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.

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.

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.

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.

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).

The two membrane isoforms of human IgE assemble into functionally distinct B cell antigen receptors.

The human C epsilon gene expresses two membrane IgE heavy chain mRNAs which differ in the sequence that encodes their extracellular membrane-proximal domain. In the long IgE isoform (mLIgE), this domain contains a stretch of 52 amino acids which are absent in the short variant (mSIgE). We have now generated B cell transfectoma cell lines that express these two isoforms and show that both types of mIgE form functional B cell antigen receptors (BCR). Both receptors associate with the Ig-alpha/Ig-beta heterodimer, as well as with protein kinases that are capable of phosphorylating this complex. Upon their cross-linking, both receptors can activate protein tyrosine kinases that phosphorylate the same substrate proteins. Both IgE receptors also associate with two novel proteins that do not bind to mIgM. Apart from these similarities, the two IgE-BCRs show several differences of which some are analogous to the differences between the IgM- and IgD-BCRs. First, the mSIgE is transported to the cell surface at a higher rate than the mLIgE. Second, the two IgE-BCRs associate with differently glycosylated Ig-alpha proteins, the mLIgE associates with the completely glycosylated form, whereas the mSIgE associates with an Ig-alpha glycoform that is partially sensitive to endoglycosidase H. Third, the kinetics of protein tyrosine phosphorylation induced by receptor cross-linking is significantly different for the two IgE-BCRs. Finally, cross-linking of the mSIgE-BCR leads to growth inhibition of the B cell transfectoma, whereas signaling through the mLIgE-BCR does not affect the cellular proliferation. These data show that the two human membrane IgE isoforms assemble into functionally distinct antigen receptors which can induce different cellular responses.

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.

IgM-producing chronic lymphocytic leukemia cells undergo immunoglobulin isotype-switching without acquiring somatic mutations.

The malignant B cells in chronic lymphocytic leukemia (CLL) typically express low-density membrane IgM or IgM/IgD. In vitro experiments have shown that the CLL cells can be induced to differentiate into cells that secrete immunoglobulin (Ig) and can occasionally undergo heavy (H) chain class switching. We now show that the CLL cells also undergo isotype-switching in vivo, since gamma and/or alpha H chain transcripts with identical FW3/CDR3/FW4 regions as the mu CLL transcripts were detected in all of the 13 investigated patients with IgM+ CLL. In most cases switching had occurred to alpha1 and gamma3, but CLL transcripts corresponding to the other gamma chain isotypes were also detected. In one case both the productively and nonproductively rearranged allele were found to undergo H chain class switching. CLL gamma transcripts were also present in surface IgG+ sorted B cells, demonstrating that a small subset of the CLL cells express membrane IgG. In addition, transcripts encoding secretary gamma2 and gamma3 H chains were detected in two cases, which suggests that some serum IgG could be produced by the leukemic clone. Analysis of sorted PBL showed that isotype-switching occurs in CLL cells that express the CD5 antigen. Finally, nucleotide sequence analysis showed that the mu, alpha, and gamma CLL transcripts are identical, demonstrating that the CLL cells do not accumulate somatic mutations in their variable region genes after the H chain class switching. These data provide in vivo evidence that isotype-switching is a frequent phenomenon in CLL, and indicate that a subset of the CLL lymphocytes progress to later stages of B cell differentiation.

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.

Multiple transcripts of the murine immunoglobulin epsilon membrane locus are generated by alternative splicing and differential usage of two polyadenylation sites.

The human C epsilon gene produces a number of alternatively spliced heavy chain transcripts of which some encode functional IgE isoforms. We now show that differentially processed epsilon mRNA variants also exist in the mouse and are generated by differential polyadenylation and alternative splicing of primary epsilon chain transcripts. The two poly(A) sites of the mouse membrane transcripts were identified in the present study by RACE-PCR analysis. The first poly(A) site is located 743 nt downstream from the beginning of the second membrane exon (M2) and contains the same non-consensus AGTAAA signal sequence as the single poly(A) site of the human membrane transcripts. The second poly(A) site is located almost 500nt further downstream and is characterized by an AAGAAA hexamer. This poly(A) site contains a (G+T) rich element downstream to the site of cleavage and polyadenylation and is preferentially utilized by the membrane epsilon transcripts. Additional diversity of epsilon transcripts is generated by alternative splicing between the last constant region exon (CH4) and the two membrane exons (M1 and M2). The alternatively spliced transcripts include two variants that skip the first membrane exon and encode epsilon heavy chains that lack the transmembrane domain. The third variant is generated by splicing to an internal site in M2 and codes for a membrane isoform that is 10 amino acids shorter in the cytoplasmic domain than the classical membrane IgE. Although little amino-acid sequence homology exists between the murine epsilon chain isoforms and their human counterparts, the pattern of splicing is rather conserved between the two species.

A novel mechanism of resistance to alpha-difluoromethylornithine induced by cycloheximide. Growth with abnormally low levels of putrescine and spermidine.

Treatment of the chemically transformed fibroblasts BP-A31 and other cell lines with low concentrations of cycloheximide (CHM) for 72 h followed by the removal of the protein synthesis inhibitor leads to the proliferation of alpha-difluoromethylornithine (DFMO)-resistant phenotypes. These drug-resistant cells contain almost no ornithine decarboxylase (ODC) activity and concomitantly very low levels of putrescine and spermidine. Southern blot analysis and measurements of ODC activity and intracellular polyamine levels showed that the described mechanism of inducing resistance to DFMO triggered by CHM does not involve ODC gene amplification, altered transport of the drug or reduced affinity of the enzyme for DFMO.

Identification of toxigenic Vibrio cholerae from the Argentine outbreak by PCR for ctx A1 and ctx A2-B.

