Transcriptome of human neuroblastoma SH-SY5Y cells in response to 2B protein of enterovirus-A71.

Infection with enterovirus-A71 (EV-A71) can cause hand-foot-mouth disease associated with fatal neurological complications. The host response to EV-A71 has not yet been fully elucidated, thus, hampering the development of a precise therapeutic approach. A nonstructural 2B protein of EV-A71 has been reported to involve with calcium dysregulation and apoptosis induction in human neuroblastoma SH-SY5Y cells. However, the molecular mechanism has not been delineated. To address this, comprehensive study of the gene expression from SH-SY5Y cells transfected with EV-A71 2B was carried out by RNA sequencing and transcriptomic analysis. It was found that the signature of the upregulated genes of SH-SY5Y cells expressing EV-A71 2B involved the Ca2+-related signaling pathways participating gene expression, inflammatory response, apoptosis, and long-term potentiation of the neuron. Protein-protein interaction network analysis revealed that the products encoded by CCL2, RELB, BIRC3, and TNFRSF9 were the most significant hub proteins in the network. It indicated that EV-A71 2B protein might play a role in immunopathogenesis of the central nervous system (CNS) which probably associated with the non-canonical NF-κB pathway. The data suggest that transcriptomic profiling can provide novel information source for studying the neuropathogenesis of EV-A71 infection leading to development of an effective therapeutic measure for CNS complications.

Role of Chikungunya nsP3 in Regulating G3BP1 Activity, Stress Granule Formation and Drug Efficacy.

BACKGROUND: Ras-GTPase activating protein SH3-domain-binding proteins (G3BP) are a small family of RNA-binding proteins implicated in regulating gene expression. Changes in expression of G3BPs are correlated to several cancers including thyroid, colon, pancreatic and breast cancer. G3BPs are important regulators of stress granule (SG) formation and function. SG are ribonucleoprotein (RNP) particles that respond to cellular stresses to triage mRNA resulting in transcripts being selectively degraded, stored or translated resulting in a change of gene expression which confers a survival response to the cell. These changes in gene expression contribute to the development of drug resistance. Many RNA viruses, including Chikungunya (and potentially Coronavirus), dismantle SG so that the cell cannot respond to the viral infection. Non-structural protein 3 (nsP3), from the Chikungunya virus, has been shown to translocate G3BP away from SG. Interestingly in cancer cells, the formation of SG is correlated to drug-resistance and blocking SG formation has been shown to reestablish the efficacy of the anticancer drug bortezomib. METHODS: Chikungunya nsP3 was transfected into breast cancer cell lines T47D and MCF7 to disrupt SG formation. Changes in the cytotoxicity of bortezomib were measured. RESULTS: Bortezomib cytotoxicity in breast cancer cell lines changed with a 22 fold decrease in its IC50 for T47D and a 7 fold decrease for MCF7 cells. CONCLUSIONS: Chikungunya nsP3 disrupts SG formation. As a result, it increases the cytotoxicity of the FDA approved drug, bortezomib. In addition, the increased cytotoxicity appears to correlate to improved bortezomib selectivity when compared to control cell lines.

Bovine serum albumin nanoparticles induce histopathological changes and inflammatory cell recruitment in the skin of treated mice.

Albumin is a natural, biocompatible, biodegradable and nontoxic polymer and due to these features, nanoparticles made of albumin are a good system for drug or antigen delivery. Polymeric nanoparticles are being widely explored as new vaccines platforms due to the capacity of those nanoparticles to prime the immune system by providing sustained release of the antigen after injection. Biodegradable nanoparticles associated with proteins represent a promising method for in vivo delivery of vaccines. In our previous studies, bovine serum albumin nanoparticles (BSA-NPs) were identified as a promising system for in vivo delivery of microbial antigens. The aim of this work was to show the effect of BSA-NPs on skin after nanoparticles administration. The pro-inflammatory activity of BSA-NPs was evaluated using in vivo models. BSA-NPs are easily uptake by macrophagic RAW 264.7 and BHK-21 cells without any significant cytotoxicity. Histological examination of skin sections from BSA-NPs-treated mice revealed intense cellular infiltration, increased skin thickness, follicular hypertrophy, vascular congestion and marked collagenesis. Mice immunized with recombinant non-structural protein 1 (rNS1) from Dengue virus 1 and BSA-NPs showed a high seroconversion rate if compared to animals immunized only with rNS1. Therefore, the effect of BSA-NPs on skin after BSA-NPs administration has a biotechnological relevance to the rational design of vaccine formulations based on albumin nanocarriers. However in the next years future studies should be carried out to best characterize the effect of BSA-NPs on dendritic cells and establish the role of these nanoparticles as a new vaccine platform for infectious diseases or cancer.

