The impact of fibrosis and steatosis on early viral kinetics in HCV genotype 1-infected patients treated with Peg-IFN-alfa-2a and ribavirin.

Summary.  Hepatitis C viral (HCV) kinetics after initiation of interferon-based therapy provide valuable insights for understanding virus pathogenesis, evaluating treatment antiviral effectiveness and predicting treatment outcome. Adverse effects of liver fibrosis and steatosis on sustained virological response have been frequently reported, yet their impacts on the early viral kinetics remain unclear. In this study, associations between histology status and early viral kinetics were assessed in 149 HCV genotype 1-infected patients treated with pegylated interferon alfa-2a and ribavirin (DITTO trial). In multivariate analyses adjusted for critical factors such as IL28B genotype and baseline viral load, presence of significant fibrosis (Ishak stage > 2) was found to independently reduce the odds of achieving an initial reduction (calculated from day 0 to day 4) in HCV RNA of ≥2 logIU/mL (adjusted OR 0.03, P = 0.004) but was not associated with the second-phase slope of viral decline (calculated from day 8 to day 29). On the contrary, presence of liver steatosis was an independent risk factor for not having a rapid second-phase slope, that is, ≥0.3 logIU/mL/week (adjusted OR 0.22, P = 0.012) but was not associated with the first-phase decline. Viral kinetic modelling theory suggests that significant fibrosis primarily impairs the treatment antiviral effectiveness in blocking viral production by infected cells, whereas the presence of steatosis is associated with a lower net loss of infected cells. Further studies will be necessary to identify the biological mechanisms underlain by these findings.

Superantigens of mouse mammary tumor virus.

Superantigens (SAgs) are proteins of microbial origin that bind to major histocompatibility complex (MHC) class II molecules and stimulate T cells via interaction with the V beta domain of the T cell receptor (TCR). Mouse mammary tumor virus (MMTV) is a milk-transmitted type B retrovirus that encodes a SAg in its 3' long terminal repeat. Upon MMTV infection, B cells present SAg to the appropriate T cell subset, which leads to a strong "cognate" T-B interaction. This immune reaction results in preferential clonal expansion of infected B cells and differentiation of some of these cells into long-lived memory cells. In this way a stable MMTV infection is achieved that ultimately results in infection of the mammary gland and virus transmission via milk. Thus, in contrast to many microorganisms that attempt to evade the host immune system (reviewed in 1), MMTV depends upon a strong SAg-induced immune response for its survival. Because of their ability to stimulate very strong T cell responses in MHC-identical mice, minor lymphocyte stimulatory (Mls) antigens, discovered more than 20 years ago, are now known to be SAgs encoded by endogenous MMTV proviruses that have randomly integrated into germ cells. The aim of this review is to combine the extensive biology of Mls SAgs with our current understanding of the life cycle of MMTV.

Estimating the basic reproductive number from viral sequence data.

Epidemiological processes leave a fingerprint in the pattern of genetic structure of virus populations. Here, we provide a new method to infer epidemiological parameters directly from viral sequence data. The method is based on phylogenetic analysis using a birth-death model (BDM) rather than the commonly used coalescent as the model for the epidemiological transmission of the pathogen. Using the BDM has the advantage that transmission and death rates are estimated independently and therefore enables for the first time the estimation of the basic reproductive number of the pathogen using only sequence data, without further assumptions like the average duration of infection. We apply the method to genetic data of the HIV-1 epidemic in Switzerland.

A novel interferon-gamma regulated human melanoma-associated antigen, gp33-38, defined by monoclonal antibody Me14-D12. II. Molecular cloning of a genomic probe.

The human Me14-D12 antigen is a cell surface glycoprotein regulated by interferon-gamma (IFN-gamma) on tumor cell lines of neuroectodermal origin. It consists of two non-convalently linked subunits with apparent mol. wt sizes of 33,000 and 38,000. Here we describe the molecular cloning of a genomic probe for the Me14-D12 gene using the gene transfer approach. Mouse Ltk- cells were stably cotransfected with human genomic DNA and the Herpes Simplex virus thymidine kinase (TK) gene. Primary and secondary transfectants expressing the Me14-D12 antigen were isolated after selection in HAT medium by repeated sorting on a fluorescence activated cell sorter (FACS). A recombinant phage harboring a 14.3 kb insert of human DNA was isolated from a genomic library made from a positive secondary transfectant cell line. A specific probe derived from the phage DNA insert allowed the identification of two mRNAs of 3.5 kb and 2.2 kb in primary and secondary L cell transfectants, as well as in human melanoma cell lines expressing the Me14-D12 antigen. The regulation of Me14-D12 antigen by INF-gamma was retained in the L cell transfectants and could be detected both at the level of protein and mRNA expression.

