Showing papers in "Viral Immunology in 2004"

Inverse interference: how viruses fight the interferon system.

Abstract: Viruses need to multiply extensively in the infected host in order to ensure transmission to new hosts and survival as a population. This is a formidable task, given the powerful innate and adaptive immune responses of the host. In particular, the interferon (IFN) system plays an important role in limiting virus spread at an early stage of infection. It has become increasingly clear that viruses have evolved multiple strategies to escape the IFN system. They either inhibit IFN synthesis, bind and inactivate secreted IFN molecules, block IFN-activated signaling, or disturb the action of IFN-induced antiviral proteins. The molecular mechanisms involved range from a broad shut-off of the host cell metabolism to fine-tuned elimination of key components of the IFN system. Type I (alpha/beta) IFNs are produced in direct response to virus infection and double-stranded RNA (dsRNA) molecules that are sensed as a danger signal by infected cells. IFNs induce the expression of a number of antiviral proteins, some of which are again activated by dsRNA. Therefore, many viruses produce dsRNA-binding proteins to sequester the danger signal or express virulence genes that target specific components of the IFN system, such as members of the IFN regulatory factor (IRF) family or components of the JAK-STAT signaling pathway. Finally, some viruses have adopted means to directly suppress the very antiviral effector proteins of the IFN-induced antiviral state directed against them. Evidently, viruses and their host's innate immune responses have coevolved, leading to a subtle balance between virus-promoting and virus-inhibiting factors. A better understanding of virus-host interactions is now emerging with great implications for vaccine development and drug design.

Hantaviruses: Immunology, Treatment, and Prevention

Abstract: Hantaviruses are rodent-borne bunyaviruses that are associated with two main clinical diseases in humans: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. It has been suggested that host-related immune mechanisms rather than direct viral cytopathology may be responsible for the principal abnormality (vascular dysfunction) in these syndromes. This review summarizes the current knowledge on hantaviral host immune responses, immune abnormalities, laboratory diagnosis, and antiviral therapy as well as the current approaches in vaccine development.

Depletion of peripheral blood T lymphocytes and NK cells during the course of ebola hemorrhagic Fever in cynomolgus macaques.

Abstract: During the course of an experimentally induced Ebola virus (EBOVA) infection of cynomolgus macaques, peripheral blood mononuclear cells were isolated and characterized by multi-color flow cytometry...

Vesicular Stomatitis Virus as an Oncolytic Vector

Abstract: Recent data has shown that viruses such as vesicular stomatitis virus (VSV), a relatively non-pathogenic, negative-stranded RNA virus, can preferentially replicate in malignant cells and less so in normal cells. VSV appears able to carry out this function in transformed cells since these hosts exhibit the hallmarks of flawed host defense, probably involving the interferon system, which is essential for preventing virus replication. The simple genetic constitution of VSV, lack of any known transforming, integrating or reassortment properties, extensive knowledge relating to its interaction with the immune system and the ability to genetically manipulate this agent affords an ideal opportunity to exploit the oncolytic and gene targeting potential of this innocuous virus. Thus, aside from preferentially targeting malignant cells VSV recombinants could be generated that could increase a tumor's susceptibility to chemotherapeutic agents and/ or importantly, the host immune response. Collectively, our data and others demonstrate that VSV as well as other RNA viruses could provide a promising and exciting approach to cancer therapy.

Coxsackievirus-Induced Pancreatitis

Abstract: In humans, infections with the group B coxsackieviruses (CVBs) range from asymptomatic infections to chronic, debilitating diseases. The CVBs are associated with chronic inflammatory diseases of th...

Aedes aegypti salivary gland extracts modulate anti-viral and TH1/TH2 cytokine responses to sindbis virus infection.

Abstract: Vector-borne viruses are naturally transmitted when a vector salivates during feeding on a vertebrate host. Most laboratory studies of infection disregard the role that the vector plays in the path...

Pathogenesis of respiratory syncytial virus infection.