Design and testing of a highly conserved human rotavirus VP8* immunogenic peptide with potential for vaccine development.

Rotavirus infection of young children particularly below five years of age resulting in severe diarhoea, is the cause of a large number of infant deaths all over the world, more so in developing countries like India. Vaccines developed against this infection in the last two decades have shown mixed results with some of them leading to complications. Oral vaccines have not been very effective in India. Significant diversity has been found in circulating virus strains in India. Development of a vaccine against diverse genetic variants of the different strains would go a long way in reducing the incidence of infection in developing countries. Success of such a vaccine would depend to a large extent on the antigenic peptide to be used in antibody production. The non-glycosylated protein VP4 on the surface capsid of the virus is important in rota viral immunogenicity and the major antigenic site(s) responsible for neutralization of the virus via VP4 is in the VP8* subunit of VP4. It is necessary that the peptide should be very specific and a peptide sequence which would stimulate both the T and B immunogenic cells would provide maximum protection against the virus. Advanced computational techniques and existing databases of sequences of the VP4 protein of rotavirus help in identification of such specific sequences. Using an in silico approach we have identified a highly conserved VP8* subunit of the VP4 surface protein of rotavirus which shows both T and B cell processivity and is also non-allergenic. This sub-unit could be used in in vivo models for induction of antibodies.

Development of Monoclonal Antibodies Specifically Recognizing the Nonstructural Protein 12 of Type 2 Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens that has caused tremendous economic losses to the swine industry worldwide. Although extensive research has been focused on PRRSV, little is known about the structure and biological functions of individual nonstructural viral proteins, especially the nonstructural protein 12 (Nsp12). In this study, we generated and identified the monoclonal antibody (mAb) against PRRSV Nsp12. Six strains of hybridoma cells named 2B10, 2B12, 5E1, 5G6, 5E7, and 8B2 secreting anti-Nsp12 mAbs were obtained by the hybridoma technique. All the mAbs were specifically reacted with PRRSV by indirect immunofluorescence assay and four of them (2B12, 5E1, 5G6, and 5E7) were specifically reacted by Western blot. Furthermore, the 5E7 specifically recognized multiple type 2 PRRSV strains, including highly pathogenic and classical PRRSV strains, but not type 1 PRRSV strain. Taken together, the mAbs against Nsp12 provide a valuable tool to specifically recognize type 2 PRRSV as a diagnostic reagent and study the biological function of Nsp12 in the future.

Canine Parvovirus ns1 gene and Chicken Anemia vp3 gene induce partial oncolysis of Canine Transmissible Venereal Tumor.

The oncolytic effect of Canine Parvovirus ns1 gene and Chicken Anemia vp3 gene in naturally occurring cases of Canine Transmissible Venereal Tumor (CTVT) is being reported. Dogs suffering from CTVT (N = 18) were systematically randomized into three groups viz. A, B, and C (n = 6). Animals of the groups A, B, and C received 100 µg of the ns1 gene, vp3 gene, and ns1 + vp3 gene combination, respectively, for three weeks intratumorally at weekly intervals; results were normalized against base values before commencement of therapy and after complete remission that were taken as negative and positive controls, respectively. Initiation of oncolytic gene therapy arrested the further progression of the tumor but most of the animals in the study underwent incomplete remission, indicating incomplete activity of ns1 and vp3 genes. The oncolytic effect of the treatments was in the order ns1 > vp3 > ns1 + vp3. Oncolysis was accompanied by decreased mitotic index and AgNOR count, and increased TUNEL positive cells and CD4+ lymphocyte counts. Our findings show that Canine Parvovirus ns1 may eventually find an important role as an oncolytic agent.

Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen.

Dengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed in Escherichia coli at large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses.

Priming with two DNA vaccines expressing hepatitis C virus NS3 protein targeting dendritic cells elicits superior heterologous protective potential in mice.

