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1.  Displaying Fel d1 on virus-like particles prevents reactogenicity despite greatly enhanced immunogenicity: a novel therapy for cat allergy 
The Journal of Experimental Medicine  2009;206(9):1941-1955.
Allergen-specific desensitization is the only disease-modifying therapy currently available for the treatment of allergies. These therapies require application of allergen over several years and some may induce life-threatening anaphylactic reactions. An ideal vaccine for desensitization should be highly immunogenic and should alleviate allergic symptoms upon few injections while being nonreactogenic. We describe such a vaccine for the treatment of cat allergy, consisting of the major cat allergen Fel d1 coupled to bacteriophage Qβ-derived virus-like particles (Qβ–Fel d1). Qβ–Fel d1 was highly immunogenic, and a single vaccination was sufficient to induce protection against type I allergic reactions. Allergen-specific immunoglobulin G antibodies were shown to be the critical effector molecules and alleviated symptoms by two distinct mechanisms. Although allergen-induced systemic basophil degranulation was inhibited in an FcγRIIb-dependent manner, inhibition of local mast cell degranulation in tissues occurred independently of FcγRIIb. In addition, treatment with Qβ–Fel d1 abolished IgE memory responses upon antigen recall. Despite high immunogenicity, the vaccine was essentially nonreactogenic and vaccination induced neither local nor systemic anaphylactic reactions in sensitized mice. Moreover, Qβ–Fel d1 did not induce degranulation of basophils derived from human volunteers with cat allergies. These data suggest that vaccination with Qβ–Fel d1 may be a safe and effective treatment for cat allergy.
PMCID: PMC2737174  PMID: 19667059
2.  VSIG4, a B7 family–related protein, is a negative regulator of T cell activation 
Journal of Clinical Investigation  2006;116(10):2817-2826.
T cell activation by APCs is positively and negatively regulated by members of the B7 family. We have identified a previously unknown function for B7 family–related protein V-set and Ig domain–containing 4 (VSIG4). In vitro experiments using VSIG4-Ig fusion molecules showed that VSIG4 is a strong negative regulator of murine and human T cell proliferation and IL-2 production. Administration to mice of soluble VSIG4-Ig fusion molecules reduced the induction of T cell responses in vivo and inhibited the production of Th cell–dependent IgG responses. Unlike that of B7 family members, surface expression of VSIG4 was restricted to resting tissue macrophages and absent upon activation by LPS or in autoimmune inflammatory foci. The specific expression of VSIG4 on resting macrophages in tissue suggests that this inhibitory ligand may be important for the maintenance of T cell unresponsiveness in healthy tissues.
PMCID: PMC1578631  PMID: 17016562
3.  Complement receptors regulate differentiation of bone marrow plasma cell precursors expressing transcription factors Blimp-1 and XBP-1 
The Journal of Experimental Medicine  2005;201(6):993-1005.
Humoral immune responses are thought to be enhanced by complement-mediated recruitment of the CD21–CD19–CD81 coreceptor complex into the B cell antigen receptor (BCR) complex, which lowers the threshold of B cell activation and increases the survival and proliferative capacity of responding B cells. To investigate the role of the CD21–CD35 complement receptors in the generation of B cell memory, we analyzed the response against viral particles derived from the bacteriophage Qβ in mice deficient in CD21–CD35 (Cr2−/−). Despite highly efficient induction of early antibody responses and germinal center (GC) reactions to immunization with Qβ, Cr2−/− mice exhibited impaired antibody persistence paralleled by a strongly reduced development of bone marrow plasma cells. Surprisingly, antigen-specific memory B cells were essentially normal in these mice. In the absence of CD21-mediated costimulation, Qβ-specific post-GC B cells failed to induce the transcriptional regulators Blimp-1 and XBP-1 driving plasma cell differentiation, and the antiapoptotic protein Bcl-2, which resulted in failure to generate the precursor population of long-lived plasma cells residing in the bone marrow. These results suggest that complement receptors maintain antibody responses by delivery of differentiation and survival signals to precursors of bone marrow plasma cells.
