Long-term stability of antibody responses elicited by Dengue virus envelope DIII-based DNA vaccines.

Dengue virus (DENV) is one the most important viral pathogens worldwide. Currently there is an imperative need for a reliable vaccine capable of inducing durable protection against all four serotypes. We have previously reported strongly neutralizing and highly specific antibody responses from all four serotypes to a DNA vaccine based on an engineered version of DENV E protein's domain III (DIII). Here, we show that monovalent and tetravalent immunizations with the DIII-based DNA vaccines are also capable of inducing highly stable antibody responses that remain strongly neutralizing over long periods of time. Our results demonstrate that DNA-vaccinated mice maintain a strong antibody response in terms of titre, avidity and virus-neutralizing capability 1 year after immunization.

Temperature-dependent folding allows stable dimerization of secretory and virus-associated E proteins of Dengue and Zika viruses in mammalian cells.

Dengue and Zika are two of the most important human viral pathogens worldwide. In both cases, the envelope glycoprotein E is the main target of the antibody response. Recently, new complex quaternary epitopes were identified which are the consequence of the arrangement of the antiparallel E dimers on the viral surface. Such epitopes can be exploited to develop more efficient cross-neutralizing vaccines. Here we describe a successful covalent stabilization of E dimers from Dengue and Zika viruses in mammalian cells. Folding and dimerization of secretory E was found to be strongly dependent on temperature but independent of PrM co-expression. In addition, we found that, due to the close relationship between flaviviruses, Dengue and Zika viruses E proteins can form heterodimers and assemble into mosaic viral particles. Finally, we present new virus-free analytical platforms to study and screen antibody responses against Dengue and Zika, which allow for differentiation of epitopes restricted to specific domains, dimers and higher order arrangements of E.

Role of N-glycosylation on Zika virus E protein secretion, viral assembly and infectivity.

Zika virus has rapidly spread reaching a global distribution pattern similar to that of dengue virus, and has been associated with serious neurological and developmental pathologies, like congenital malformation during pregnancy and Guillain-Barré syndrome. Sequence analysis of different clinical and laboratory isolates has shown the existence of mutants with loss of the conserved N-glycosylation motif on domain I of protein E that is common to all flaviviruses. We found that loss of E N-linked glycosylation leads to compromised expression and secretion of E ectodomain from mammalian cells. For both, wild type and glycosylation-negative mutant, secretion was independent of co-expression of the PrM viral protein, but highly dependent on temperature. Low temperature (28 °C) favoured secretion, although the glycosylation mutant E ectodomain showed impaired secretion and membrane display compared to the wild type. Production of pseudoviral particles with a West Nile virus replicon packaged with the Zika virus structural proteins C-PrM-E was significantly reduced with the non-glycosylated E. Similarly, glycosylation-negative pseudoviral particles showed impaired infectivity of Vero cells and reduced ability to infect K562 cells upon particles opsonisation with anti-E antibodies.

Lack of evidence of rotavirus-dependent molecular mimicry as a trigger of coeliac disease.

New data suggest the involvement of rotavirus (RV) in triggering autoimmunity in coeliac disease (CD) by molecular mimicry between the human-transglutaminase protein and the dodecapeptide (260-271 aa) of the RV protein VP7 (pVP7). To assess the role of RV in the onset of CD, we measured anti-pVP7 antibodies in the sera of children with CD and of control groups. We analysed serum samples of 118 biopsy-proven CD patients and 46 patients with potential CD; 32 children with other gastrointestinal diseases; 107 no-CD children and 107 blood donors. Using enzyme-linked immunosorbent assay (ELISA) assay, we measured immunoglobulin (Ig)A-IgG antibodies against the synthetic peptides pVP7, the human transglutaminase-derived peptide (476-487 aa) which shows a homology with VP7 protein and a control peptide. The triple-layered RV particles (TLPs) containing the VP7 protein and the double-layered RV-particles (DLPs) lacking the VP7 protein were also used as antigens in ELISA assay. Antibody reactivity to the RV-TLPs was positive in 22 of 118 (18%) CD patients and in both paediatric (17 of 107, 16%) and adult (29 of 107, 27%) control groups, without showing a statistically significant difference among them (P = 0·6, P = 0·1). Biopsy-proven CD patients as well as the adult control group demonstrated a high positive antibody reactivity against both pVP7 (34 of 118, 29% CD patients; 66 of 107, 62% adult controls) and control synthetic peptides (35 of 118, 30% CD patients; 56 of 107, 52% adult controls), suggesting a non-specific response against RV pVP7. We show that children with CD do not have higher immune reactivity to RV, thus questioning the molecular mimicry mechanism as a triggering factor of CD.

