OmpL1 Is an Extracellular Matrix- and Plasminogen-Interacting Protein of Leptospira spp.

Leptospirosis is a zoonosis with multisystem involvement caused by pathogenic strains of the genus Leptospira. OmpL1 is an outer membrane protein of Leptospira spp. that is expressed during infection. In this work, we investigated novel features of this protein. We describe that OmpL1 is a novel leptospiral extracellular matrix (ECM)-binding protein and a plasminogen (PLG) receptor. The recombinant protein was expressed in Escherichia coli BL21(DE3) Star/pLysS as inclusion bodies, refolded, and purified by metal-chelating chromatography. The protein presented a typical beta-strand secondary structure, as evaluated by circular dichroism spectroscopy. The recombinant protein reacted with antibodies in serum samples from convalescent leptospirosis patients with a high specificity compared to serum samples from individuals with unrelated diseases. These data strengthen the usefulness of OmpL1 as a diagnostic marker of leptospirosis. The characterization of the immunogenicity of recombinant OmpL1 in inoculated BALB/c mice showed that the protein has the capacity to elicit humoral and cellular immune responses, as denoted by high antibody titers and the proliferation of lymphocytes. We demonstrate that OmpL1 has the ability to mediate attachment to laminin and plasma fibronectin, with KD (equilibrium dissociation constant) values of 2,099.93 +/- 871.03 nM and 1,239.23 +/- 506.85 nM, respectively. OmpL1 is also a PLG receptor, with a KD of 368.63 +/- 121.23 nM, capable of generating enzymatically active plasmin. This is the first report that shows and characterizes OmpL1 as an ECM-interacting and a PLG-binding protein of Leptospira spp. that may play a role in bacterial pathogenesis when expressed during infection.

A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells

Abstract Background A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. These findings have attracted considerable interest in the field of DNA vaccination. We have previously described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis. As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses. Methods To address this question, we analysed the activation of both human macrophages and dendritic cells (DCs) cultured with DNA-HSP65. Then, these cells stimulated with the DNA vaccine were evaluated regarding the expression of surface markers, cytokine production and microbicidal activity. Results It was observed that DCs and macrophages presented different ability to uptake DNA vaccine. Under DNA stimulation, macrophages, characterized as CD11b+/CD86+/HLA-DR+, produced high levels of TNF-alpha, IL-6 (pro-inflammatory cytokines), and IL-10 (anti-inflammatory cytokine). Besides, they also presented a microbicidal activity higher than that observed in DCs after infection with M. tuberculosis. On the other hand, DCs, characterized as CD11c+/CD86+/CD123-/BDCA-4+/IFN-alpha-, produced high levels of IL-12 and low levels of TNF-alpha, IL-6 and IL-10. Finally, the DNA-HSP65 vaccine was able to induce proliferation of peripheral blood lymphocytes. Conclusion Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans. These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.

Targeting the non-structural protein 1 from dengue virus to a dendritic cell population confers protective immunity to lethal virus challenge

Dengue is the most prevalent arboviral infection, affecting millions of people every year. Attempts to control such infection are being made, and the development of a vaccine is a World Health Organization priority. Among the proteins being tested as vaccine candidates in preclinical settings is the non-structural protein 1 (NS1). In the present study, we tested the immune responses generated by targeting the NS1 protein to two different dendritic cell populations. Dendritic cells (DCs) are important antigen presenting cells, and targeting proteins to maturing DCs has proved to be an efficient means of immunization. Antigen targeting is accomplished by the use of a monoclonal antibody (mAb) directed against a DC cell surface receptor fused to the protein of interest. We used two mAbs (αDEC205 and αDCIR2) to target two distinct DC populations, expressing either DEC205 or DCIR2 endocytic receptors, respectively, in mice. The fusion mAbs were successfully produced, bound to their respective receptors, and were used to immunize BALB/c mice in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)), as a DC maturation stimulus. We observed induction of strong anti-NS1 antibody responses and similar antigen binding affinity irrespectively of the DC population targeted. Nevertheless, the IgG1/IgG2a ratios were different between mouse groups immunized with αDEC-NS1 and αDCIR2-NS1 mAbs. When we tested the induction of cellular immune responses, the number of IFN-γ producing cells was higher in αDEC-NS1 immunized animals. In addition, mice immunized with the αDEC-NS1 mAb were significantly protected from a lethal intracranial challenge with the DENV2 NGC strain when compared to mice immunized with αDCIR2-NS1 mAb. Protection was partially mediated by CD4(+) and CD8(+) T cells as depletion of these populations reduced both survival and morbidity signs. We conclude that targeting the NS1 protein to the DEC205(+) DC population with poly (I:C) opens perspectives for dengue vaccine development.