Viral-mediated overexpression of mutant huntingtin to model HD in various species.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expansion of CAG repeats in the huntingtin (Htt) gene. Despite intensive efforts devoted to investigating the mechanisms of its pathogenesis, effective treatments for this devastating disease remain unavailable. The lack of suitable models recapitulating the entire spectrum of the degenerative process has severely hindered the identification and validation of therapeutic strategies. The discovery that the degeneration in HD is caused by a mutation in a single gene has offered new opportunities to develop experimental models of HD, ranging from in vitro models to transgenic primates. However, recent advances in viral-vector technology provide promising alternatives based on the direct transfer of genes to selected sub-regions of the brain. Rodent studies have shown that overexpression of mutant human Htt in the striatum using adeno-associated virus or lentivirus vectors induces progressive neurodegeneration, which resembles that seen in HD. This article highlights progress made in modeling HD using viral vector gene transfer. We describe data obtained with of this highly flexible approach for the targeted overexpression of a disease-causing gene. The ability to deliver mutant Htt to specific tissues has opened pathological processes to experimental analysis and allowed targeted therapeutic development in rodent and primate pre-clinical models.

Vaccination-induced functional competence of circulating human tumor-specific CD8 T-cells.

T-cells specific for foreign (e.g., viral) antigens can give rise to strong protective immune responses, whereas self/tumor antigen-specific T-cells are thought to be less powerful. However, synthetic T-cell vaccines composed of Melan-A/MART-1 peptide, CpG and IFA can induce high frequencies of tumor-specific CD8 T-cells in PBMC of melanoma patients. Here we analyzed the functionality of these T-cells directly ex vivo, by multiparameter flow cytometry. The production of multiple cytokines (IFNγ, TNFα, IL-2) and upregulation of LAMP-1 (CD107a) by tumor (Melan-A/MART-1) specific T-cells was comparable to virus (EBV-BMLF1) specific CD8 T-cells. Furthermore, phosphorylation of STAT1, STAT5 and ERK1/2, and expression of CD3 zeta chain were similar in tumor- and virus-specific T-cells, demonstrating functional signaling pathways. Interestingly, high frequencies of functionally competent T-cells were induced irrespective of patient's age or gender. Finally, CD8 T-cell function correlated with disease-free survival. However, this result is preliminary since the study was a Phase I clinical trial. We conclude that human tumor-specific CD8 T-cells can reach functional competence in vivo, encouraging further development and Phase III trials assessing the clinical efficacy of robust vaccination strategies.

A pathogen-specific epitope inserted into recombinant secretory immunoglobulin A is immunogenic by the oral route.

Oral administration of rabbit secretory IgA (sIgA) to adult BALB/c mice induced IgA+, IgM+, and IgG+ lymphoblasts in the Peyer's patches, whose fusion with myeloma cells resulted in hybridomas producing IgA, IgM, and IgG1 antibodies to the secretory component (SC). This suggests that SC could serve as a vector to target protective epitopes into mucosal lymphoid tissue and elicit an immune response. We tested this concept by inserting a Shigella flexneri invasin B epitope into SC, which, following reassociation with IgA, was delivered orally to mice. To identify potential insertion sites at the surface of SC, we constructed a molecular model of the first and second Ig-like domains of rabbit SC. A surface epitope recognized by an SC-specific antibody was mapped to the loop connecting the E and F beta strands of domain I. This 8-amino acid sequence was replaced by a 9-amino acid linear epitope from S. flexneri invasin B. We found that cellular trafficking of recombinant SC produced in mammalian CV-1 cells was drastically altered and resulted in a 50-fold lower rate of secretion. However, purification of chimeric SC could be achieved by Ni2+-chelate affinity chromatoraphy. Both wild-type and chimeric SC bound to dimeric IgA, but not to monomeric IgA. Reconstituted sIgA carrying the invasin B epitope within the SC moiety triggers the appearance of seric and salivary invasin B-specific antibodies. Thus, neo-antigenized sIgA can serve as a mucosal vaccine delivery system inducing systemic and mucosal immune responses.