Abstract: Respiratory syncytial virus (RSV) is recognized as the most important cause of serious lower respiratory tract illness in infants and young children worldwide causing repeat infections throughout life with serious complications occurring in the elderly and immune compromised patient. The level of disease pathogenesis associated with RSV infection is balanced between virus elimination and the nature of the immune response to infection. The innate and adaptive immune responses to RSV infection are not fully elucidated; however, significant progress has been made in understanding the virus-host relationship and mechanisms associated with disease pathogenesis. This review summarizes important aspects of these findings, and provides current perspective on processes that may contribute to RSV disease pathogenesis.

Adenoviral vectors for gene replacement therapy.

Abstract: Adenovirus-based vectors are promising vehicles for gene replacement therapy due to their ability to efficiently transduce a wide variety of proliferating and non-proliferating cells. Over the past decade, different versions of adenoviral vectors (Ads) have been developed. These vectors can be classified into two major categories, based on whether the viral coding sequences are partially (first or second-generation Ads) or completely deleted (helper-dependent or gutted Ads). Both types of Ads have been tested in a variety of gene delivery studies, and major obstacles to their clinical application have been identified. Currently, innate and adaptive host immune responses to Ads remain major challenges, limiting both the initial viral dose and the effectiveness of subsequent administrations. Recent developments in vector design and delivery methods have improved the potential of Ads for successful gene therapy application.

Single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) for influenza A virus subtype H5N1 detection.

Abstract: Influenza A virus subtype H5N1 causes a rapidly fatal systemic disease in domestic poultry and spreads directly from poultry to humans. The aim of this study was to develop a rapid, cost-saving and effective method for influenza A virus subtype H5N1 detection. The selected primer set was used in single-step RT-PCR for simultaneous detection in multiplex format of the 276-, 189-, and 131-bp fragments, corresponding to sequences specific for M, H5 and N1. The amplified DNA fragments were clearly separated by agarose gel electrophoresis. The sensitivity of this assay was about 103copies/µL. Moreover, this method can be applied to detect not only avian but also human influenza A virus subtype H5N1. In conclusion, the highlights of this particular method are its rapidity and cost-effectiveness, thus rendering it feasible and attractive for large-scale screening at times of influenza A virus subtype H5N1 outbreak.

T Cell Responses to Influenza Virus Infection: Effector and Memory Cells

Abstract: New approaches to visualizing antigen-specific primary responses to influenza and the development of memory subsets in distinct sites suggest that both CD4 and CD8 T cells play complex roles in primary viral clearance and have the potential to contribute to protection from secondary infection.

Increased Plasma Transforming Growth Factor-β1 Is Associated with Disease Progression in HIV-1-Infected Patients

Abstract: Transforming growth factor-beta(1) (TGF-beta(1)) is a pleiotropic cytokine with a variety of effects on a wide range of cells in the immune system. Evidence suggests that TGF-beta(1) is also involved in the pathogenesis of human immunodeficiency virus type 1 infections. The aim of this study was to explore possible relationships between circulating TGF-beta(1) and immune as well as clinical HIV infection parameters with special impact on disease progression. TGF-beta(1) concentrations were measured by ELISA in the plasma of 66 patients in different stages of HIV infection and 20 healthy controls. HIV infection resulted in a significant increase of plasma TGF-beta(1) concentration compared to healthy individuals (11.4 +/- 6.8 vs. 6.1 +/- 1.5 ng/mL, p < 0.01). TGF-beta(1) values showed a significant negative correlation with CD4 cells count (r = -0.42, p = 0.001), as well as with CD8 cells count (r = -0.031, p < 0.05). Moreover, patients with the symptomatic phase of HIV infection presented an almost twofold increase of plasma TGF-beta(1) concentration in comparison to asymptomatic patients and healthy individuals. Our results demonstrate the relationship between TGF-beta(1) concentrations and HIV infection advancement with marked elevation in the late stages of the disease. These findings support in vitro observations suggesting an important, immunosuppressive role of TGF-beta(1) in HIV infection pathogenesis.