Secretion of dengue virus envelope protein ectodomain from mammalian cells is dependent on domain II serotype and affects the immune response upon DNA vaccination.

Dengue virus (DENV) is currently among the most important human pathogens and affects millions of people throughout the tropical and subtropical regions of the world. Although it has been a World Health Organization priority for several years, there is still no efficient vaccine available to prevent infection. The envelope glycoprotein (E), exposed on the surface on infective viral particles, is the main target of neutralizing antibodies. For this reason it has been used as the antigen of choice for vaccine development efforts. Here we show a detailed analysis of factors involved in the expression, secretion and folding of E ectodomain from all four DENV serotypes in mammalian cells, and how this affects their ability to induce neutralizing antibody responses in DNA-vaccinated mice. Proper folding of E domain II (DII) is essential for efficient E ectodomain secretion, with DIII playing a significant role in stabilizing soluble dimers. We also show that the level of protein secreted from transfected cells determines the strength and efficiency of antibody responses in the context of DNA vaccination and should be considered a pivotal feature for the development of E-based DNA vaccines against DENV.

Recombinant AAV-mediated in vivo long-term expression and antitumour activity of an anti-ganglioside GM3(Neu5Gc) antibody.

The ganglioside GM3(Neu5Gc) has gained increasing attention as therapeutic target because of its selective expression in various human tumours, such as melanoma, breast and lung cancer. 14F7 is a mouse IgG1 with specific reactivity to GM3(Neu5Gc)-positive tumours. The therapeutic activity of 14F7 has also been demonstrated in vivo, through its repetitive passive administration in tumour-bearing animals. In this work we used an alternative strategy to deliver recombinant 14F7 in vivo and analysed the therapeutic efficacy of this approach. We engineered a recombinant adeno-associated vector to direct the expression of secretable recombinant 14F7 in BALB/c animals. A single administration of the rAAV induced efficient production and secretion of the antibody in the bloodstream, with an expression level reaching plateau at ∼3 weeks after injection and persisting for almost a year. Strikingly, upon challenge with GM3(Neu5Gc)-positive X63-AG8.653 myeloma cells, tumour development was significantly delayed in animals treated with rAAV-14F7 with respect to animals treated with a control rAAV codifying for an irrelevant antibody. Finally, no significant differences in survival proportion were detected in animals injected with rAAV-14F7 or treated by standard administration of repetitive doses of purified monoclonal antibody 14F7.

Production of in vivo-biotinylated rotavirus particles.

Although inserting exogenous viral genome segments into rotavirus particles remains a hard challenge, this study describes the in vivo incorporation of a recombinant viral capsid protein (VP6) into newly assembled rotavirus particles. In vivo biotinylation technology was exploited to biotinylate a recombinant VP6 protein fused to a 15 aa biotin-acceptor peptide (BAP) by the bacterial biotin ligase BirA contextually co-expressed in mammalian cells. To avoid toxicity of VP6 overexpression, a stable HEK293 cell line was constructed with tetracycline-inducible expression of VP6-BAP and constitutive expression of BirA. Following tetracycline induction and rotavirus infection, VP6-BAP was biotinylated, recruited into viroplasms and incorporated into newly assembled virions. The biotin molecules in the capsid allowed the use of streptavidin-coated magnetic beads as a purification technique instead of CsCl gradient ultracentrifugation. Following transfection, double-layered particles attached to beads were able to induce viroplasm formation and to generate infective viral progeny.

Rotavirus replication requires a functional proteasome for effective assembly of viroplasms.

The ubiquitin-proteasome system has been shown to play an important role in the replication cycle of different viruses. In this study, we describe a strong impairment of rotavirus replication upon inhibition of proteasomal activity. The effect was evidenced at the level of accumulation of viral proteins, viral RNA, and yield of infective particles. Kinetic studies revealed that the early steps of the replicative cycle following attachment, entry, and uncoating were clearly more sensitive to proteasome inhibition. We ruled out a direct inhibition of the viral polymerase activities and stability of viral proteins and found that the crucial step that was impaired by blocking proteasome activity was the assembly of new viroplasms. This was demonstrated by using chemical inhibitors of proteasome and by gene silencing using small interfering RNAs (siRNAs) specific for different proteasomal subunits and for the ubiquitin precursor RPS27A. In addition, we show that the effect of proteasome inhibition on virus infection is not due to increased levels of beta interferon (IFN-ß).