RNA-basierte Impfstoffe zur Induktion optimaler Immunantworten

Antigen-kodierende RNA wird als eine sichere und effiziente Alternative zu traditionellen Impfstoff-Formulierungen, wie Peptid-, Protein-, rekombinanten viralen oder DNA basierten Impfstoffen betrachtet. Der endgültige klinische Nutzen RNA-basierter Impfstoffe wird von der Optimierung verschiedener Parameter abhängig sein, die zur Induktion und effizienten Expansion der humoralen und zellvermittelten Immunantwort beitragen. Vor diesem Hintergrund war die Zielsetzung der vorliegenden Arbeit, die Etablierung pharmakologischer und immunologischer Parameter für die Generierung effektiver Immunantworten durch RNA-Impfstoffe sowie deren Wirksamkeit in vitro und im Mausmodell unter Nutzung von Modellantigenen zu testen. Zur Untersuchung und Optimierung der RNA-Pharmakokinetik, als einem Schlüsselaspekt der klinischen Medikamentenentwicklung, wurde der Einfluss von strukturellen Modifikationen auf die Transkriptstabilität und Translationseffizienz von Reporter-Proteinen in einer zeitabhängigen Kinetik evaluiert. Es wurde gezeigt, dass ein poly(A) Schwanz von 120 Adenosinen, verglichen mit einem kürzeren, ein freies 3´ poly(A) Ende, verglichen mit einem verdeckten und eine doppelte β-globin 3´ UTR, unabhängig voneinander zu einer Erhöhung der IVT-RNA Stabilität und zu einer Verbesserung der Translationseffizienz beitrugen und dadurch insgesamt zu einer erhöhten Proteinexpression führten. Antigen-kodierende IVT-RNA mit diesen molekularen Merkmalen in Kombination führte, im Vergleich zur Standard IVT-RNA, zu einer erhöhten Dichte und Stabilität von Peptid/MHC-Komplexen auf der Zelloberfläche transfizierter DCs und dadurch zu einer verbesserten Stimulation von CD4+ und CD8+ T-Zellen im murinen und humanen System. Mit dem Ziel, die RNA kodierte Antigenform für die Induktion einer verstärkten Antikörperantwort zu modifizieren, wurde im zweiten Teil der Arbeit ein Antigen-IgM Fusionskonstrukt hergestellt und hinsichtlich seiner Eignung als neues Impfstoff-Format untersucht. Die Ausgangshypothese, dass die RNA kodierten Antigen-IgM Fusionsproteine polymerisieren, von transfizierten Zellen sezerniert werden und aufgrund der repetitiven Antigenstruktur im Vergleich mit dem monomeren Antigen zu einer Verstärkung der Antikörperantwort führen, wurde in vitro und in vivo im Mausmodell bestätigt. Die Entwicklung und Evaluierung von Zytokinfusionsproteinen zur selektiven Verstärkung der antigenspezifischen Immunantworten bildeten den dritten Schwerpunkt der vorliegenden Arbeit. Zur weiteren Verstärkung der Antikörperantwort wurde basierend auf den Resultaten aus dem zweiten Teil ein IL2-IgM Fusionskonstrukt hergestellt. Die Ko-Transfektion von Antigen-IgM und IL2-IgM kodierender IVT-RNA führte zu einer signifikant stärkeren Antikörperantwort als die Ko-Transfektion von Antigen-IgM und IL2. Für die Initiierung einer erfolgreichen anti-Tumor-Immunantwort ist das Priming antigenspezifischer T-Zellen essentiell. Um die Effizienz dieses Prozesses zu steigern, wurde ein bifunktionelles IL2-mCD40L Fusionskonstrukt hergestellt und sein Einfluss auf die Effektorfunktion von DCs in vitro und in vivo untersucht. Es wurde gezeigt, dass ein RNA kodiertes IL2-mCD40L Fusionsprotein als genetisches Adjuvanz zu einer Effizienzsteigerung des Priming zytotoxischer T-Zellen führt. Somit wurden in dieser Arbeit durch die Optimierung der Pharmakokinetik, die Modifikation der Antigenform und die Herstellung und Evaluierung von Zytokinfusionskonstrukten als genetische Adjuvantien, RNA-basierte Impfstoffe für eine optimierte Induktion von antigenspezifischen Immunantworten weiter verbessert.