Impact of Immunizations with Porcine Reproductive and Respiratory Syndrome Virus on Lymphoproliferative Recall Responses of CD8+ T Cells

Abstract: The objective of this study was to investigate the immune responses elicited by either a modified-live (MLV) or a killed virus (KV) porcine reproductive and respiratory syndrome virus (PRRSV) vaccine. Specifically, we investigated the effects of multiple vaccinations on antigen-specific cellular and antibody responses against PRRSV. Twelve sows were obtained from herds with either a history of repeated MLV or KV PRRSV vaccination and a non-vaccinated, PRRSV-negative herd. Within herd, sows were divided into three groups and vaccinated with MLV, KV, or injected with saline. On day 0, 27, and 38, recall responses of peripheral blood mononuclear cells (PBMC) to the parent strains of the vaccines (e.g., MLV-VR2332 or KV-ISUP) were examined. The concentrations of total PRRSV-specific and virus-neutralizing serum antibodies were determined by ELISA and serum neutralization assays. Following immunization, the antigen-specific proliferation of CD8alphabeta(+), CD4(+)CD8alphaalpha(+) T cells in the naive sows was greater than in sows repeatedly vaccinated with KV or MLV. This diminished lymphoproliferative responses of CD8alphabeta(+) and CD4(+)CD8alphaalpha(+) T cells could be partially overcome by heterologous immunization. However, B cell proliferation, PRRSV antibody concentrations and virus neutralizing antibody titers were not enhanced by heterologous immunization and only KV vaccination increased antibody levels in previously immunized (MLV or KV) sows.

Respiratory syncytial virus-induced immunoprotection and immunopathology.

Abstract: Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family, is a major clinical problem causing yearly epidemics of severe lower airway disease in both infants and the elderly. Attem...

Host evasion by emerging paramyxoviruses : hendra virus and nipah virus V proteins inhibit interferon signaling

Abstract: Interferon (IFN) can activate Signal Transducer and Activator of Transcription (STAT) proteins to establish a cellular antiviral response and inhibit virus replication. Many viruses have evolved strategies to inhibit this antiviral mechanism, but paramyxoviruses are unique in their abilities to directly target the IFN-responsive STAT proteins. Hendra virus and Nipah virus (Henipaviruses) are recently emerged paramyxoviruses that are the causative agents of fatal disease outbreaks in Australia and peninsular Malaysia. Similar to other paramyxoviruses, Henipaviruses inhibit IFN signal transduction through a virus-encoded protein called V. Recent studies have shown that Henipavirus V proteins target STAT proteins by inducing the formation of cytoplasmically localized high molecular weight STAT-containing complexes. This sequestration of STAT1 and STAT2 prevents STAT activation and blocks antiviral IFN signaling. As the V proteins are important factors for host evasion, they represent logical targets for therapeutics directed against Henipavirus epidemics.

Influenza pandemics: can we prepare for the unpredictable?

Abstract: Although no viruses are better understood or more intensively studied than the viruses of influenza, if the next influenza pandemic occurs within the next 5-10 years its control will depend on innovations in vaccine production developed more than 40 years ago, but not yet applied to the full extent demanded by our present hard-won knowledge of the epidemiology of the disease. We have become so enamored of the brilliant advances made in the interim in understanding the molecular biology of both virus and host that common sense and inexpensive implementation of proven and older methods of control have been neglected as an interim barricade. In this review, I have advocated a return to first principles, while embracing the promise and returns of contemporary research. With the assumption that the next pandemic virus will contain one of the 13 influenza A virus hemagglutinin subtypes not currently causing epidemic human disease, high-yield reassortant viruses of each of these subtypes should be produced with all dispatch and, in collaboration with industry, tested for production stability and immunogenicity in humans. From this archive, an appropriate reassortant could be selected within days or weeks, and production could ensue. If not a perfect match with the imminent pandemic virus, this "barricade vaccine" could stand as a first line of defense until supplanted by a definitive "rampart vaccine," matching better the emergent, potentially pandemic virus.