PMCID: PMC2213108  PMID: 15767369
4.  Bacterially Produced Recombinant Influenza Vaccines Based on Virus-Like Particles 
PLoS ONE  2013;8(11):e78947.
Although current influenza vaccines are effective in general, there is an urgent need for the development of new technologies to improve vaccine production timelines, capacities and immunogenicity. Herein, we describe the development of an influenza vaccine technology which enables recombinant production of highly efficient influenza vaccines in bacterial expression systems. The globular head domain of influenza hemagglutinin, comprising most of the protein's neutralizing epitopes, was expressed in E. coli and covalently conjugated to bacteriophage-derived virus-like particles produced independently in E.coli. Conjugate influenza vaccines produced this way were used to immunize mice and found to elicit immune sera with high antibody titers specific for the native influenza hemagglutinin protein and high hemagglutination-inhibition titers. Moreover vaccination with these vaccines induced full protection against lethal challenges with homologous and highly drifted influenza strains.
PMCID: PMC3832520  PMID: 24260136
5.  Enhanced Neutralizing Antibody Titers and Th1 Polarization from a Novel Escherichia coli Derived Pandemic Influenza Vaccine 
PLoS ONE  2013;8(10):e76571.
Influenza pandemics can spread quickly and cost millions of lives; the 2009 H1N1 pandemic highlighted the shortfall in the current vaccine strategy and the need for an improved global response in terms of shortening the time required to manufacture the vaccine and increasing production capacity. Here we describe the pre-clinical assessment of a novel 2009 H1N1 pandemic influenza vaccine based on the E. coli-produced HA globular head domain covalently linked to virus-like particles derived from the bacteriophage Qβ. When formulated with alum adjuvant and used to immunize mice, dose finding studies found that a 10 µg dose of this vaccine (3.7 µg globular HA content) induced antibody titers comparable to a 1.5 µg dose (0.7 µg globular HA content) of the licensed 2009 H1N1 pandemic vaccine Panvax, and significantly reduced viral titers in the lung following challenge with 2009 H1N1 pandemic influenza A/California/07/2009 virus. While Panvax failed to induce marked T cell responses, the novel vaccine stimulated substantial antigen-specific interferon-γ production in splenocytes from immunized mice, alongside enhanced IgG2a antibody production. In ferrets the vaccine elicited neutralizing antibodies, and following challenge with influenza A/California/07/2009 virus reduced morbidity and lowered viral titers in nasal lavages.
PMCID: PMC3799843  PMID: 24204639
6.  Plasmodium vivax malaria vaccines 
Human Vaccines & Immunotherapeutics  2013;9(12):2558-2565.
Malaria is one of the few diseases in which morbidity is still measured in hundreds of millions of cases every year. Plasmodium vivax and Plasmodium falciparum are responsible for nearly all the malaria cases in the world and despite difficulties in obtaining an exact number, estimates indicate an astonishing 349–552 million clinical cases of malaria due to P. falciparum in 2007 and between 132–391 million clinical episodes due to P. vivax in 2009. It is becoming evident that eradication of malaria will be an arduous task and P. vivax will be one of the most difficult species to eliminate and perhaps become the last standing malaria parasite. Indeed, in countries that succeed in decreasing the disease burden, nearly all the remaining malaria cases are caused by P. vivax. Such resilience is mainly due to the sophisticated mechanism that the parasite has evolved to remain dormant for months or years forming hypnozoites, a small structure in the liver that will be a major hurdle in the efforts toward malaria eradication. Furthermore, while clinical trials of vaccines against P. falciparum are making fast progress, a very different picture is seen with P. vivax, where only few candidates are currently active in clinical trials.
PMCID: PMC4162059  PMID: 23978931
Plasmodium vivax; vaccine; clinical trials; malaria; human trials
7.  CD8+ T Cells Mediate CD40-independent Maturation of Dendritic Cells In Vivo  
The Journal of Experimental Medicine  1999;189(12):1875-1884.
