterium leprae were investigated. Using a B10 series of congenic mice we found that the repertoire of antibody responses was under H-2 gene control, and that non-H2 genes were also involved. By Western blotting, differences in the number and m.w. of proteins recognised by mice of different genetic background were apparent. Such differences were also reflected in the total antibody response to a soluble extract of M. leprae (M. leprae sonicate), as measured by ELISA. Concentrating on one particular Ag, the 65-kDa heat shock protein, we found that all strains of mice developed antibodies following immunization with the purified recombinant protein, although there was a continuous distribution in the titer of antibodies obtained, with differences between individual strains indicating both H-2 and non-H-2 effects. Using a library of overlapping peptides based on the amino acid sequence of this protein, we have mapped the B cell epitopes in the different strains of mice. H-2 genes had no effect on the structure and number of epitopes recognized, although this was influenced by non-H-2 genes. There was a high level of concordance between actual epitopes recognized and those predicted by calculations of antigenic index, and B cell epitopes were located in similar positions to previously determined T cell epitopes.     

Carrier-induced epitopic suppression, a major issue for future synthetic vaccines

Synthetic antigens have been shown, in experimental models, to induce protective immunity against a variety of pathogens. These studies have demonstrated that, due to their low immunogenicity, these synthetic antigens required conjugation to carrier molecules. Therefore, the choice of appropriate carriers for human immunization by future synthetic vaccines is a major issue. Tetanus toxoid is generally considered to be an effective potential carrier devoid of side-effects. However, the present study performed in mice with two synthetic vaccine models demonstrates that the immune response against the synthetic epitopes conjugated to tetanus toxoid can be suppressed by pre-existing immunity against this same carrier. Because most humans have been exposed to this antigen, this effect may have important implications for the development of synthetic vaccines.     

Expression of a functional recombinant chimeric protein of human hepatitis A virus VP1 and an Fc antibody fragment in transgenic tomato plants

Transgenic tomato plants expressing the gene of a chimeric protein (HAV VP1-Fc) consisting of human hepatitis A virus (HAV) VP1 and an Fc antibody fragment have been obtained. Recombinant VP1-Fc protein with a molecular mass of approximately 68 kDa was purified from transgenic tomato plants using Protein A Sepharose affinity chromatography. The recombinant protein elicited production of specific IgG antibodies in the serum after intraperitoneal immunization of BALB/c mice. The antibodies produced by mice against transgenic plant-derived recombinant VP1-Fc most likely recognize epitopes in the HAV viral antigen. Following vaccination with recombinant VP1-Fc protein, expression of IFN-γ and IL-4 were increased in splenocytes at the time of sacrifice. Our findings indicate that transgenic tomato plants can provide a useful system for the production of HAV antigens.     

Recombinant scFv antibodies against E protein and N protein of severe acute respiratory syndrome virus

Severe acute respiratory syndrome (SARS) brought aglobal outbreak in spring of 2003 [1–3], and more andmore attention has been paid on it when a new caseresurfaced in Singapore last September [4]. By the endof May in 2003, WHO reported a cumulative total of 8202infected cases with 725 deaths from 28 countries.Because of the high transmission and morality rate ofSARS, scientists in many countries have made theirefforts in studying SARS coronavirus (SARS-CoV)[5, 6]. Several genomes of…     

Murine polyomavirus virus-like particles carrying full-length human PSA protect BALB/c mice from outgrowth of a PSA expressing tumor