Innate immune responses in respiratory syncytial virus infections

Abstract: Respiratory syncytial virus (RSV) is the most important viral respiratory pathogen of early life. Studies of the immune response in general (and the innate response in particular) to this agent are of interest for a number of reasons. First, severe forms of illness may be a result of enhanced immunologic responsiveness to viral constituents at the time of infection. Secondly, the immune response to RSV may consist principally of innate immune responses at the time of maximum severity of illness. Third, RSV infection in infancy may be linked via immune mechanisms to the development of childhood wheezing. Finally there are no meaningfully effective forms of therapy for RSV infection, and elucidation of the immune response may suggest new therapeutic approaches. This review will summarize our current knowledge of innate immune responses to RSV infection. Specifically we will review early interactions of the virus with surfactant proteins and Toll-like receptors, chemokine release from infected cells, cytokine release from activated inflammatory cells, activation of neuroimmune pathways, generation of dendritic cells, the release of soluble mediators of airway obstruction, and genetic polymorphisms associated with RSV-related illness.

Modulation of dengue virus infection of dendritic cells by Aedes aegypti saliva.

Abstract: Dengue virus (DV) is a flavivirus carried by the Aedes aegypti mosquito that causes a spectrum of illnesses in the tropics, including dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Dendritic cells (DCs) are professional antigen presenting cells recently shown to be permissive for DV, and implicated as the primary targets of initial DV infection. DV is transmitted to human host by infected mosquitoes during a blood meal, but it is currently unknown whether transmission is modified by vector saliva that is also deposited in the host's skin during feeding. Previous studies evaluated only the outcome of DV infection of DCs, and did not address the influence of mosquito saliva. To more fully characterize natural transmission of DV, we evaluated the effects of Ae. aegypti saliva on DV infection of human myeloid DCs. We found that saliva inhibited DV infection in DCs. Moreover, pre-sensitization of DCs with saliva, prior to DV infection, enhanced inhibition. In addition, enhanced production o...

Segments of puumala hantavirus nucleocapsid protein inserted into chimeric polyomavirus-derived virus-like particles induce a strong immune response in mice.

Abstract: Insertion of a short-sized epitope at four different sites of yeast-expressed hamster polyomavirus major capsid protein VP1 has been found to result in the formation of chimeric virus-like particle...

Mouse strain differences in the chemokine response to acute lung infection with a murine gammaherpesvirus.

Abstract: Numerous mouse strain-based differences in the immune response and in susceptibility to numerous pathogens have been described, but it is not known if these differences extend to chemokine responses to viral infection of the lungs. To define mouse strain-based differences in the host chemokine response and susceptibility to infection with murine gammaherpesvirus-68 (MHV-68), we compared the induced chemokine response to MHV-68 infection in the lungs of BALB/c and C57BL/6 mice at 1-15 days post-infection. CC and CXC chemokines were induced in both BALB/c and C57BL/6 following infection but the level of chemokine induction was significantly higher in the BALB/c mice for all chemokines measured. In addition, interferon-γ (IFN-γ) was also induced to a significantly higher level in the lungs of BALB/c infected mice compared to C57BL/6 mice. Interestingly, viral gene expression was lower in the lungs of C57BL/6 mice during the acute phase of replication. Titers of infectious virus were also greater in BALB/c lu...

Induction of T Lymphocytes Specific for Bovine Viral Diarrhea Virus in Calves with Maternal Antibody

Abstract: Passive antibody to bovine viral diarrhea virus (BVDV) acquired through colostrum intake may interfere with the development of a protective immune response by calves to this virus. The objective of this study was to determine if calves, with a high level of maternal antibody to bovine viral diarrhea virus (BVDV), develop CD4(+), CD8(+), or gammadelta T lymphocyte responses to BVDV in the absence of a measurable humoral immune response. Colostrum or milk replacer fed calves were challenged with virulent BVDV at 2-5 weeks of age and/or after maternal antibody had waned. Calves exposed to BVDV while passive antibody levels were high did not mount a measurable humoral immune response to BVDV. However, compared to nonexposed animals, these animals had CD4(+), CD8(+), and gammadelta T lymphocytes that were activated by BVDV after exposure to in vitro BVDV. The production of IFNgamma by lymphocytes after in vitro BVDV exposure was also much greater in lymphocytes from calves exposed to BVDV in the presence of maternal antibody compared to the nonexposed calves. These data indicate that calves exposed to BVDV while maternal antibody levels are high can develop antigen specific CD4(+), CD8(+), and gammadelta T lymphocytes in the absence of an active antibody response. A manuscript presented separately demonstrates that the calves with T lymphocytes specific for BVDV in this study were also protected from virulent BVDV genotype 2 challenge after maternal antibody became undetectable.