Inhibitory Receptor Expression Depends More Dominantly on Differentiation and Activation than "Exhaustion" of Human CD8 T Cells.

Under conditions of chronic antigen stimulation, such as persistent viral infection and cancer, CD8 T cells may diminish effector function, which has been termed "exhaustion." Expression of inhibitory Receptors (iRs) is often regarded as a hallmark of "exhaustion." Here we studied the expression of eight different iRs by CD8 T cells of healthy humans, including CTLA-4, PD1, TIM3, LAG3, 2B4, BTLA, CD160, and KLRG1. We show that many iRs are expressed upon activation, and with progressive differentiation to effector cells, even in absence of long-term ("chronic") antigenic stimulation. In particular, we evaluated the direct relationship between iR expression and functionality in CD8 T cells by using anti-CD3 and anti-CD28 stimulation to stimulate all cells and differentiation subsets. We observed a striking up-regulation of certain iRs following the cytokine production wave, in agreement with the notion that iRs function as a negative feedback mechanism. Intriguingly, we found no major impairment of cytokine production in cells positive for a broad array of iRs, as previously shown for PD1 in healthy donors. Rather, the expression of the various iRs strongly correlated with T cell differentiation or activation states, or both. Furthermore, we analyzed CD8 T cells from lymph nodes (LNs) of melanoma patients. Interestingly, we found altered iR expression and lower cytokine production by T cells from metastatic LNs, but also from non-metastatic LNs, likely due to mechanisms which are not related to exhaustion. Together, our data shows that expression of iRs per se does not mark dysfunctional cells, but is rather tightly linked to activation and differentiation. This study highlights the importance of considering the status of activation and differentiation for the study and the clinical monitoring of CD8 T cells.

Immunology taught by lung dendritic cells.

Dendritic cells (DCs) are leukocytes specialised in the uptake, processing, and presentation of antigen and fundamental in regulating both innate and adaptive immune functions. They are mainly localised at the interface between body surfaces and the environment, continuously scrutinising incoming antigen for the potential threat it may represent to the organism. In the respiratory tract, DCs constitute a tightly enmeshed network, with the most prominent populations localised in the epithelium of the conducting airways and lung parenchyma. Their unique localisation enables them to continuously assess inhaled antigen, either inducing tolerance to inoffensive substances, or initiating immunity against a potentially harmful pathogen. This immunological homeostasis requires stringent control mechanisms to protect the vital and fragile gaseous exchange barrier from unrestrained and damaging inflammation, or an exaggerated immune response to an innocuous allergen, such as in allergic asthma. During DC activation, there is upregulation of co-stimulatory molecules and maturation markers, enabling DC to activate naïve T cells. This activation is accompanied by chemokine and cytokine release that not only serves to amplify innate immune response, but also determines the type of effector T cell population generated. An increasing body of recent literature provides evidence that different DC subpopulations, such as myeloid DC (mDC) and plasmacytoid DC (pDC) in the lungs occupy a key position at the crossroads between tolerance and immunity. This review aims to provide the clinician and researcher with a summary of the latest insights into DC-mediated pulmonary immune regulation and its relevance for developing novel therapeutic strategies for various disease conditions such as infection, asthma, COPD, and fibrotic lung disease.

Identification of naturally processed hepatitis C virus-derived major histocompatibility complex class I ligands.

Fine mapping of human cytotoxic T lymphocyte (CTL) responses against hepatitis C virus (HCV) is based on external loading of target cells with synthetic peptides which are either derived from prediction algorithms or from overlapping peptide libraries. These strategies do not address putative host and viral mechanisms which may alter processing as well as presentation of CTL epitopes. Therefore, the aim of this proof-of-concept study was to identify naturally processed HCV-derived major histocompatibility complex (MHC) class I ligands. To this end, continuous human cell lines were engineered to inducibly express HCV proteins and to constitutively express high levels of functional HLA-A2. These cell lines were recognized in an HLA-A2-restricted manner by HCV-specific CTLs. Ligands eluted from HLA-A2 molecules isolated from large-scale cultures of these cell lines were separated by high performance liquid chromatography and further analyzed by electrospray ionization quadrupole time of flight mass spectrometry (MS)/tandem MS. These analyses allowed the identification of two HLA-A2-restricted epitopes derived from HCV nonstructural proteins (NS) 3 and 5B (NS3₁₄₀₆₋₁₄₁₅ and NS5B₂₅₉₄₋₂₆₀₂). In conclusion, we describe a general strategy that may be useful to investigate HCV pathogenesis and may contribute to the development of preventive and therapeutic vaccines in the future.