Virus-like particles (VLPs) consist of capsid proteins from viruses and have been shown to be usable as carriers of protein and peptide antigens for immune therapy. In this study, we have produced and assayed murine polyomavirus (MPyV) VLPs carrying the entire human Prostate Specific Antigen (PSA) (PSA-MPyVLPs) for their potential use for immune therapy in a mouse model system. BALB/c mice immunized with PSA-MPyVLPs were only marginally protected against outgrowth of a PSA-expressing tumor. To improve protection, PSA-MPyVLPs were co-injected with adjuvant CpG, either alone or loaded onto murine dendritic cells (DCs). Immunization with PSA-MPyVLPs loaded onto DCs in the presence of CpG was shown to efficiently protect mice from tumor outgrowth. In addition, cellular and humoral immune responses after immunization were examined. PSA-specific CD4(+) and CD8(+) cells were demonstrated, but no PSA-specific IgG antibodies. Vaccination with DCs loaded with PSA-MPyVLPs induced an eight-fold lower titre of anti-VLP antibodies than vaccination with PSA-MPyVLPs alone. In conclusion, immunization of BALB/c mice with PSA-MPyVLPs, loaded onto DCs and co-injected with CpG, induces an efficient PSA-specific tumor protective immune response, including both CD4(+) and CD8(+) cells with a low induction of anti-VLP antibodies.     

Induction of virus-neutralizing antibodies by bacteria expressing the C3 poliovirus epitope in the periplasm. The route of immunization influences the isotypic distribution and the biologic activity of the antipoliovirus antibodies

Two viral epitopes (C3 neutralization epitope from poliovirus type 1 and the 132-145 peptide from the PreS2 region from hepatitis B virus) have been expressed in the Escherichia coli periplasm as protein fusion with the maltose binding protein (MalE protein). Immunization of mice with live bacteria expressing the foreign viral epitopes in their periplasm elicited high antibody titers against the viral peptide as well as against the corresponding virus. This demonstrates for the first time in the case of defined epitopes that, when live bacteria are used as immunogens, presentation at the cell surface is not a prerequisite to obtain an antibody response. On the other hand, the induction of antiviral antibody responses by these recombinant bacteria depended dramatically on the route of immunization: a response was induced by live bacteria through the i.v. route but not through the s.c. route. However, when bacteria were heat killed or when the MalE hybrid protein was released under a soluble form from the cell, a response was induced even upon s.c. immunization. From these results, we suggest that in order to induce high levels of antibodies by the s.c. route, a major parameter for bacterial Ag would be their capacity to be released into a soluble form before the interaction of the bacteria with the APC. This would permit the presentation by B cells rather than by phagocytic cells. Finally, we demonstrate that the route of immunization influences the isotypic distribution and the neutralizing activity of the antipoliovirus antibodies. Such results may have major implications for the development of bacterial vaccines based on fusion proteins.     

Generation of recombinant monoclonal antibodies to study structure-function of envelope protein VP28 of white spot syndrome virus from shrimp

White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP28 is one of the most important envelope proteins of WSSV. In this study, a recombinant antibody library, as single-chain fragment variable (scFv) format, displayed on phage was constructed using mRNA from spleen cells of mice immunized with full-length VP28 expressed in Escherichia coli. After several rounds of panning, six scFv antibodies specifically binding to the epitopes in the N-terminal, middle, and C-terminal regions of VP28, respectively, were isolated from the library. Using these scFv antibodies as tools, the epitopes in VP28 were located on the envelope of the virion by immuno-electron microscopy. Neutralization assay with these antibodies in vitro suggested that these epitopes may not be the attachment site of WSSV to host cell receptor. This study provides a new way to investigate the structure and function of the envelope proteins of WSSV.     

Generation of monoclonal antibodies against human prion proteins in PrP0/0 mice.