Regulation of cellular gene expression in endothelial cells by sin nombre and prospect hill viruses.

Abstract: Mechanisms of hantavirus-induced vascular leakage remain unknown. This study was initiated to determine whether hantavirus-induced changes in endothelial cell gene expression may provide insight into disease mechanisms. Additionally, by using pathogenic Sin Nombre virus (SNV) and non-pathogenic Prospect Hill virus (PHV), we wanted to identify cellular responses that are likely to differentiate pathogenic from nonpathogenic hantaviruses. Using the Affymetrix DNA Array, we found that PHV and SNV did not significantly differ in the number of activated genes (18 versus 14 genes) in infected endothelial cells at 4 h PI. However, a smaller group of genes (36) were up-regulated by PHV compared to SNV (175) at 12 h PI. Only two genes were down-regulated in SNV-infected cells. Expression of the functionally diverse group of genes was altered at an early stage of infection (4 and 12 h PI). The genes affected include putative anti-viral factors, transcription factors, growth factors, chemokines, receptors, structura...

Modifying adenoviral vectors for use as gene-based cancer vaccines.

Abstract: The past decade has produced significant advances in our understanding of antigen-presenting cells, tumor antigens, and other components of the immune response to cancer. Gene-based vaccination is emerging as one of the more promising approaches for loading dendritic cells (DC) with tumor-associated antigens. In this respect, it is proposed that adenoviral (AdV) vectors can deliver high antigen concentrations, promote effective processing and MHC expression, and stimulate potent cell-mediated immunity. While AdV vectors have performed well in pre-clinical vaccine models, their application to patient care has limitations. The in vivo administration of AdV vectors is associated with both innate and adaptive host responses that result in tissue inflammation and injury, viral neutralization, and premature clearance of AdV-transduced cells. A variety of strategies have been developed to address these limitations. The ideal vaccine would avoid vector-related immune responses, have relative specificity for transducing DC, and induce high levels of transgene expression. This review describes the range of host responses to AdV vaccines, identifies strategies to reduce viral recognition and enhance transgene antigen expression, and suggests future approaches to vector development and administration. There is every reason to believe that safer and more effective forms of AdV-based vaccines can be developed and applied to patient therapy.

CMV Antigen-Specific CD4+ and CD8+ T Cell IFNγ Expression and Proliferation Responses in Healthy CMV-Seropositive Individuals

Abstract: CMV-specific CD4+ and CD8+ T cell IFNgamma expression and proliferation were measured in healthy volunteers by flow cytometry after CMV lysate or CMV pp65 or IE peptide pool stimulation. Cutoff values were set to maximize specificity (i.e., no false positive CMV-seronegatives). Sensitivity (defined as a positive response in CMV-seropositives to at least one of the 3 antigen preparations used) was 100% for CMV-specific CD4+ and CD8+ T cell IFN expression and CD4+ T cell proliferation and 95.4% for CMV-specific CD8+ T cell proliferation. All 22 CMV-seropositive individuals had positive responses by at least three of these four measurements. These findings support the concept that a multiplicity of antigen-specific functional immune responses and persistence of robust virus-specific CD4+T cells are important components of protective immunity in this chronic viral infection.

Temporary CD8+ T-cell depletion in pigs does not exacerbate infection with porcine reproductive and respiratory syndrome virus (PRRSV).