Optimierung einer partikulären Cytomegalovirus-Vakzine

Pränatale Infektionen mit dem humanen Cytomegalovirus (HCMV) sind die häufigste Ursache frühkindlicher Schädigung, noch vor dem Down-Syndrom oder dem fetalen Alkoholsyndrom. Reaktivierung dieses Herpesvirus ist darüber hinaus als lebensbedrohliche Komplikation in der Transplantationsmedizin gefürchtet. Von Experten wurde daher die Entwicklung einer Vakzine vielfach angemahnt. Trotz unterschiedlicher Ansätze zu ihrer Entwicklung ist bisher jedoch kein Impfstoff verfügbar. Die Verwendung von subviralen Dense Bodies (DB) des Virus als Vakzinegrundlage stellt eine vielversprechende Strategie zur HCMV-Impfstoffentwicklung dar. DB enthalten bereits in ihrer natürlichen Form wichtige Zielantigene der humoralen und zellulären Immunantwort gegen HCMV. Durch gezielte Mutation des 230.000 Basenpaare umfassenden Genoms des HCMV konnte in Vorarbeiten der Beweis erbracht werden, dass DB hinsichtlich ihres antigenen Repertoires optimierbar sind. Allerdings waren Immunogenität und erzielte Ausbeuten noch unbefriedigend. Ziel der vorliegenden Arbeit war es, den Ansatz der Verwendung modifizierter DB als Impfstoff-Grundlage weiter zu entwickeln und Erkenntnisse über die für die Partikelbildung entscheidenden molekularen Mechanismen zu erarbeiten. In einem ersten Abschnitt wurde der Ansatz der Modifikation von DB durch Insertion heterologer Peptidantigene in das virale Tegumentprotein pp65 verfeinert. Das pp65 ist die mengenmäßig dominante Komponente von DB. Durch Herstellung und Austestung definierter HCMV Mutanten konnte die Position 175 des pp65 als geeignete Insertionsstelle für virale wie für nicht-virale Antigene identifiziert werden. In einem zweiten Schritt der Arbeit wurde die Rolle des pp65 im Verlauf der viralen Vermehrung und Morphogenese näher untersucht. Grundlage für diese Analysen war eine Virusmutante, die eine dominant-negative Variante des pp65 exprimierte. Vergleichende massenspektrometrische Untersuchungen unter Einbeziehung von pp65-kompetenten und pp65-negativen Virusmutanten zeigten, dass pp65 in der spät-infizierten Zelle mit dem viralen RNA-Exportfaktor pUL69 und der virale Kinase pUL97 komplexiert vorkommt. Das pp65 wurde als Substrat von pUL97 identifiziert. Daneben wurden essentielle Proteine des viralen Replikationsapparates, sowie zelluläre Proteine des RNA-Metabolismus und Transports und virale DNA in diesen Komplexen gefunden. Die Ergebnisse deuteten darauf hin, dass pp65 zu späten Zeitpunkten der viralen Infektion zu Stellen viraler DNA rekrutiert wird und dort regulatorisch in posttranskriptionelle Vorgänge von RNA Prozessierung oder RNA Transport eingreift. Die Hypothese, dass pp65 einen regulatorischen Einfluss auf die RNA-Exportfunktion von pUL69 nimmt, liegt nahe und ist nun in weiteren Analysen prüfbar.