Induction of cytotoxic T lymphocyte (CTL) responses against minor histocompatibility antigens is dependent upon the presence of T cell help and requires the interaction of CD40 on dendritic cells (DCs) with CD40 ligand on activated T helper cells (Th). This study demonstrates that CD40 is neither involved in Th-dependent nor Th-independent antiviral CTL responses. Moreover, the data show that DC maturation occurs in vivo after viral infection in the absence of CD40 and Th. This maturation did not require viral infection of  DCs but was mediated by peptide-specific CD8+ T cells. Surprisingly, naive CD8+ T cells were able to trigger DC maturation within 24 h after activation in vivo and in vitro. Moreover, peptide-activated CD8+ T cells were able to induce maturation in trans, as DCs that failed to present the relevant antigen in vivo also underwent maturation. Upon isolation, the in vivo–stimulated DCs were able to convert a classically Th-dependent CTL response (anti-HY) into a Th-independent response in vitro. Thus, antiviral CD8+ T cells are sufficient for the maturation of DCs in the absence of CD40.
PMCID: PMC2192972  PMID: 10377183
virus; infection; cytotoxic T lymphocyte; dendritic cell
8.  Developmental Regulation of Lck Targeting to the CD8 Coreceptor Controls Signaling in Naive and Memory T Cells  
The Journal of Experimental Medicine  1999;189(10):1521-1530.
The question of whether enhanced memory T cell responses are simply due to an increased frequency of specific cells or also to an improved response at the single cell level is widely debated. In this study, we analyzed T cell receptor (TCR) transgenic memory T cells and bona fide memory T cells isolated from virally infected normal mice using the tetramer technology. We found that memory T cells are qualitatively different from naive T cells due to a developmentally regulated rearrangement of the topology of the signaling machinery. In naive cytotoxic T cells, only a few CD8 molecules are associated with Lck and the kinase is homogeneously distributed inside the cell. However, in vivo priming of naive T cells induces the targeting of Lck to the CD8 coreceptor in the cell membrane and the consequent organization of a more efficient TCR signaling machinery in effector and memory cells.
PMCID: PMC2193636  PMID: 10330431
memory; virus; costimulation
9.  TRANCE, a Tumor Necrosis Factor Family Member Critical for CD40 Ligand–independent T Helper Cell Activation  
The Journal of Experimental Medicine  1999;189(7):1025-1031.
CD40 ligand (CD40L), a tumor necrosis factor (TNF) family member, plays a critical role in antigen-specific T cell responses in vivo. CD40L expressed on activated CD4+ T cells stimulates antigen-presenting cells such as dendritic cells, resulting in the upregulation of costimulatory molecules and the production of various inflammatory cytokines required for CD4+ T cell priming in vivo. However, CD40L- or CD40-deficient mice challenged with viruses mount protective CD4+ T cell responses that produce normal levels of interferon γ, suggesting a CD40L/CD40-independent mechanism of CD4+ T cell priming that to date has not been elucidated. Here we show that CD4+ T cell responses to viral infection were greatly diminished in CD40-deficient mice by administration of a soluble form of TNF-related activation-induced cytokine receptor (TRANCE-R) to inhibit the function of another TNF family member, TRANCE. Thus, the TRANCE/TRANCE-R interaction provides costimulation required for efficient CD4+ T cell priming during viral infection in the absence of CD40L/CD40. These results also indicate that not even the potent inflammatory microenvironment induced by viral infections is sufficient to elicit efficient CD4+ T cell priming without proper costimulation provided by the TNF family (CD40L or TRANCE). Moreover, the data suggest that TRANCE/TRANCE-R may be a novel and important target for immune intervention.
PMCID: PMC2193017  PMID: 10190893
TRANCE; CD40 ligand; T cell; dendritic cell; virus
10.  Therapeutic vaccines for chronic diseases: successes and technical challenges 
Chronic, non-communicable diseases are the major cause of death and disability worldwide and have replaced infectious diseases as the major burden of society in large parts of the world. Despite the complexity of chronic diseases, relatively few predisposing risk factors have been identified by the World Health Organization. Those include smoking, alcohol abuse, obesity, high cholesterol and high blood pressure as the cause of many of these chronic conditions. Here, we discuss several examples of vaccines that target these risk factors with the aim of preventing the associated diseases and some of the challenges they face.