Abstract: Several studies have demonstrated a consistent increase in the CD8+ T-cell subset of pigs following infection with porcine reproductive and respiratory virus (PRRSV). Consequently, it has been suggested that CD8+ T-cells may play an important role in protection against this infection. In order to test this hypothesis, we examined five 5-week-old pigs, which had been depleted for CD8+ T-cells by treatment with anti-CD8 mAb injections, starting 2 days before inoculation with PRRSV. Virus-inoculated and sham-inoculated age-matched pigs served as controls. Blood samples were collected continuously, together with organ material at necropsy, to study kinetics of leukocyte subpopulations, antibody production and virus persistence in individual pigs. Significant lower CD8+ T-cell counts on day 0, that is, before virus challenge, in the anti-CD8 mAb treated pigs compared to the control pigs, confirmed the depletion effect of specific mAb therapy. Almost complete depletion of cell subsets expressing the CD8+ antigen was obtained on day 2 and 5 post infection (PI) with nadir less than 1% of peripheral blood mononuclear cells (PBMC). One week PI, an increase in T-cell subsets was observed for both anti-CD8 mAb treated pigs and virus-inoculated control pigs. T-memory cells and cytotoxic T-cells reached levels comparative with the virus-inoculated control pigs on days 8 and 12 PI, respectively, whereas NKcells remained suppressed for the rest of the experimental period. An extraordinary increase of T-helper cells in the anti-CD8 mAb treated pigs with a significantly higher level than in the virus-inoculated and sham-inoculated control pigs, was observed from day 12 PI. In conclusion, it was established that CD8+ T-cell depletion in the early phase of PRRSV infection neither caused increased disease nor influenced the ability to clear virus in the treated pigs.

Structural constraints on viral escape from HIV- and SIV-specific cytotoxic T-lymphocytes.

Abstract: Cytotoxic T lymphocytes (CTL) play a central role in controlling lentiviral infections in both humans and monkeys. While they contain the spread of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), CTL are not capable of fully eradicating virus following infection. Ongoing viral replication can therefore lead to the accumulation of viral mutations within CTL epitopes that can undermine cellular immune control of virus. Here we review the importance of CTL in controlling HIV/SIV infection and how immunologic pressure exerted by effector T cells selects for viral variants that escape CTL recognition. We review two examples of viral escape from CTL at highly conserved epitopes that illustrate the extraordinary capacity of lentiviruses to adapt to their immunologic environment despite structural constraints on the ability of the virus to accommodate mutations.

Yeast-expressed hantavirus Dobrava nucleocapsid protein induces a strong, long-lasting, and highly cross-reactive immune response in mice.

Abstract: In Europe, Dobrava virus (DOBV) carried by the yellow-necked field mouse Apodemus flavicollis is one of the hantaviruses that can cause severe hemorrhagic fever with renal syndrome in humans. For several hantaviruses, the nucleocapsid (N) protein has proven to be very immunogenic in humans and rodents and even can protect rodents against a virus challenge. To investigate the immunogenicity of DOBV N protein, BALB/c and C57BL/6 mice were immunized three times with a DOBV recombinant N (rN) protein expressed in yeast Saccharomyces cerevisiae together with complete Freund's, with incomplete Freund's, and without adjuvant, respectively. Mice of both strains elicited N-specific antibodies with end-point titers being as high as 1:1,000,000 in C57BL/6 mice. The antibodies induced by DOBV rN protein were highly cross-reactive to the rN proteins of hantaviruses Puumala and Hantaan. In both mice strains, DOBV rN protein induced N-specific antibodies of all IgG subclasses (IgG1, IgG2a, IgG2b, and IgG3), suggesting a...

Structural similarities in the cellular receptors used by adenovirus and reovirus.

Abstract: Adenovirus and reovirus are nonenveloped viruses that engage cell-surface receptors using filamentous attachment proteins with head-and-tail morphology. The coxsackievirus and adenovirus receptor (CAR) and reovirus receptor junctional adhesion molecule 1 (JAM1) are immunoglobulin superfamily members that form homodimers stabilized by ionic and hydrophobic contacts between their N-terminal immunoglobulin-like domains. Both proteins are expressed at regions of cell-cell contact and contain sequences in their cytoplasmic tails that anchor the proteins to the actin cytoskeleton. Like CAR and JAM1, the attachment proteins of adenovirus and reovirus, fiber and sigma1, respectively, also share key structural features. Both fiber and sigma1 have defined regions of flexibility within the tail, which is constructed in part using an unusual triple beta-spiral motif. The head domains of both proteins are formed by an 8-stranded beta-barrel with identical beta-strand connectivity. Strikingly, both adenovirus fiber and reovirus 1 engage their receptors by interacting with sequences that also mediate formation of receptor homodimers. Therefore, while adenovirus and reovirus belong to different virus families and have few overall properties in common, the observed similarities between the receptors and attachment proteins of these viruses suggest a conserved mechanism of attachment and an evolutionary relationship.