Constrained pattern of viral evolution in acute and early HCV infection limits viral plasticity

Cellular immune responses during acute Hepatitis C virus (HCV) and HIV infection are a known correlate of infection outcome. Viral adaptation to these responses via mutation(s) within CD8+ T-cell epitopes allows these viruses to subvert host immune control. This study examined HCV evolution in 21 HCV genotype 1-infected subjects to characterise the level of viral adaptation during acute and early HCV infection. Of the total mutations observed 25% were within described CD8+ T-cell epitopes or at viral adaptation sites. Most mutations were maintained into the chronic phase of HCV infection (75%). The lack of reversion of adaptations and high proportion of silent substitutions suggests that HCV has structural and functional limitations that constrain evolution. These results were compared to the pattern of viral evolution observed in 98 subjects during a similar phase in HIV infection from a previous study. In contrast to HCV, evolution during acute HIV infection is marked by high levels of amino acid change relative to silent substitutions, including a higher proportion of adaptations, likely reflecting strong and continued CD8+ T-cell pressure combined with greater plasticity of the virus. Understanding viral escape dynamics for these two viruses is important for effective T cell vaccine design.

Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines

CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.

Detection of latent tuberculosis in immunosuppressed patients with autoimmune diseases: performance of a Mycobacterium tuberculosis antigen-specific interferon gamma assay

OBJECTIVE: To analyse the performance of a new M. tuberculosis-specific interferon gamma (IFNgamma) assay in patients with chronic inflammatory diseases who receive immunosuppressive drugs, including tumour necrosis factor alpha (TNFalpha) inhibitors. METHODS: Cellular immune responses to the M. tuberculosis-specific antigens ESAT-6, CFP-10, TB7.7 were prospectively studied in 142 consecutive patients treated for inflammatory rheumatic conditions. Results were compared with tuberculin skin tests (TSTs). Association of both tests with risk factors for latent M. tuberculosis infection (LTBI) and BCG vaccination were determined and the influence of TNFalpha inhibitors, corticosteroids, and disease modifying antirheumatic drugs (DMARDs) on antigen-specific and mitogen-induced IFNgamma secretion was analysed. RESULTS: 126/142 (89%) patients received immunosuppressive therapy. The IFNgamma assay was more closely associated with the presence of risk factors (odds ratio (OR) = 23.8 (95% CI 5.14 to 110) vs OR = 2.77 (1.22 to 6.27), respectively; p = 0.009), but less associated with BCG vaccination than the TST (OR = 0.47 (95% CI 0.15 to 1.47) vs OR = 2.44 (0.74 to (8.01), respectively; p = 0.025). Agreement between the IFNgamma assay and TST results was low (kappa = 0.17; 95% CI 0.02 to 0.32). The odds for a positive IFNgamma assay strongly increased with increasing prognostic relevance of LTBI risk factors. Neither corticosteroids nor conventional DMARDs significantly affected IFNgamma responses, but the odds for a positive IFNgamma assay were decreased in patients treated with TNFalpha inhibitors (OR = 0.21 (95% CI 0.07 to 0.63), respectively; p = 0.006). CONCLUSIONS: These results demonstrate that the performance of the M. tuberculosis antigen-specific IFNgamma ELISA is better than the classic TST for detection of LTBI in patients receiving immunosuppressive therapy for treatment of systemic autoimmune disorders.

Design, expression, and processing of epitomized hepatitis C virus-encoded CTL epitopes

Hepatitis C virus (HCV) vaccine efficacy may crucially depend on immunogen length and coverage of viral sequence diversity. However, covering a considerable proportion of the circulating viral sequence variants would likely require long immunogens, which for the conserved portions of the viral genome, would contain unnecessarily redundant sequence information. In this study, we present the design and in vitro performance analysis of a novel "epitome" approach that compresses frequent immune targets of the cellular immune response against HCV into a shorter immunogen sequence. Compression of immunological information is achieved by partial overlapping shared sequence motifs between individual epitopes. At the same time, sequence diversity coverage is provided by taking advantage of emerging cross-reactivity patterns among epitope variants so that epitope variants associated with the broadest variant cross-recognition are preferentially included. The processing and presentation analysis of specific epitopes included in such a compressed, in vitro-expressed HCV epitome indicated effective processing of a majority of tested epitopes, although re-presentation of some epitopes may require refined sequence design. Together, the present study establishes the epitome approach as a potential powerful tool for vaccine immunogen design, especially suitable for the induction of cellular immune responses against highly variable pathogens.