PMCID: PMC3146783  PMID: 21893545
therapeutic vaccines; chronic diseases; smoking cessation vaccine
11.  Selective utilization of Toll-like receptor and MyD88 signaling in B cells for enhancement of the anti-viral germinal center response 
Immunity  2011;34(3):375-384.
The contribution of Toll-like receptor (TLR) signaling to T cell-dependent (TD) antibody responses was assessed by using mice lacking the TLR signaling adaptor MyD88 in individual cell types. When a soluble TLR9 ligand was used as adjuvant for a protein antigen, MyD88 was required in dendritic cells but not in B cells to enhance the TD antibody response, regardless of the inherent immunogenicity of the antigen. In contrast, a TLR9 ligand contained within a virus-like particle substantially augmented the TD germinal center IgG antibody response, and this augmentation required B cell MyD88. The ability of B cells to discriminate between antigens based the physical form of a TLR ligand likely reflects an adaptation to facilitate strong anti-viral antibody responses.
PMCID: PMC3064721  PMID: 21353603
Toll-like receptor; MyD88; T cell-dependent antibody response; dendritic cells; B cells; virus-like particle; virus
12.  A VLP-based vaccine targeting domain III of the West Nile virus E protein protects from lethal infection in mice 
Virology Journal  2010;7:146.
Since its first appearance in the USA in 1999, West Nile virus (WNV) has spread in the Western hemisphere and continues to represent an important public health concern. In the absence of effective treatment, there is a medical need for the development of a safe and efficient vaccine. Live attenuated WNV vaccines have shown promise in preclinical and clinical studies but might carry inherent risks due to the possibility of reversion to more virulent forms. Subunit vaccines based on the large envelope (E) glycoprotein of WNV have therefore been explored as an alternative approach. Although these vaccines were shown to protect from disease in animal models, multiple injections and/or strong adjuvants were required to reach efficacy, underscoring the need for more immunogenic, yet safe DIII-based vaccines.
We produced a conjugate vaccine against WNV consisting of recombinantly expressed domain III (DIII) of the E glycoprotein chemically cross-linked to virus-like particles derived from the recently discovered bacteriophage AP205. In contrast to isolated DIII protein, which required three administrations to induce detectable antibody titers in mice, high titers of DIII-specific antibodies were induced after a single injection of the conjugate vaccine. These antibodies were able to neutralize the virus in vitro and provided partial protection from a challenge with a lethal dose of WNV. Three injections of the vaccine induced high titers of virus-neutralizing antibodies, and completely protected mice from WNV infection.
The immunogenicity of DIII can be strongly enhanced by conjugation to virus-like particles of the bacteriophage AP205. The superior immunogenicity of the conjugate vaccine with respect to other DIII-based subunit vaccines, its anticipated favourable safety profile and low production costs highlight its potential as an efficacious and cost-effective prophylaxis against WNV.
PMCID: PMC2914671  PMID: 20604940
13.  Versatile Virus-Like Particle Carrier for Epitope Based Vaccines 
PLoS ONE  2010;5(3):e9809.
Recombinant proteins and in particular single domains or peptides are often poorly immunogenic unless conjugated to a carrier protein. Virus-like-particles are a very efficient means to confer high immunogenicity to antigens. We report here the development of virus-like-particles (VLPs) derived from the RNA bacteriophage AP205 for epitope-based vaccines.
Methodology/Principal Findings
Peptides of angiotensin II, S.typhi outer membrane protein (D2), CXCR4 receptor, HIV1 Nef, gonadotropin releasing hormone (GnRH), Influenza A M2-protein were fused to either N- or C-terminus of AP205 coat protein. The A205-peptide fusions assembled into VLPs, and peptides displayed on the VLP were highly immunogenic in mice. GnRH fused to the C-terminus of AP205 induced a strong antibody response that inhibited GnRH function in vivo. Exposure of the M2-protein peptide at the N-terminus of AP205 resulted in a strong M2-specific antibody response upon immunization, protecting 100% of mice from a lethal influenza infection.
AP205 VLPs are therefore a very efficient and new vaccine system, suitable for complex and long epitopes, of up to at least 55 amino acid residues in length. AP205 VLPs confer a high immunogenicity to displayed epitopes, as shown by inhibition of endogenous GnRH and protective immunity against influenza infection.