Mutation of the "a" determinant of HBsAg with discordant HBsAg diagnostic kits.

Abstract: Sera from 1003 in- and out-patients were investigated for hepatitis B surface antigen (HBsAg) mutation at Siriraj Hospital, Bangkok, Thailand. Individual samples were screened using two commercial HBsAg kits on automatic machine-based assays set up in parallel. The first kit was a sandwich ELISA kit that used monoclonal capture/monoclonal conjugate and color detection whereas the second was a sandwich MEIA, using monoclonal capture/polyclonal indicator and fluorochrome determination. Six specimens were found discordant by negative EIA and positive MEIA; two specimens of which were detectable of HBV DNA. Three out of four discordant results were confirmed by an anti-HBs neutralization assay. Based on direct sequencing, one HBsAg/anti-HBs sample with multiple mutations in the S gene was found. The mutation included the common glycine to arginine escape mutation at amino acid position 145 of the "a" determinant. The observation should alert the blood bank to the necessity of using screening kits capable of detecting HBV mutant carriers as well as providing verification for the phenomenon of vaccine-escape mutation in Thailand.

Prion diseases and the spleen.

Abstract: Transmissible spongiform encephalopathies are fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy and scrapie in sheep and goats. Transmissible spongiform encephalopathies are thought by some to result from changes in the conformation of a membrane glycoprotein called PrPC (prion protein) into a pathogenic form, PrPSc, which constitutes the major component of an unprecedented type of infectious particle supposedly devoid of nucleic acid. Although there is no primary immunological response to the infectious agent, several lines of evidence indicate an involvement of the lymphoreticular system in the development of prion diseases. Studies in rodents have shown that after peripheral infection, uptake of the scrapie agent is followed by an initial phase of replication in the lymphoreticular system, particularly the spleen and lymph nodes. Moreover, infectivity titers in lymphoreticular organs reach a maximum relatively quickly, well before those in the brain, and then maintain a plateau for the remainder of the disease progression. The presence of PrPSc in peripheral lymphoid organs of all cases of variant Creutzfeldt-Jakob disease strongly underscores the importance of the lymphoreticular system. Thus, a better understanding of the cells participating in PrPSc replication and dissemination into the central nervous system is of particular interest. This review will therefore discuss the present knowledge of the role of the spleen in transmissible spongiform encephalopathies as well as the participation of the different spleen cell types in the disease process.

Flavivirus immunization with capsid-deletion mutants: basics, benefits, and barriers.

Abstract: The flaviviruses comprise a number of arthropod-transmitted human disease agents that cause significant and increasing health threats in major parts of the world. The development of new vaccines is of vital importance, but the stringent need for safety, efficacy and cost-effectiveness together with the problems associated with the specific immune pathogenesis of some flavivirus infections impose significant challenges to innovative vaccine research. Using tick-borne encephalitis virus (TBEV) as a model, the viral capsid protein gene was recently identified as a novel target for generating flavivirus vaccines. This approach can be applied to produce either attenuated strains that can serve as live vaccines or to make a new type of a genetic vaccine consisting of non-infectious RNA replicons from which subviral particles are synthesized in vivo. Flaviviruses are small, enveloped viruses with an unsegmented positive-stranded RNA genome encoding a single polyprotein that is cleaved into the individual viral proteins. The specific introduction of various deletions and other mutations into the genomic segment coding for the capsid protein C and the biochemical and immunological characterization of the resulting mutants in cell culture and an animal model have revealed remarkable properties of this building block of the nucleocapsid and yielded information that opened the way for new vaccine approaches. In this review the in vitro and in vivo findings with various capsid deletion mutants of TBEV are summarized and discussed in the context of recent structural and biochemical data obtained for protein C of various flaviviruses. Potential benefits of this new strategy for generating flavivirus vaccines as well as hurdles that still have to be overcome are discussed in comparison to conventional or other experimental approaches. Capsid-deletion mutants can be used to rationally design safe and effective vaccine strains or to create new vaccines that combine advantages of genetic vaccination, conventional inactivated, and live vaccines.