Encéphalites virales [Viral encephalitis: update]

Suspicion of viral encephalitis should always be considered as a medical emergency and the prognosis depend on both the immune status of the host and the virulence of the virus. Among them, the herpes simplex virus is by far the most important one since it can be associated with severe encephalitis in immunocompetent host, and because a good response to acyclovir can be expected when rapidly initiated. Nevertheless, confirmation of the diagnosis requires exclusion of both metabolic or toxic encephalopathy and inflammatory encephalitis of non-infectious origin. In addition, other germs than viruses can mimic viral encephalitis and must be taken into consideration. The purpose of this review is to update the investigation that should be performed in clinical practice for any patient with suspicion of acute viral encephalitis.

Retrovirus-induced target cell activation in the early phases of infection: the mouse mammary tumor virus model.

Mouse mammary tumor virus (MMTV) infects B lymphocytes and expresses a superantigen on the cell surface after integration of its reverse-transcribed genome. Superantigen-dependent B- and T-cell activation becomes detectable 2 to 3 days after infection. We show here that before this event, B cells undergo a polyclonal activation which does not involve massive proliferation. This first phase of B-cell activation is T cell independent. Moreover, during the first phase of activation, when only a small fraction of B cells is infected by MMTV(SW), viral DNA is detected only in activated B cells. Such a B-cell activation is also seen after injection of murine leukemia virus but not after injection of vaccinia virus, despite the very similar kinetics and intensity of the immune response. Since retroviruses require activated target cells to induce efficient infection, these data suggest that the early polyclonal retrovirus-induced target cell activation might play an important role in the establishment of retroviral infections.

Transmitted drug resistant HIV-1 and association with virologic and CD4 cell count response to combination antiretroviral therapy in the EuroSIDA Study.

OBJECTIVES: To investigate prevalence of transmitted drug-resistant human immunodeficiency virus (TDR) and factors associated with TDR and to compare virological and CD4 count response to combination antiretroviral therapy. METHODS: In this study, 525 mostly chronically infected EuroSIDA patients were included who had genotypic resistance tests performed on plasma samples collected while antiretroviral therapy naive. TDR was defined as at least one resistance mutation from a list proposed for genotypic TDR surveillance. Multivariable logistic regression was used to analyze factors associated with detection of TDR, with virological (viral load<500 copies/mL) and CD4 count response (>or=50% increase) to combination antiretroviral therapy at months 6-12. RESULTS: The overall prevalence of TDR was 11.4%, which was stable over 1996-2004. There were no significant differences in virological suppression (those resistant to at least one drug prescribed versus susceptible), adjusted odds ratio: 0.68 (95% confidence interval: 0.27 to 1.71; P=0.408) or CD4 count response, adjusted odds ratio: 1.65 (95% confidence interval: 0.73 to 3.73; P=0.231). CONCLUSIONS: Prevalence of TDR in antiretroviral-naive patients was found to be in line with other European studies. No significant differences were found in virological and CD4 count response after initiation of first-line combination antiretroviral therapy between resistant and susceptible patients, possibly due to the small number of patients with resistance and consequently low power.

Siglec-1 is a novel dendritic cell receptor that mediates HIV-1 trans-infection through recognition of viral membrane gangliosides.

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of immunity against pathogens. However, HIV-1 spread is strongly enhanced in clusters of DCs and CD4(+) T cells. Uninfected DCs capture HIV-1 and mediate viral transfer to bystander CD4(+) T cells through a process termed trans-infection. Initial studies identified the C-type lectin DC-SIGN as the HIV-1 binding factor on DCs, which interacts with the viral envelope glycoproteins. Upon DC maturation, however, DC-SIGN is down-regulated, while HIV-1 capture and trans-infection is strongly enhanced via a glycoprotein-independent capture pathway that recognizes sialyllactose-containing membrane gangliosides. Here we show that the sialic acid-binding Ig-like lectin 1 (Siglec-1, CD169), which is highly expressed on mature DCs, specifically binds HIV-1 and vesicles carrying sialyllactose. Furthermore, Siglec-1 is essential for trans-infection by mature DCs. These findings identify Siglec-1 as a key factor for HIV-1 spread via infectious DC/T-cell synapses, highlighting a novel mechanism that mediates HIV-1 dissemination in activated tissues.