PMCID: PMC2843720  PMID: 20352110
14.  mTOR regulates memory CD8 T cell differentiation 
Nature  2009;460(7251):108-112.
Memory CD8 T cells are a critical component of protective immunity and inducing effective memory T cell responses is a major goal of vaccines against chronic infections and tumors 1-3. Considerable effort has gone into designing vaccine regimens that will increase the magnitude of the memory response but there has been minimal emphasis on developing strategies to improve the functional qualities of memory T cells 4. In this study we show that mTOR, the mammalian target of rapamycin 5, is a major regulator of memory CD8 T cell differentiation and in contrast to what we expected the mTOR specific inhibitor rapamycin, an immunosuppressive drug, had surprising immunostimulatory effects on the generation of memory CD8 T cells. Treatment of mice with rapamycin following acute lymphocytic choriomeningitis virus (LCMV) infection enhanced not only the quantity but also the quality of virus specific CD8 T cells. Similar effects were seen after immunization of mice with a non-replicating VLP based vaccine. In addition, rapamycin treatment also enhanced memory T cell responses in non-human primates following vaccination with MVA (modified vaccinia virus - Ankara). Rapamycin was effective during both the expansion and contraction phases of the T cell response; during the expansion phase it increased the number of memory precursors and during the contraction phase (effector to memory transition) it accelerated the memory T cell differentiation program. Experiments using RNAi to inhibit mTOR, raptor or FKBP12 expression in antigen specific CD8 T cells showed that mTOR acts intrinsically through the mTORC1 pathway to regulate memory T cell differentiation. Thus, these studies identify a molecular pathway regulating memory formation and provide an effective strategy for improving the functional qualities of vaccine or infection induced memory T cells.
PMCID: PMC2710807  PMID: 19543266
15.  Prophylactic and therapeutic activity of fully human monoclonal antibodies directed against Influenza A M2 protein 
Virology Journal  2009;6:224.
Influenza virus infection is a prevalent disease in humans. Antibodies against hemagglutinin have been shown to prevent infection and hence hemagglutinin is the major constituent of current vaccines. Antibodies directed against the highly conserved extracellular domain of M2 have also been shown to mediate protection against Influenza A infection in various animal models. Active vaccination is generally considered the best approach to combat viral diseases. However, passive immunization is an attractive alternative, particularly in acutely exposed or immune compromized individuals, young children and the elderly. We recently described a novel method for the rapid isolation of natural human antibodies by mammalian cell display. Here we used this approach to isolate human monoclonal antibodies directed against the highly conserved extracellular domain of the Influenza A M2 protein. The identified antibodies bound M2 peptide with high affinities, recognized native cell-surface expressed M2 and protected mice from a lethal influenza virus challenge. Moreover, therapeutic treatment up to 2 days after infection was effective, suggesting that M2-specific monoclonals have a great potential as immunotherapeutic agents against Influenza infection.
PMCID: PMC2804611  PMID: 20025741
16.  Vaccination against GIP for the Treatment of Obesity 
PLoS ONE  2008;3(9):e3163.
According to the WHO, more than 1 billion people worldwide are overweight and at risk of developing chronic illnesses, including cardiovascular disease, type 2 diabetes, hypertension and stroke. Current therapies show limited efficacy and are often associated with unpleasant side-effect profiles, hence there is a medical need for new therapeutic interventions in the field of obesity. Gastric inhibitory peptide (GIP, also known as glucose-dependent insulinotropic polypeptide) has recently been postulated to link over-nutrition with obesity. In fact GIP receptor-deficient mice (GIPR−/−) were shown to be completely protected from diet-induced obesity. Thus, disrupting GIP signaling represents a promising novel therapeutic strategy for the treatment of obesity.
Methodology/Principal Findings
In order to block GIP signaling we chose an active vaccination approach using GIP peptides covalently attached to virus-like particles (VLP-GIP). Vaccination of mice with VLP-GIP induced high titers of specific antibodies and efficiently reduced body weight gain in animals fed a high fat diet. The reduction in body weight gain could be attributed to reduced accumulation of fat. Moreover, increased weight loss was observed in obese mice vaccinated with VLP-GIP. Importantly, despite the incretin action of GIP, VLP-GIP-treated mice did not show signs of glucose intolerance.