Rotavirus NSP5 orchestrates recruitment of viroplasmic proteins.

Rotavirus genome replication and the first steps of virus morphogenesis take place in cytoplasmic viral factories, called viroplasms, containing four structural (VP1, VP2, VP3 and VP6) and two non-structural (NSP2 and NSP5) proteins. NSP2 and NSP5 have been shown to be essential for viroplasm formation and, when co-expressed in uninfected cells, to form viroplasm-like structures (VLS). In the present work, VLS formation was shown upon co-expression of NSP5 with the core protein VP2 despite the absence of NSP2, indicating a central role for NSP5 in VLS assembly. Since VP2 and NSP2 also induce NSP5 hyperphosphorylation, the possible correlation between VLS formation and the NSP5 phosphorylation status was investigated without evidence of a direct link. In VLS induced by NSP2, the polymerase VP1 was recruited, while the middle layer protein VP6 was not, forming instead tubular structures. On the other hand, VLS induced by VP2 were able to recruit both VP1 and VP6. More importantly, in VLS formed when NSP5 was expressed with both inducers, all viroplasmic proteins were found co-localized, resembling their distribution in viroplasms. Our results suggest a key role for NSP5 in architectural assembly of viroplasms and in recruitment of viroplasmic proteins. A new role for VP2 as an inducer of viroplasms and of NSP5 hyperphosphorylation is also described. These data may contribute to the understanding of rotavirus morphogenesis.

Boosting anti-idiotype immune response with recombinant AAV enhances tumour protection induced by gene gun vaccination.

Thanks to the safety of administration, efficiency of in vivo transduction and persistence of transgene expression, vectors based on the adeno-associated virus (AAV) are extensively utilized in both preclinical and clinical experimentation. Here we thoroughly explore the potential of AAV-mediated antigen delivery for tumour vaccination. A recombinant AAV vector (rAAV) encoding a lymphoma idiotype (Id) in a single-chain variable fragment format was found to induce an efficient anti-Id immune response upon injection in immunocompetent animals. The intensity of the immune response and the protective effect of rAAV administration in vivo were systematically compared with those elicited by simple injection of naked DNA or biolistic immunization. The results indicate that Id delivery via rAAV enhances the intensity of immune response compared with injection of naked DNA, while anti-idiotypic antibodies titres are not considerably increased compared with biolistic vaccination. On the contrary, a prime-boost vaccination strategy combining biolistic and AAV DNA delivery results in a major increase in anti-Id antibody response compared with the repetitive biolistic immunization. This increased anti-Id humoral response strictly correlated with a significant improvement on tumour protection in vivo.

Interaction of rotavirus polymerase VP1 with nonstructural protein NSP5 is stronger than that with NSP2.

Rotavirus morphogenesis starts in intracellular inclusion bodies called viroplasms. RNA replication and packaging are mediated by several viral proteins, of which VP1, the RNA-dependent RNA polymerase, and VP2, the core scaffolding protein, were shown to be sufficient to provide replicase activity in vitro. In vivo, however, viral replication complexes also contain the nonstructural proteins NSP2 and NSP5, which were shown to be essential for replication, to interact with each other, and to form viroplasm-like structures (VLS) when coexpressed in uninfected cells. In order to gain a better understanding of the intermediates formed during viral replication, this work focused on the interactions of NSP5 with VP1, VP2, and NSP2. We demonstrated a strong interaction of VP1 with NSP5 but only a weak one with NSP2 in cotransfected cells in the absence of other viral proteins or viral RNA. By contrast, we failed to coimmunoprecipitate VP2 with anti-NSP5 antibodies or NSP5 with anti-VP2 antibodies. We constructed a tagged form of VP1, which was found to colocalize in viroplasms and in VLS formed by NSP5 and NSP2. The tagged VP1 was able to replace VP1 structurally by being incorporated into progeny viral particles. When applying anti-tag-VP1 or anti-NSP5 antibodies, coimmunoprecipitation of tagged VP1 with NSP5 was found. Using deletion mutants of NSP5 or different fragments of NSP5 fused to enhanced green fluorescent protein, we identified the 48 C-terminal amino acids as the region essential for interaction with VP1.