CHARACTERIZATION OF ALPHA-GALACTOSYLCERAMIDE AS A MUCOSAL ADJUVANT

Adjuvants are essential components of vaccine formulations that enhance adaptive immune responses to antigens, particularly for immunizations targeting the tolerogenic mucosal tissues, which are more biologically relevant for protective immunity against pathogens transmitted by the mucosal routes. Adjuvants possess the inherent capacity to bridge innate and adaptive immune responses through activating innate immune mediators. Here evidence is presented in support of the effectiveness of a synthetic glycolipid, alpha-Galactosylceramide (-GalCer), as an adjuvant for mucosal immunization with peptide and protein antigens, by oral and intranasal routes, to prime antigen-specific immune responses in multiple systemic and mucosal compartments. The adjuvant activity of -GalCer delivered by the intranasal route was manifested in terms of potent activation of NKT cells, an important innate immunity mediator, along with the activation of dendritic cells (DC) which serve as the professional antigen-presenting cells. Data from this investigation provide the first evidence for mucosal delivery as an effective means to harness the adjuvant potential of α-GalCer for priming as well as boosting cellular immune responses to co-administered immunogens. Unlike systemic administration where a single dose of α-GalCer leads to anergy of responding NKT cells and thus hinders delivery of booster immunizations, we demonstrated that administration of multiple doses of α-GalCer by the intranasal route affords repeated activation of NKT cells and the induction of broad systemic and mucosal immunity. This is specifically advantageous, and may be even essential, for vaccination regimens against mucosal pathogens such as the human immunodeficiency virus (HIV) and the human papillomavirus (HPV), where priming of durable protective immunity at the mucosal portals of pathogen entry would be highly desirable.

Suppression of peripheral blood mononuclear cell proliferation by a periodontopathic bacteria {\it Actinobacillus actinomycetemcomitans\/}

Actinobacillus actinomycetemcomitans (Aa) is a gram-negative coccobacillus implicated as a major pathogen in juvenile periodontitis. The immunosuppressive activity of a sonic extract (designated 100SN) derived from Aa was investigated. 100SN suppressed spontaneous proliferation as well as proliferative response to the mitogens, PHA and PWM, of human peripheral blood mononuclear cells (PBMC). 100SN-induced suppression of PHA-stimulated proliferation was heat-sensitive, inactivated by pronase and trypsin, dose-dependent and non-cytotoxic. There were no significant changes in the CD4$\sp+$ or CD8$\sp+$ subsets of PBMC after 7-day incubation with 100SN. There was a trend toward increased levels of the CD4$\sp+$CD45R$\sp{\rm hi}$CDw29$\sp{\rm lo}$ (naive cells, associated with suppressor-inducer activity) and CD4$\sp+$CDw29$\sp{\rm hi}$CD45R$\sp{\rm lo}$ (memory cells, associated with helper-inducer activity) subsets. The target of 100SN appeared to be the non-adherent cells and suppression by 100SN could not be reversed by indomethacin (IDM), the cyclo-oxygenase inhibitor of prostaglandin (PG) synthesis. The mechanism of 100SN-induced suppression was studied in terms of inhibition involving IL-2-regulated T cell proliferation and the results point to the possibility that suppression occurred subsequent to IL-2 receptor binding.^ The suppressive activity observed could occur through multiple mechanisms including cell-cell; contact or release of soluble factors. Supernatants derived from 7-day cultures of PBMC and 100SN (designated CSN-A) were able to suppress proliferative response of PBMC to PHA without affecting cell viability. Analysis of CSN-A showed that it contained PGE2 and soluble IL-2 receptors. Suppression by CSN-A could be partially overcome by either IDM or exogenous IL-2. Significant suppression was also maintained when both IDM and exogenous IL-2 were added at the same time. These findings suggest that PGE2 and soluble IL-2 receptors contribute to the suppression observed but other suppressive cytokine(s) may be involved. Collectively, the data indicate that a factor derived from oral bacteria associated with juvenile periodontitis have profound effects on cellular immune responses, and that these effects may be partially mediated by secondary factors produced by the host in response to the bacteria. ^

Classical swine fever virus replicon particles lacking the Erns gene: a potential marker vaccine for intradermal application.