This study shows that vaccination against GIP was safe and effective. Thus active vaccination may represent a novel, long-lasting treatment for obesity. However further preclinical safety/toxicology studies will be required before the therapeutic concept can be addressed in humans.
PMCID: PMC2525840  PMID: 18779862
17.  A Vaccine against Nicotine for Smoking Cessation: A Randomized Controlled Trial 
PLoS ONE  2008;3(6):e2547.
Tobacco dependence is the leading cause of preventable death and disabilities worldwide and nicotine is the main substance responsible for the addiction to tobacco. A vaccine against nicotine was tested in a 6-month randomized, double blind phase II smoking cessation study in 341 smokers with a subsequent 6-month follow-up period.
Methodology/Principal Findings
229 subjects were randomized to receive five intramuscular injections of the nicotine vaccine and 112 to receive placebo at monthly intervals. All subjects received individual behavioral smoking cessation counseling. The vaccine was safe, generally well tolerated and highly immunogenic, inducing a 100% antibody responder rate after the first injection. Point prevalence of abstinence at month 2 showed a statistically significant difference between subjects treated with Nicotine-Qβ (47.2%) and placebo (35.1%) (P = 0.036), but continuous abstinence between months 2 and 6 was not significantly different. However, in subgroup analysis of the per-protocol population, the third of subjects with highest antibody levels showed higher continuous abstinence from month 2 until month 6 (56.6%) than placebo treated participants (31.3%) (OR 2.9; P = 0.004) while medium and low antibody levels did not increase abstinence rates. After 12 month, the difference in continuous abstinence rate between subjects on placebo and those with high antibody response was maintained (difference 20.2%, P = 0.012).
Whereas Nicotine-Qβ did not significantly increase continuous abstinence rates in the intention-to-treat population, subgroup analyses of the per-protocol population suggest that such a vaccination against nicotine can significantly increase continuous abstinence rates in smokers when sufficiently high antibody levels are achieved. Immunotherapy might open a new avenue to the treatment of nicotine addiction.
Trial Registration
Swiss Medical Registry 2003DR2327; NCT00369616
PMCID: PMC2432028  PMID: 18575629
18.  Potent T cell agonism mediated by a very rapid TCR/pMHC interaction 
European Journal of Immunology  2007;37(3):798-806.
The interaction between T cell receptors (TCR) and peptide-major histocompatibility complex (pMHC) antigens can lead to varying degrees of agonism (T cell activation), or antagonism. The P14 TCR recognises the lymphocytic choriomeningitis virus (LCMV)-derived peptide, gp33 residues 33–41 (KAVYNFATC), presented in the context of H-2Db. The cellular responses to various related H-2Db peptide ligands are very well characterised, and P14 TCR-transgenic mice have been used extensively in models of virus infection, autoimmunity and tumour rejection. Here, we analyse the binding of the P14 soluble TCR to a broad panel of related H-2Db-peptide complexes by surface plasmon resonance, and compare this with their diverse cellular responses. P14 TCR binds H-2Db-gp33 with a KD of 3 µM (±0.5 µM), typical of an immunodominant antiviral TCR, but with unusually fast kinetics (koff=1 s−1), corresponding to a half-life of 0.7 s at 25°C, outside the range previously observed for murine agonist TCR/pMHC interactions. The most striking feature of these data is that a very short half-life does not preclude the ability of a TCR/pMHC interaction to induce antiviral immunity, autoimmune disease and tumour rejection.
PMCID: PMC2435421  PMID: 17295390
Biophysics; Protein-protein interactions; TCR
19.  Interleukin-1 Is Responsible for Acute Lung Immunopathology but Increases Survival of Respiratory Influenza Virus Infection 
Journal of Virology  2005;79(10):6441-6448.