Rotavirus vp7 antigen produced by Lactococcus lactis induces neutralizing antibodies in mice.

AIMS: To determine if live recombinant Lactococcus lactis strains expressing rotavirus VP7 antigen are immunogenic in mice. METHODS AND RESULTS: Using the food-grade lactic acid bacterium L. lactis as a carrier, we expressed VP7, the major rotavirus outer shell protein and one of the main components of the infective particle, as a cytoplasmic, secreted or cell wall anchored forms. Our results showed that recombinant L. lactis strains secreting VP7 proved to be more immunogenic than strains containing the antigen in the cytoplasm or anchored to the cell wall. CONCLUSIONS: This is the first demonstration that recombinant L. lactis producing VP7 can induce the production of a neutralizing antibody response against rotavirus by the intragastric route. SIGNIFICANCE AND IMPACT OF THE STUDY: Rotaviruses are the single most important aetiological agents of severe diarrhoea of infants and young children worldwide and have been estimated to be responsible for 650 000-800 000 deaths per year of children younger than 5 years old in development countries. Thus, the development of a safe and effective vaccine has been a global public health goal. Although two of five mice orally inoculated with L. lactis strains secreting VP7 elicited a specific-antibody response, these strains could be very useful to be used as a prototype to develop a new generation of protective rotavirus vaccines.

Genetic vaccination for the immunotherapy of B-cell malignancies.

Vaccination protocols based on targeting of the idiotype expressed on malignant B cells have so far provided encouraging results in clinical trials. The essential requirement to induce an immune response is the inclusion of carriers to overcome T-cell tolerance. Chemical cross-linking of idiotypic protein is so far the method of choice to induce protective responses in human studies. Meanwhile, a flurry of alternative strategies to simplify vaccine production is being tested in murine model. Thanks to the advance in antibody engineering the two relevant antigenic domains of the lymphoma immunoglobulin can be assembled into an appropriate format, genetically linked to molecules that act as immunological adjuvants and directly delivered as plasmid DNA. Upon immunization, rejection of tumor cells may depend on cellular or humoral mechanisms, whose relative importance has not been entirely estimated. We have recently analyzed the specificity of anti-idiotypic antibodies induced by DNA vaccination and characterised the elements contributing to optimal anti-idiotypic responses.

Anti-idiotypic antibodies induced by genetic immunisation are directed exclusively against combined V(L)/V(H) determinants.

DNA vaccines encoding the idiotype of immunoglobulins of tumour B cells were shown to induce protection in several mouse lymphoma models. The mechanism of rejection of tumour cells has not been fully understood, but there is strong evidence suggesting that engagement of the idiotype by anti-idiotypic antibodies may directly result in inhibition of tumour growth. In this study, we have investigated the structural basis of the idiotypic/anti-idiotypic interaction following immunisation with DNA vaccines. scFvs containing only one of the two tumour-derived V regions recombined to an irrelevant V region partner were generated. These constructs encoding a secretory form of the scFv were used as immunogens to induce anti-Id antibodies. The same scFvs were expressed as membrane-bound molecules on the surface of mammalian cells. Analysis of immune sera on the membrane-displayed idiotypes revealed that DNA immunisation induced a polyclonal antibody response restricted to conformational combined epitopes formed by the parental V(L)/V(H) association. Immune sera raised by scFv DNA vaccination did not show any detectable reactivity towards chimeric scFvs containing only one of the two immunising V regions, indicating that the response against combined V(L)/V(H)determinants is highly dominant. Remarkably, the same immunogen, delivered as scFv protein, induced antibodies also directed against chain-specific determinants. These findings indicate that presentation of properly folded idiotypes results in a highly specific antibody response directed exclusively to private idiotypic determinants of the V(L)/V(H) combination of the immunogen.

Membrane immunoglobulins are stabilized by interchain disulfide bonds occurring within the extracellular membrane-proximal domain.