Development an effective vaccine may offer an alternative preventive and therapeutic strategy against HCV infection. DNA vaccination has been shown to induce robust humoral and cellular immunity and overcome many problems associated with conventional vaccines. In this study, mice were primed with either conventional pVRC-based or suicidal pSC-based DNA vaccines carrying DEC-205-targeted NS3 antigen (DEC-NS3) and boosted with type 5 adenoviral vectors encoding the partial NS3 and core antigens (C44P). The prime boost regimen induced a marked increase in antigen-specific humoral and T-cell responses in comparison with either rAd5-based vaccines or DEC-205-targeted DNA immunization in isolation. The protective effect against heterogeneous challenge was correlated with high levels of anti-NS3 IgG and T-cell-mediated immunity against NS3 peptides. Moreover, priming with a suicidal DNA vaccine (pSC-DEC-NS3), which elicited increased TNF-α-producing CD4+ and CD8+ T-cells against NS3-2 peptides (aa 1245-1461), after boosting, showed increased heterogeneous protective potential compared with priming with a conventional DNA vaccine (pVRC-DEC-NS3). In conclusion, a suicidal DNA vector (pSC-DEC-NS3) expressing DEC-205-targeted NS3 combined with boosting using an rAd5-based HCV vaccine (rAd5-C44P) is a good candidate for a safe and effective vaccine against HCV infection.

[Immunogenicity and heterologous protection in mice with a recombinant adenoviral-based vaccine carrying a hepatitis C virus truncated NS3 and core fusion protein].

To develop a safe and broad-spectrum effective hepatitis C virus (HCV) T cell vaccine,we constructed the recombinant adenovirus-based vaccine that carried the hepatitis C virus truncated NS3 and core fusion proteins. The expression of the fusion antigen was confirmed by in vitro immunofluorescence and western blotting assays. Our results indicated that this vaccine not only stimulated antigen-specific antibody responses,but also activated strong NS3-specific T cell immune responses. NS3-specific IFN-γ+ and TNF-α+ CD4+ T cell subsets were also detected by a intracellular cytokine secretion assay. In a surrogate challenge assay based on a recombinant heterologous HCV (JFH1,2a) vaccinia virus,the recombinant adenovirus-based vaccine was capable of eliciting effective levels of cross-protection. These findings have im- portant implications for the study of HCV immune protection and the future development of a novel vaccine.

A Multiantigenic DNA Vaccine That Induces Broad Hepatitis C Virus-Specific T-Cell Responses in Mice.

UNLABELLED: There are 3 to 4 million new hepatitis C virus (HCV) infections annually around the world, but no vaccine is available. Robust T-cell mediated responses are necessary for effective clearance of the virus, and DNA vaccines result in a cell-mediated bias. Adjuvants are often required for effective vaccination, but during natural lytic viral infections damage-associated molecular patterns (DAMPs) are released, which act as natural adjuvants. Hence, a vaccine that induces cell necrosis and releases DAMPs will result in cell-mediated immunity (CMI), similar to that resulting from natural lytic viral infection. We have generated a DNA vaccine with the ability to elicit strong CMI against the HCV nonstructural (NS) proteins (3, 4A, 4B, and 5B) by encoding a cytolytic protein, perforin (PRF), and the antigens on a single plasmid. We examined the efficacy of the vaccines in C57BL/6 mice, as determined by gamma interferon enzyme-linked immunosorbent spot assay, cell proliferation studies, and intracellular cytokine production. Initially, we showed that encoding the NS4A protein in a vaccine which encoded only NS3 reduced the immunogenicity of NS3, whereas including PRF increased NS3 immunogenicity. In contrast, the inclusion of NS4A increased the immunogenicity of the NS3, NS4B, andNS5B proteins, when encoded in a DNA vaccine that also encoded PRF. Finally, vaccines that also encoded PRF elicited similar levels of CMI against each protein after vaccination with DNA encoding NS3, NS4A, NS4B, and NS5B compared to mice vaccinated with DNA encoding only NS3 or NS4B/5B. Thus, we have developed a promising "multiantigen" vaccine that elicits robust CMI. IMPORTANCE: Since their development, vaccines have reduced the global burden of disease. One strategy for vaccine development is to use commercially viable DNA technology, which has the potential to generate robust immune responses. Hepatitis C virus causes chronic liver infection and is a leading cause of liver cancer. To date, no vaccine is currently available, and treatment is costly and often results in side effects, limiting the number of patients who are treated. Despite recent advances in treatment, prevention remains the key to efficient control and elimination of this virus. Here, we describe a novel DNA vaccine against hepatitis C virus that is capable of inducing robust cell-mediated immune responses in mice and is a promising vaccine candidate for humans.