Classical swine fever virus replicon particles (CSF-VRP) deficient for E(rns) were evaluated as a non-transmissible marker vaccine. A cDNA clone of CSFV strain Alfort/187 was used to obtain a replication-competent mutant genome (replicon) lacking the sequence encoding the 227 amino acids of the glycoprotein E(rns) (A187delE(rns)). For packaging of A187delE(rns) into virus particles, porcine kidney cell lines constitutively expressing E(rns) of CSFV were established. The rescued VRP were infectious in cell culture but did not yield infectious progeny virus. Single intradermal vaccination of two pigs with 10(7) TCID(50) of VRP A187delE(rns) elicited neutralizing antibodies, anti-E2 antibodies, and cellular immune responses determined by an increase of IFN-gamma producing cells. No anti-E(rns) antibodies were detected in the vaccinees confirming that this vaccine represents a negative marker vaccine allowing differentiation between infected and vaccinated animals. The two pigs were protected against lethal challenge with the highly virulent CSFV strain Eystrup. In contrast, oral immunization resulted in only partial protection, and neither CSFV-specific antibodies nor stimulated T-cells were found before challenge. These data represent a good basis for more extended vaccination/challenge trials including larger numbers of animals as well as more thorough analysis of virus shedding using sentinel animals to monitor horizontal spread of the challenge virus.

Polyethylenimine-based polyplex delivery of self-replicating RNA vaccines

Self-amplifying replicon RNA (RepRNA) are large molecules (12-14kb); their self-replication amplifies mRNA template numbers, affording several rounds of antigen production, effectively increasing vaccine antigen payloads. Their sensitivity to RNase-sensitivity and inefficient uptake by dendritic cells (DCs) - absolute requirements for vaccine design - were tackled by condensing RepRNA into synthetic, nanoparticulate, polyethylenimine (PEI)-polyplex delivery vehicles. Polyplex-delivery formulations for small RNA molecules cannot be transferred to RepRNA due to its greater size and complexity; the N:P charge ratio and impact of RepRNA folding would influence polyplex condensation, post-delivery decompaction and the cytosolic release essential for RepRNA translation. Polyplex-formulations proved successful for delivery of RepRNA encoding influenza virus hemagglutinin and nucleocapsid to DCs. Cytosolic translocation was facilitated, leading to RepRNA translation. This efficacy was confirmed in vivo, inducing both humoral and cellular immune responses. Accordingly, this paper describes the first PEI-polyplexes providing efficient delivery of the complex and large, self-amplifying RepRNA vaccines.

Prophylactic DNA vaccine for hepatitis C virus (HCV) infection: HCV-specific cytotoxic T lymphocyte induction and protection from HCV-recombinant vaccinia infection in an HLA-A2.1 transgenic mouse model

DNA vaccines express antigens intracellularly and effectively induce cellular immune responses. Because only chimpanzees can be used to model human hepatitis C virus (HCV) infections, we developed a small-animal model using HLA-A2.1-transgenic mice to test induction of HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs) and protection against recombinant vaccinia expressing HCV-core. A plasmid encoding the HCV-core antigen induced CD8+ CTLs specific for three conserved endogenously expressed core peptides presented by human HLA-A2.1. When challenged, DNA-immunized mice showed a substantial (5–12 log10) reduction in vaccinia virus titer compared with mock-immunized controls. This protection, lasting at least 14 mo, was shown to be mediated by CD8+ cells. Thus, a DNA vaccine expressing HCV-core is a potential candidate for a prophylactic vaccine for HLA-A2.1+ humans.

Upregulation of class I major histocompatibility complex antigens by interferon gamma is necessary for T-cell-mediated elimination of recombinant adenovirus-infected hepatocytes in vivo.

Recombinant adenoviruses are attractive vehicles for liver-directed gene therapy because of the high efficiency with which they transfer genes to hepatocytes in vivo. First generation recombinant adenoviruses deleted of E1 sequences also express recombinant and early and late viral genes, which lead to development of destructive cellular immune responses. Previous studies indicated that class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTLs) play a major role in eliminating virus-infected cells. The present studies utilize mouse models to evaluate the role of T-helper cells in the primary response to adenovirus-mediated gene transfer to the liver. In vivo ablation of CD4+ cells or interferon gamma (IFN-gamma) was sufficient to prevent the elimination of adenovirus-transduced hepatocytes, despite the induction of a measurable CTL response. Mobilization of an effective TH1 response as measured by in vitro proliferation assays was associated with substantial upregulation of MHC class I expression, an effect that was prevented in IFN-gamma-deficient animals. These results suggest that elimination of virus-infected hepatocytes in a primary exposure to recombinant adenovirus requires both induction of antigen-specific CTLs as well as sensitization of the target cell by TH1-mediated activation of MHC class I expression.