Membrane-bound immunoglobulins have, in addition to the transmembrane and cytoplasmic portions, an extracellular membrane-proximal domain (EMPD), absent in the secretory forms. EMPDs of immunoglobulin isotypes alpha, gamma, and epsilon contain cysteines whose role has so far not been elucidated. Using a genetic strategy, we investigated the ability of these cysteines to form disulfide bridges. Shortened versions of human membrane immunoglobulins, depleted of cysteines known to form intermolecular disulfide bonds, were constructed and expressed on the surface of a B-cell line. The resulting membrane proteins contain a single chain fragment of variable regions (scFv) linked to the dimerizing domain from the immunoglobulin heavy chains (CH3 for alpha and gamma or CH4 for epsilon isotypes), followed by the corresponding EMPD and the transmembrane and cytoplasmic domains. The two functional membrane versions of the epsilon chain, containing the short and long EMPD, were analyzed. Our results show that the single cysteine within alpha1L and gamma1 EMPD and the short version of epsilon EMPD form an interchain disulfide bond. Conversely, the cysteine resident in the epsilon transmembrane domain remains unreacted. epsilon-long EMPD contains four cysteines; two are involved in interchain bonds while the remaining two are likely forming an intrachain bridge. Expression of a full-length membrane epsilon heavy chain mutant, in which Cys(121) and Cys(209) within domain CH2 (involved in interchain bridges) were mutated to alanines, confirmed that, within the complete IgE, EMPD cysteines form interchain disulfide bonds. In conclusion, we unveil evidence for additional covalent stabilization of membrane-bound immunoglobulins.

Epitope mapping of trans-sialidase from Trypanosoma cruzi reveals the presence of several cross-reactive determinants.

Trypanosoma cruzi, the agent of Chagas' disease, expresses trans-sialidase, a unique enzyme activity that enables the parasite to invade host cells by transferring sialyl residues from host glyconjugates to the parasite's surface acceptor molecules. The enzyme is also shed into the surrounding environment, causing apoptosis in cells from the immune system. During infections, an antibody response against the catalytic region of the trans-sialidase that is coincident with the control of the parasitemia and survival of the host is observed. This low-titer humoral response is characterized by its persistence for many years in benznidazole-treated patients. Here we analyzed the antigenic structure of the molecule by phage-displayed peptide combinatorial libraries and SPOT synthesis. Several epitopes were defined and located on the three-dimensional model of the enzyme. Unexpectedly, cross-reaction was found among several epitopes distributed in different locations displaying nonconsensus sequences. This finding was confirmed by the reactivity of three monoclonal antibodies able to recognize non-sequence-related peptides that together constitute the surface surrounding the catalytic site of the enzyme. The presence of cross-reacting epitopes within a single molecule suggests a mechanism developed to avoid a strong humoral response by displaying an undefined target to the immune system.

Anti-idiotypic DNA vaccines for lymphoma immunotherapy require the presence of both variable region genes for tumor protection.

Vaccination with immunogenic formulations of lymphoma-derived immunoglobulin can elicit strong anti-idiotypic immune responses which have proved effective in murine B cell tumor challenge experiments and suggested possible benefits in recent human clinical trials. Naked plasmid DNA vaccines encoding the Id determinants as scFv fragments provide the most promising alternative to protein immunization. With this approach the addition of an immunogenic domain linked to the scFv has proved essential for the induction of a protective immune response. In this study we have produced a scFv gene construct linked to the CH3 exon of the human IgG1 constant region and tested its efficacy in inducing protective immunity against the mouse BCL1 lymphoma. We have also generated a second construct in which the BCL1 VL gene was deleted to investigate whether the VH region domain contains sufficient antigenic determinants for a protective immune response. Both constructs induced anti-idiotypic antibodies that specifically reacted with the BCL1 IgM protein in ELISA and with BCL1 tumor cells in flow cytometry assays. Protection against tumor challenge was fully achieved with the complete scFv construct whereas immunization with the construct lacking the VL gene resulted in only a slight prolongation of the survival. We therefore conclude that a plasmid DNA vaccine containing the VH and VL genes of the lymphoma Ig linked to the human IgG1 CH3 exon is highly effective in inducing a protective immune response in the BCL1 model. We also demonstrated that VH gene immunization can induce strong anti-idiotypic antibody responses.

Molecular cloning and chimerisation of CDI 315B monoclonal antibody.