Interleukin-1α (IL-1α) and IL-1β are proinflammatory cytokines, which induce a plethora of genes and activities by binding to the type 1 IL-1 receptor (IL-1R1). We have investigated the role of IL-1 during pulmonary antiviral immune responses in IL-1R1−/− mice infected with influenza virus. IL-1R1−/− mice showed markedly reduced inflammatory pathology in the lung, primarily due to impaired neutrophil recruitment. Activation of CD4+ T cells in secondary lymphoid organs and subsequent migration to the lung were impaired in the absence of IL-1R1. In contrast, activation of virus-specific cytotoxic T lymphocytes and killing of virus-infected cells in the lung were intact. Influenza virus-specific immunoglobulin G (IgG) and IgA antibody responses were intact, while the IgM response was markedly reduced in both serum and mucosal sites in IL-1R1−/− mice. We found significantly increased mortality in the absence of IL-1R1; however, lung viral titers were only moderately increased. Our results demonstrate that IL-1α/β mediate acute pulmonary inflammatory pathology while enhancing survival during influenza virus infection. IL-1α/β appear not to influence killing of virus-infected cells but to enhance IgM antibody responses and recruitment of CD4+ T cells to the site of infection.
PMCID: PMC1091664  PMID: 15858027
20.  Virus-Like Particles as Carriers for T-Cell Epitopes: Limited Inhibition of T-Cell Priming by Carrier-Specific Antibodies 
Journal of Virology  2005;79(2):717-724.
Virus-like particles (VLPs) are able to induce cytotoxic T-cell responses in the absence of infection or replication. This makes VLPs promising candidates for the development of recombinant vaccines. However, VLPs are also potent inducers of B-cell responses, and it is generally assumed that such VLP-specific antibodies interfere with the induction of protective immune responses, a phenomenon summarized as carrier suppression. In this study, we investigated the impact of preexisting VLP-specific antibodies on the induction of specific cytotoxic T-cell and Th-cell responses in mice. The data show that VLP-specific antibodies did not measurably reduce antigen presentation in vitro or in vivo. Nevertheless, T-cell priming was slightly reduced by antigen-specific antibodies; however, the overall reduction was limited and vaccination with VLPs in the presence of VLP-specific antibodies still resulted in protective T-cell responses. Thus, carrier suppression is unlikely to be a limiting factor for VLP-based T-cell vaccines.
PMCID: PMC538584  PMID: 15613299
21.  CpG Oligodeoxynucleotides Block Human Immunodeficiency Virus Type 1 Replication in Human Lymphoid Tissue Infected Ex Vivo 
Journal of Virology  2004;78(22):12344-12354.
Oligodeoxynucleotides (ODNs) with immunomodulatory motifs control a number of microbial infections in animal models, presumably by acting through toll-like receptor 9 (TLR9) to induce a number of cytokines (e.g., alpha interferon and tumor necrosis factor alpha). The immunomodulatory motif consists of unmethylated sequences of cytosine and guanosine (CpG motif). ODNs without CpG motifs do not trigger TLR9. We hypothesized that triggering of TLR9 generates a cellular environment unfavorable for human immunodeficiency virus (HIV) replication. We tested this hypothesis in human lymphocyte cultures and found that phosphorothioate-modified ODN CpG2006 (type B ODNs) inhibited HIV replication nearly completely and prevented the loss of CD4+ T cells. ODNs CpG2216 and CpG10 (type A ODNs) were less effective. CpG2006 blocked HIV replication in purified CD4+ T cells and T-cell lines; CpG10 was ineffective in this setting, indicating that type A ODNs may inhibit HIV replication in CD4+ T-cell lines indirectly through a separate cell subset. However, control ODNs without CpG motifs also showed anti-HIV effects, indicating that these effects are nonspecific and not due to TLR9 triggering. The mechanism of action is not clear. CpG2006 and its control ODN blocked syncytium formation in a cell fusion-based assay, but CpG10, CpG2216, and their control ODNs did not. The latter types interfered with the HIV replication cycle during disassembly or reverse transcription. In contrast, CpG2006 and CpG2216 specifically induced cytokines critical to initiation of the innate immune response. In summary, the nonspecific anti-HIV activity of CpG ODNs, their ability to stimulate HIV replication in latently infected cells, potentially resulting in their elimination, and their documented ability to link the innate and adaptive immune responses make them attractive candidates for further study as anti-HIV drugs.