BACKGROUND: Prion diseases belong to a group of neurodegenerative disorders affecting humans and animals. The human diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). The pathogenic mechanisms of the prion diseases are not yet understood. Monoclonal antibodies provide valuable tools in the diagnosis, as well as in the basic research, of several diseases; however, monospecific antisera or monoclonal antibodies (mAbs) against human prion proteins were, until now, not available. MATERIALS AND METHODS: We have developed an immunization protocol based on nucleic acid injection into nontolerant PrP0/0 mice. DNA or RNA coding for different human prion proteins including the mutated sequences associated with CJD, GSS, and FFI were injected into muscle tissue. Mice were primarily inoculated with DNA plasmids encoding the prion protein (PRNP) gene and boosted either with DNA, RNA, or recombinant Semliki Forest Virus particles expressing PRNP. Hybridomas were then prepared. RESULTS: Different mAbs against human prion proteins were obtained, and their binding behavior was analyzed by peptide enzyme-linked immunosorbent assay, Western blot, immunofluorescence, and immunoprecipitation. Their cross-reactivity with prion protein from other species was also determined. Our mAbs are directed against four different linear epitopes and may also recognize discontinuous regions of the native prion protein. CONCLUSIONS: These antibodies should allow us to address questions concerning the nature of the prion protein as well as the initiation and progression of prion diseases. Moreover, these mAbs can now be used for the diagnosis of prion diseases of humans and animals.     

Passive protection against rotavirus-induced diarrhea by monoclonal antibodies to surface proteins vp3 and vp7.

Monoclonal antibodies directed against two rotavirus surface proteins (vp3 and vp7) as well as a rotavirus inner capsid protein (vp6) were tested for their ability to protect suckling mice against virulent rotavirus challenge. Monoclonal antibodies to two distinct epitopes of vp7 of simian rotavirus strain RRV neutralized RRV in vitro and passively protected suckling mice against RRV challenge. A monoclonal antibody directed against vp3 of porcine rotavirus strain OSU neutralized three distinct serotypes in vitro (OSU, RRV, and UK) and passively protected suckling mice against OSU, RRV, and UK virus-induced diarrhea. The role of vp3 in eliciting protection against heterotypic rotavirus challenge should be considered when developing a vaccine with cloned rotavirus genes. Alternatively, immunization with a reassortant rotavirus containing vp3 and vp7 from two antigenically distinct rotavirus parents might protect against diarrhea induced by two or more rotavirus serotypes.     

Impact of epitope permutations in the antibody response of mice to a multi-epitope polypeptide of the V3 loop of human immunodeficiency virus type 1

Our group have produced in Escherichia coli and evaluated the immunogenicity of different multi-epitope polypeptides (MEPs) bearing one copy of V3 loop sequential B cell epitopes from several isolates of human immunodeficiency virus type 1 (HIV-1) gp120. One of these MEPs called TAB9 comprises the 15 central amino acids of the V3 loop from isolates LR150, JY1, RF, MN, BRVA and IIIB in this order. Antibodies against all V3 regions were elicited after immunization of rabbits, macaques and humans with TAB9. In contrast, mice immunized with this protein only developed antibodies against epitopes JY1, LR150 and MN in that order (JY1>LR150>MN>RF, BRVA, IIIB) resembling an immunodominant gradient from the N-terminus to the C-terminal portion of this construction. To assess what role the location of the V3 epitopes in TAB9 could play, we constructed the protein TAB16, by altering the position of V3 epitopes in TAB9 primary structure and compared the pattern of antibodies elicited by both MEPs in H-2(d) Balb/c mice. The MEP TAB16 elicited antibody titers comparable to that of the sera from mice immunized with TAB9. There were no statistical differences in antibody titers between both groups (P>0.05). JY1, LR150 and MN V3 epitopes were again immunodominant in mice immunized with TAB16 fusion protein. The highest antibody titers detected in both groups among V3 epitopes corresponded to JY1, now located at the C-terminus of the permuted chimera. Antibodies against V3 epitopes RF, BRVA and IIIB were again not detected. Additionally, the MN V3 epitope showed to be significantly more immunogenic in its new orientation in TAB16, possibly as a result of a higher degree of accessibility in the surface of the protein. The results of the present investigation strongly suggest that the sequential order or the intramolecular position of V3 epitopes inside the primary structure of TAB9 and TAB16 MEPs does not interfere with the global immunogenicity or with the hierarchy of immunodominance of these regions.