Transcriptome analysis of rainbow trout in response to non-virion (NV) protein of viral haemorrhagic septicaemia virus (VHSV).

The non-virion (NV) protein of viral haemorrhagic septicaemia virus (VHSV), an economically important fish novirhabdovirus, has been implicated in the interference of some host innate mechanisms (i.e. apoptosis) in vitro. This work aimed to characterise the immune-related transcriptome changes in rainbow trout induced by NV protein that have not yet been established in vivo. For that purpose, immune-targeted microarrays were used to analyse the transcriptomes from head kidney and spleen of rainbow trout (Oncorhynchus mykiss) after injection of recombinant NV (rNV). Results showed the extensive downregulation (and in some cases upregulation) of many innate and adaptive immune response genes not related previously to VHSV infection. The newly identified genes belonged to VHSV-induced genes (vigs), tumour necrosis factors, Toll-like receptors, antigen processing and presentation, immune co-stimulatory molecules, interleukins, macrophage chemotaxis, transcription factors, etc. Classification of differentially downregulated genes into rainbow trout immune pathways identified stat1 and jun/atf1 transcription factor genes as the most representative of the multipath gene targets of rNV. Altogether, these results contribute to define the role and effects of NV in trout by orchestrating an immunosuppression of the innate immune responses for favouring viral replication upon VHSV infection. Finally, these transcriptome results open up the possibility to find out new strategies against VHSV and better understand the interrelationships between some immune pathways in trout.

Strong vaccine-induced CD8 T-cell responses have cytolytic function in a chimpanzee clearing HCV infection.

A single correlate of effective vaccine protection against chronic HCV infection has yet to be defined. In this study, we analyzed T-cell responses in four chimpanzees, immunized with core-E1-E2-NS3 and subsequently infected with HCV1b. Viral clearance was observed in one animal, while the other three became chronically infected. In the animal that cleared infection, NS3-specific CD8 T-cell responses were observed to be more potent in terms of frequency and polyfunctionality of cytokine producing cells. Unique to this animal was the presence of killing-competent CD8 T-cells, specific for NS3 1258-1272, being presented by the chimpanzee MHC class I molecule Patr-A*03∶01, and a high affinity recognition of this epitope. In the animals that became chronically infected, T-cells were able to produce cytokines against the same peptide but no cytolysis could be detected. In conclusion, in the animal that was able to clear HCV infection not only cytokine production was observed but also cytolytic potential against specific MHC class I/peptide-combinations.

Amino acids 78 and 79 of Mammalian Orthoreovirus protein µNS are necessary for stress granule localization, core protein λ2 interaction, and de novo virus replication.

At early times in Mammalian Orthoreovirus (MRV) infection, cytoplasmic inclusions termed stress granules (SGs) are formed as a component of the innate immune response, however, at later times they are no longer present despite continued immune signaling. To investigate the roles of MRV proteins in SG modulation we examined non-structural protein µNS localization relative to SGs in infected and transfected cells. Using a series of mutant plasmids, we mapped the necessary µNS residues for SG localization to amino acids 78 and 79. We examined the capacity of a µNS(78-79) mutant to associate with known viral protein binding partners of µNS and found that it loses association with viral core protein λ2. Finally, we show that while this mutant cannot support de novo viral replication, it is able to rescue replication following siRNA knockdown of µNS. These data suggest that µNS association with SGs, λ2, or both play roles in MRV replication.

Effects of route of inoculation and viral genetic variation on antibody responses to polyomavirus SV40 in Syrian golden hamsters.