PMCID: PMC525063  PMID: 15507621
22.  The Human Membrane Cofactor CD46 Is a Receptor for Species B Adenovirus Serotype 3 
Journal of Virology  2004;78(9):4454-4462.
Many human adenovirus (Ad) serotypes use the coxsackie B virus-Ad receptor (CAR). Recently, CD46 was suggested to be a receptor of species B Ad serotype 11 (Ad11), Ad14, Ad16, Ad21, Ad35, and Ad50. Using Sindbis virus-mediated cDNA library expression, we identify here the membrane cofactor protein CD46 as a surface receptor of species B Ad3. All four major CD46 transcripts and one minor CD46 transcript expressed in nucleated human cells were isolated. Rodent BHK cells stably expressing the BC1 form of CD46 bound radiolabeled Ad3 with a dissociation constant of 0.3 nM, identical to that of CD46-positive HeLa cells expressing twice as many Ad3 binding sites. Pull-down experiments with recombinant Ad3 fibers and a soluble form of the CD46 extracellular domain linked to the Fc portion of human immunoglobulin G (CD46ex-Fc) indicated direct interactions of the Ad3 fiber knob with CD46ex-Fc but not CARex-Fc (Fc-linked extracellular domain of CAR). Ad3 colocalized with cell surface CD46 in both rodent and human cells at the light and electron microscopy levels. Anti-CD46 antibodies and CD46ex-Fc inhibited Ad3 binding to CD46-expressing BHK cells more than 10-fold and to human cells 2-fold. In CD46-expressing BHK cells, wild-type Ad3 and a chimeric Ad consisting of the Ad5 capsid and the Ad3 fiber elicited dose-dependent cytopathic effects and transgene expression, albeit less efficiently than in human cells. Together, our results show that all of the major splice forms of CD46 are predominant and functional binding sites of Ad3 on CD46-expressing rodent and human cells but may not be the sole receptor of species B Ads on human cells. These results have implications for understanding viral pathogenesis and therapeutic gene delivery.
PMCID: PMC387694  PMID: 15078926
23.  New applications of alphavirus-based expression vectors 
Cytotechnology  2001;35(3):203-212.
Alphaviruses are positive stranded RNA viruses that replicate to extremely high titers. Sindbis and Semliki Forest viral vectors are widely used tools for high-level production of recombinant proteins. Recent studies have broadened their scope to vaccine production, gene therapy, and analysis of cell function. Here we discuss the development of non-cytopathic and inducible expression vectors which can be applied to bioprocess development strategies. Furthermore, a Sindbis-based expression cloning system has been developed that allows for the rapid identification of genes encoding proteins with a selected functional activity.
PMCID: PMC3449701  PMID: 22358860
alphavirus; expression; gene therapy; inducibleexpression; neurobiology; non-cytopathic replicon; screening; vaccine development
25.  Inducible Costimulator Protein (Icos) Controls T Helper Cell Subset Polarization after Virus and Parasite Infection 
It has been shown that certain pathogens can trigger efficient T cell responses in the absence of CD28, a key costimulatory receptor expressed on resting T cells. Inducible costimulator protein (ICOS) is an inducible costimulator structurally and functionally related to CD28. Here, we show that in the absence of CD28 both T helper cell type 1 (Th1) and Th2 responses were impaired but not abrogated after infection with lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus (VSV), and the nematode Nippostrongylus brasiliensis. Inhibition of ICOS in CD28-deficient mice further reduced Th1/Th2 polarization. Blocking of ICOS alone had a limited but significant capacity to downregulate Th subset development. In contrast, cytotoxic T lymphocyte (CTL) responses, which are regulated to a minor and major extent by CD28 after LCMV and VSV infection, respectively, remained unaffected by blocking ICOS. Together, our results demonstrate that ICOS regulates both CD28-dependent and CD28-independent CD4+ subset (Th1 and Th2) responses but not CTL responses in vivo.
PMCID: PMC1887704  PMID: 10880526
ICOS; CD28; Th1/Th2; Nippostrongylus brasiliensis; LCMV

Results 1-25 (31)