A polymerase chain reaction (PCR) to detect a region of the A1 cholera toxin gene was applied to the identification of 43 Vibrio cholerae strains isolated from the recent outbreak in Argentina. A good correlation was observed between the GM1-enzyme-linked immunosorbent assay (GM1-ELISA) to detect the B subunit of the enterotoxin and PCR. However, a V. cholerae non-01 strain that was negative by the ELISA test, was positive by the PCR assay for the A1 region. A second PCR test to detect the A2-B coding region was developed to solve this case. We propose that routine detection of toxigenic V. cholerae by PCR should include analysis of A2-B coding region or the whole cholera toxin operon.

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.

Sequence diversity in the kinetoplast DNA minicircles of Trypanosoma cruzi.

Minicircles are the most abundant component of the mitochondrially located kinetoplast DNA in the members of the order Kinetoplastida. Minicircle sequences differ among most trypanosomatid species. To learn about the molecular mechanisms that give rise to this diversity, we sequenced a complete minicircle (pTckAWP-2) and two homologous but polymorphic minicircle fragments isolated from different Trypanosoma cruzi clones. Comparison of these sequences revealed 23 point mutations, 19 of which were transitions. A single base pair insertion was also detected in one of the two minicircle fragments sequenced. Analysis of pTckAWP-2 sequence showed the following features: the presence of four internal 118 base pairs conserved regions with 80% or higher homology; the fact that these four conserved regions also differed mainly by point mutations, although in this case a bias in favor of transversions was observed; the existence in each of these four regions of the highly conserved 13 bp sequence 5'GGGGTTGGTGTAA3', detected in all trypanosomatid minicircles, which is thought to be the origin of replication; and the presence of several direct and inverted repeat sequences of 8 base pairs or longer, scattered throughout the minicircle molecule. Comparison of the T. cruzi conserved minicircle region with that of other trypanosomatids showed a higher homology of T. cruzi with T. lewisi, another stercorarian trypanosome, than with African trypanosomes or Leishmania.

Expression of CD44 splice variants in metastatic and non-metastatic mouse tumour cell lines.

Different splice variants of the CD44 cell-surface molecule have been linked to metastasis formation in several animal and human cancers. We have used metastatic CSML-100 and non-metastatic CSML-0 mouse adenocarcinoma cell lines to determine whether variant CD44 molecules could be implicated in the different behaviour of these cells. Two CD44 splice variants containing exons v7-v10 and v8-v10 were detected in the non-metastatic CSML-0. Two other mouse cell lines, the normal mammary gland NMuMG and the mammary pre-neoplastic CL-S1 were also found to express these exons. A short (hematopoietic) CD44 isoform was expressed in the metastatic CSML-100 and three other mouse mammary tumour cell lines. Overexpression of v7-v10 and v8-v10 CD44 variants in CSML 100 cells did not decrease their metastatic potential.

The pathologic significance of the immunoglobulins expressed by chronic lymphocytic leukemia B-cells in the development of autoimmune hemolytic anemia.

The increased number of CD5+ B-cells in some human autoimmune diseases, the frequent commitment of CD5+ B-cells to the production of natural autoantibodies, and the apparent involvement of these cells in the pathogenesis of the autoimmune hemolytic anemia (AIHA) in certain mouse models suggests a causal relationship between the CD5+ chronic lymphocytic leukemia (CLL) B-cell and the AIHA which frequently develops in this malignant disorder. In support of this conclusion is our recent finding that the VH region gene repertoire of the leukemic B-cells from CLL patients with AIHA is rather biased and characterised by the over-representation of the 51p1 VH gene. On the other hand, it appears relatively certain that the pathogenic anti-erythrocyte antibodies in CLL patients with AIHA are produced by remnant normal B-cells, and that the antibodies expressed by the leukemic CD5+ B-cells do not directly bind red blood cells (RBC). Of interest, the antibodies produced by the leukemic B-cells from CLL patients with AIHA might have in common rheumatoid factor (RF) activity. These data indicate that the antibodies produced by the leukemic B-cells from CLL patients with AIHA are not directly involved in red blood cell destruction, but may be involved in the induction or amplification of a polyclonal anti-RBC response. Finally, we discuss the possible clinical implications of our finding that CLL patients with leukemic cells expressing the 51p1 VH gene may be at a higher risk to develop autoimmune hemolytic anemia.

Hepatitis C virus and non-Hodgkin's lymphomas.

The etiology of non-Hodgkin's lymphomas (NHL) remains a controversial matter, but, in the last few years, considerable evidence suggests that aberrations of the immune system and viruses may act as etiologic agents, in at least some cases of NHL. In fact, patients with primary immuno-deficiencies, or those suffering from diseases characterized by autoimmune dysfunction, show an increased risk for the development of NHL. Several viruses have been identified as possible etiologic agents for NHL; one of the best studied is the Epstein-Barr virus, which was detected in cultures of tumor cells from patients with Burkitt's lymphoma. The pathogenetic potential of this virus is illustrated by its association with an increasing number of malignant diseases. In addition, the human T-cell lymphotropic virus family (HTLV), was also recognized as possible etiologic agents for several lymphomas, such as cutaneous T-cell lymphoma and T-cell leukemia-lymphoma syndrome (HTLV-I), and T-cell hairy cell leukemia (HTLV-II). Recently, the presence of hepatitis C virus infection has also been recognized in several hematological malignancies such as mixed cryoglobulinemia, low-grade malignant lymphomas and Waldenström's disease. The possible etiopathogenetic role of this virus in non-Hodgkin's lymphomas is discussed on the basis of molecular, clinical, and epidemiological considerations.