Genetic variants of polyomavirus SV40 are powerful agents with which to define viral effects on cells and carcinogenesis pathways. We hypothesized that differences in biologic variation among viral strains affect the process of viral infection and are reflected in antibody responses to the viral nonstructural large T-antigen (TAg) protein but not in neutralizing antibody responses against the inoculated viral particles. We analyzed the production of TAg antibody and neutralizing antibody in Syrian golden hamsters that were inoculated with SV40 viral strains by intracardiac, intravenous, or intraperitoneal routes and remained tumor free. Compared with the intraperitoneal route, intravascular (that is, intravenous, intracardiac) inoculation resulted in increased frequency of responsiveness to TAg but not in higher TAg antibody titers. The intravascular route was superior both for eliciting neutralizing antibody responses and for higher titers of those responses. Viruses with complex regulatory regions induced TAg antibody more often than did viruses with simple regulatory regions after intraperitoneal but not intravascular injections, with no differences in antibody titers. This viral genetic variation had no effect on neutralizing antibody production after intraperitoneal or intravascular inoculations or on neutralizing antibody titers achieved. These findings confirm that SV40 variants differ in their biologic properties. Route of inoculation combined with viral genetic variation significantly influence the development of serum antibodies to SV40 TAg in tumor-free hamsters. Route of inoculation-but not viral genetic variation-is an important factor in production of neutralizing antibody to SV40.

Enhanced and sustained CD8+ T cell responses with an adenoviral vector-based hepatitis C virus vaccine encoding NS3 linked to the MHC class II chaperone protein invariant chain.

Potent and broad cellular immune responses against the nonstructural (NS) proteins of hepatitis C virus (HCV) are associated with spontaneous viral clearance. In this study, we have improved the immunogenicity of an adenovirus (Ad)-based HCV vaccine by fusing NS3 from HCV (Strain J4; Genotype 1b) to the MHC class II chaperone protein invariant chain (Ii). We found that, after a single vaccination of C57BL/6 or BALB/c mice with Ad-IiNS3, the HCV NS3-specific CD8(+) T cell responses were significantly enhanced, accelerated, and prolonged compared with the vaccine encoding NS3 alone. The AdIiNS3 vaccination induced polyfunctional CD8(+) T cells characterized by coproduction of IFN-γ, TNF-α and IL-2, and this cell phenotype is associated with good viral control. The memory CD8(+) T cells also expressed high levels of CD27 and CD127, which are markers of long-term survival and maintenance of T cell memory. Functionally, the AdIiNS3-vaccinated mice had a significantly increased cytotoxic capacity compared with the AdNS3 group. The AdIiNS3-induced CD8(+) T cells protected mice from infection with recombinant vaccinia virus expressing HCV NS3 of heterologous 1b strains, and studies in knockout mice demonstrated that this protection was mediated primarily through IFN-γ production. On the basis of these promising results, we suggest that this vaccination technology should be evaluated further in the chimpanzee HCV challenge model.

A novel combined vaccine candidate containing epitopes of HCV NS3, core and E1 proteins induces multi-specific immune responses in BALB/c mice.

Hepatitis C virus (HCV) has emerged as the major pathogen of liver disease worldwide. The mechanisms of HCV infection and interaction with a host are poorly understood. What exactly is required for efficient control of HCV infection is largely unknown. Standard treatment combining interferon-alpha (IFN-alpha) and ribavirine is effective in about 50% of the treated patients, however associated with significant toxicity and cost. Therefore, the development of new drugs or vaccines is urgently needed. An efficient vaccine against HCV infection requires induction of broad cellular and humoral immune responses against several viral proteins. We have engineered the combined vaccine candidate mT+mE1, an inclusion of multiple epitopes from HCV NS3, core (C) and E1 proteins. mT contains multiple conserved CD4(+) and CD8(+) T cell epitopes from HCV NS3 and C proteins. mE1 is based on eight dominant neutralizing epitopes of E1 protein from six HCV genotypes. In current study, we showed that immunization with mT+mE1 induced high titers of IgG, IgG1 and IgG2a antibodies to mE1, and high level of NS3- or C-specific CTLs. Furthermore, mT+mE1 elicited a Th1-biased immune response with secretion of high amounts of IFN-gamma, compared with mT alone. Prophylactic as well as therapeutic administration of mT+mE1 in BALB/c mice led to protecting mice against SP2/0 tumor cells expressing HCV NS3 protein. These results suggested that mT+mE1 elicited strong humoral immune responses and multiple specific cellular immune responses. The vaccine candidate is now being tested in pre-clinical trials.

Modulation of the immune response induced by gene electrotransfer of a hepatitis C virus DNA vaccine in nonhuman primates.