A chimeric mouse-human antibody has been created that recognizes an antigen found on breast cancer cells and melanoma cells. Immunoglobulin constant domains of mouse monoclonal antibody CDI 315B Cgamma1 and CK, were substituted by the human Cgamma1 and Ckappa. The CDI 315B variable heavy and light chain regions were PCR amplified from hybridoma RNA and sequenced. Mouse variable VH and VL regions were joint to human IgG1 and kappa constant regions and subcloned into pcDNA3 expression vectors. The Sp2/0 murine myeloma cells were transfected with expression vectors pcDNA3L and pcDNA3H and the reactivity of chimeric antibodies was tested by indirect ELISA using B16F1 murine melanoma cells as well as MCF7 human breast cancer cells, as antigen.

Ethnic difference in the prevalence of monoclonal B-cell proliferation in patients affected by hepatitis C virus chronic liver disease.

BACKGROUND/AIM: In previous studies we demonstrated that all patients affected by HCV-positive type II mixed cryoglobulinaemia have a monoclonal B-cell population in peripheral blood mononuclear cells, and that a large fraction of HCV-infected patients develop a monoclonal B-cell expansion, even in the absence of dosable serum cryoglobulins. However, the prevalence of Type II mixed cryoglobulinaemia in HCV-infected individuals seems to be high in Italy, whereas it is very low in Japan. This study was performed to investigate whether there are ethnic differences in the prevalence of asymptomatic HCV-associated monoclonal B-cell expansions. METHODS: Forty-four Japanese patients affected by HCV-positive chronic liver disease (two healthy carriers, 31 chronic hepatitis and 11 cirrhosis) were compared with a group of 60 Italian patients (one healthy carrier, 49 chronic hepatitis, and 10 cirrhosis) without dosable levels of cryoglobulins. The monoclonality of peripheral blood mononuclear cells was investigated by RT/PCR analysis of Immunoglobulin gene rearrangements. Liver function tests, rheumatoid factor, cryocrit level, anti-HCV antibodies, HCV-RNA, and HCV genotype were performed according to standard methodology. RESULTS: A B-cell monoclonal population was found in 26% of Italian patients, whereas all Japanese patients were negative. No correlation was found between B-cell monoclonality and severity of liver disease, length or source of the infection, HCV genotype, sex, clinical and biochemical parameters. CONCLUSIONS: This study indicates that a monoclonal B-cell proliferation in peripheral blood mononuclear cells is common in HCV infection, but only in Italy, whereas it is absent in Japan. This explains the very low prevalence of Type II mixed cryoglobulinaemia in HCV-positive Japanese subjects, and suggests that HCV is able to determine a B-cell expansion only in the presence of, presently undetermined, host factors.

Sequence-specific antibodies against human IgE isoforms induced by an epitope display system.

BACKGROUND: Unlike other immunoglobulin isotypes, the human C epsilon gene generates by alternative splicing two types of secretory and two types of membrane epsilon chains. The two secreted epsilon heavy chains, epsilon(S1) and epsilon(S2), differ only in the sequence of the last eight C-terminal amino acids, being epsilon(S2) six amino acids longer. The two types of membrane isoforms differ in the extracellular membrane proximal domain, with the longer variant, epsilon(mL), containing 52 extra amino acids which are absent in the shorter epsilon(mS) isoform. OBJECTIVES: We wished to produce quality antibody reagents that specifically detect epitopes that are epsilon isoform-specific. STUDY DESIGN: Short sequences of seven or ten amino acids were chosen as target epitopes and expressed as part of the highly immunogenic loops of deletion variants of engineered Flock House Virus capsid protein RNA2. Chimeric proteins were expressed in E. coli, and used to immunize rabbits. Antisera were screened by immunoblotting of purified IgE isoforms expressed by murine transfectomas. RESULTS: Chimeric proteins expressing epsilon isoform-specific epitopes proved to be strong immunogens in vivo and induced highly specific rabbit antisera. Two antisera so obtained recognize specifically the IgE-S2 isoform. A third one recognizes the long membrane variant m(L)IgE and a fourth one detects an epitope specific to m(S)IgE. CONCLUSION: Here we describe a simplified and efficient protocol of immunization which does not require peptide synthesis and conjugation to carrier protein. Our results show that short peptides of unknown immunogenicity, when genetically introduced into the modified Flock House Virus epitope display system, successfully induced IgE isoform-specific polyclonal antisera in rabbits. These are valuable tools to specifically identify secretory and membrane isoforms of human IgE, and the method is potentially applicable to other variant isoforms or mutants of a given protein.