Induction of multispecific, functional CD4+ and CD8+ T cells is the immunological hallmark of acute self-limiting hepatitis C virus (HCV) infection in humans. In the present study, we showed that gene electrotransfer (GET) of a novel candidate DNA vaccine encoding an optimized version of the nonstructural region of HCV (from NS3 to NS5B) induced substantially more potent, broad, and long-lasting CD4+ and CD8+ cellular immunity than naked DNA injection in mice and in rhesus macaques as measured by a combination of assays, including IFN-gamma ELISPOT, intracellular cytokine staining, and cytotoxic T cell assays. A protocol based on three injections of DNA with GET induced a substantially higher CD4+ T cell response than an adenovirus 6-based viral vector encoding the same Ag. To better evaluate the immunological potency and probability of success of this vaccine, we have immunized two chimpanzees and have compared vaccine-induced cell-mediated immunity to that measured in acute self-limiting infection in humans. GET of the candidate HCV vaccine led to vigorous, multispecific IFN-gamma+CD8+ and CD4+ T lymphocyte responses in chimpanzees, which were comparable to those measured in five individuals that cleared spontaneously HCV infection. These data support the hypothesis that T cell responses elicited by the present strategy could be beneficial in prophylactic vaccine approaches against HCV.

Primary and memory T cell responses induced by hepatitis C virus multiepitope long peptides.

To develop a vaccine against hepatitis C virus, we synthesized four long peptides from nonstructural proteins NS3, NS4 and NS5B containing HLA-class I and class II epitopes mainly inducing responses in natural infection. In HLA-A2.1 transgenic mice, the four peptides primed higher CTL responses to 6:7 minimal HLA-A2 epitopes than those induced by the minimal epitopes. HLA-A2.1/HLA-DR1 transgenic mice immunized with one peptide, containing a class II epitope implicated in viral resolution, developed IFNgamma-producing CD4+-T and CD8+-T cells. These peptides recalled HCV-specific IFNgamma-producing cells from HCV-infected patients' PBMC. This support the selection of these domains for inclusion in a vaccine formulation.

Oral immunization with a shiga toxin B subunit: rotavirus NSP4(90) fusion protein protects mice against gastroenteritis.

A fusion protein containing the shiga toxin-1 B subunit (STB) linked to a 90 amino acid peptide (aa residues 86--175) from simian rotavirus (SA--11) nonstructural protein NSP4 was synthesized in Escherichia coli. Mice orally inoculated with 60 microg of STB::NSP4(90) fusion protein per dose generated higher humoral and intestinal antibody titers than mice inoculated with 30 microg of NSP4 alone. Serum anti-NSP4 IgG2a isotype titers were substantially greater than IgG1 titers, suggesting a dominant Th1 immune response. ELISA measurement of cytokines secreted from splenocytes isolated from immunized mice confirmed the STB::NSP4(90) fusion protein stimulation of a strong Th1 cell mediated immune response. Diarrhea in SA-11 rotavirus challenged neonates suckling from STB::NSP4 immunized dams was significantly reduced in severity and duration in comparison with virus challenged neonates from unimmunized mice. Together, our experiments demonstrate for the first time that the shiga toxin B subunit provides ligand mediated delivery of virus antigens to the gut-associated lymphoid tissues for enhanced stimulation of humoral and cellular responses against rotavirus gastroenteritis.

Human parvovirus B19 transgenic mice become susceptible to polyarthritis.

Human parvovirus B19 (B19) often causes acute polyarthritis in adults. In this paper, we analyzed nucleotide sequences of the B19 genome of patients with rheumatoid arthritis (RA), and then introduced the nonstructural protein 1 (NS1) gene of B19 into C57BL/6 mice that had a genetic origin not susceptible to arthritis. The transgenic mice developed no lesions spontaneously, but were susceptible to type II collagen (CII)-induced arthritis. B19 NS1 was expressed in synovial cells on the articular lesions that were histologically characteristic of granulomatous synovitis and pannus formation in cartilage and bone. Serum levels of anti-CII Abs and TNF-alpha increased in NS1 transgenic mice to the same levels as those of DBA/1 mice, which were susceptible to polyarthritis. Stimulation with CII increased secretion of Th1-type- and Th2-type cytokines in NS1 transgenic mice, indicating that a nonpermissive H-2(b) haplotype in the wild type of C57BL/6 mice can be made susceptible to polyarthritis through the expression of NS1. This study is the first to show that a viral agent from the joints in humans can cause CII-induced arthritis resembling RA.