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ABSTRACTThe hepatitis C virus (HCV) nonstructural (NS) protein 5A is a multifunctional protein that plays a central role in viral replication and assembly. Antiviral agents directly targeting NS5A are currently in clinical development. Although the elucidation of the mechanism of action (MOA) of NS5A inhibitors has been the focus of intensive research, a detailed understanding of how these agents exert their antiviral effect is still lacking. In this study, we observed that the downregulation of NS5A hyperphosphorylation is associated with the actions of NS5A inhibitors belonging to different chemotypes. NS5A is known to recruit the lipid kinase phosphatidylinositol 4-kinase IIIα (PI4KIIIα) to the HCV-induced membranous web in order to generate phosphatidylinositol 4-phosphate (PI4P) at the sites of replication. We demonstrate that treatment with NS5A inhibitors leads to an impairment in the NS5A-PI4KIIIα complex formation that is paralleled by a significant reduction in PI4P and cholesterol levels within the endomembrane structures of HCV-replicating cells. A similar decrease in PI4P and cholesterol levels was also obtained upon treatment with a PI4KIIIα-targeting inhibitor. In addition, both the NS5A and PI4KIIIα classes of inhibitors induced similar subcellular relocalization of the NS5A protein, causing the formation of large cytoplasmic NS5A-containing clusters previously reported to be one of the hallmarks of inhibition of the action of PI4KIIIα. Because of the similarities between the effects induced by treatment with PI4KIIIα or NS5A inhibitors and the observation that agents targeting NS5A impair NS5A-PI4KIIIα complex formation, we speculate that NS5A inhibitors act by interfering with the function of the NS5A-PI4KIIIα complex.

A nonglycosylated denatured form of human immunodeficiency virus (HIV) 1 glycoprotein gp120 (Env 2-3), which does not bind to CD4, was used with muramyl tripeptide as adjuvant to immunize HIV-seronegative healthy volunteers. In all the volunteers, three 50-micrograms injections of Env 2-3 induced priming of CD4+ T cells specific for conserved regions of the native glycosylated gp120. Moreover, we found that several major histocompatibility complex class II (DR) alleles can function as restriction molecules for presentation of conserved epitopes of gp120 to T cells, implying that a T-cell response to these epitopes can be obtained in a large fraction of the population. The possibility to prime CD4+ T cells specific for conserved epitopes of a HIV protein is particularly important in view of the lack of such cells in HIV-infected individuals and of a possible role that CD4+ T cells may play in the development of protective immunity against AIDS.

We found that three tumor patients treated with mouse mAbs have T cells that recognize processed mouse Ig on autologous APC in a class II-restricted fashion, and we have shown that mouse mAbs directed against various cell surface molecules can be used as antigens to focus these T cells on an MHC class II-positive target of choice.

Proteins to be used as vaccines are frequently treated with formaldehyde, although little is known about the effects of this treatment on protein antigenicity. To investigate the effect of formaldehyde treatment on antigen recognition by T cells, we compared the in vitro T-cell response to proteins that have been formaldehyde treated with the response to untreated proteins. We found that peripheral blood mononuclear cells from individuals vaccinated with three formaldehyde-treated proteins (pertussis toxin, filamentous hemagglutinin, pertactin) of Bordetella pertussis showed little or no response to the formaldehyde-treated proteins but proliferated very well in response to the corresponding untreated protein. These findings were further confirmed with CD4+ T-cell clones specific for defined epitopes of the bacterial proteins. We found that some epitopes are presented poorly or not at all when formaldehyde-treated proteins are used, whereas other epitopes are equally presented to T-cell clones when either formaldehyde-treated or untreated antigens are used. However, T-cell recognition could be restored by either antigen degradation before formaldehyde treatment or heat denaturation after such treatment. Parallel digestion with trypsin of both formaldehyde-treated and untreated proteins showed that fragments generated from the two forms of the same antigen were different in size. These results demonstrate that formaldehyde treatment can constrain antigen presentation to T cells and that this may be due to an altered proteolytic processing of formaldehyde-treated proteins.

Immunization of the female reproductive tract is important for protection against sexually transmitted diseases and other pathogens of the reproductive tract. However, intravaginal immunization with soluble antigens generally does not induce high levels of secretory immunoglobulin A (IgA). We recently developed safe mucosal adjuvants by genetically detoxifying Escherichia coli heat-labile enterotoxin, a molecule with a strong mucosal adjuvant activity, and here we describe the use of the nontoxic mutant LTK63 to induce a response in the mouse vagina against ovalbumin (Ova). We compared intravaginal and intranasal routes of immunization for induction of systemic and vaginal responses against LTK63 and Ova. We found that LTK63 is a potent mucosal immunogen when given by either the intravaginal or intranasal route. It induces a strong systemic antibody response and IgG and long-lasting IgA in the vagina. The appearance of vaginal IgA is delayed in the intranasally immunized mice, but the levels of vaginal anti-LTK63 IgA after repeated immunizations are higher in the intranasally immunized mice than in the intravaginally immunized mice. LTK63 also acts as a mucosal adjuvant, inducing a serum response against Ova, when given by both the intravaginal and intranasal routes. However, vaginal IgA against Ova is stimulated more efficiently when LTK63 and antigen are given intranasally. In conclusion, our results demonstrate that LTK63 can be used as a mucosal adjuvant to induce antigen-specific antibodies in vaginal secretions and show that the intranasal route of immunization is the most effective for this purpose.

Colonization of human gastric mucosa with cytotoxic strains of the bacterium Helicobacter pylori is associated with peptic ulcer and with chronic gastritis. Since little is known about the T-cell response to H. pylori, we investigated the CD4+ T-cell response both in peripheral blood mononuclear cells (PBMCs) and at the site of infection. First, we compared the bulk PBMC proliferative response to the bacterium in individuals with and without symptoms of gastroduodenal disease. We found that the PBMCs from virtually all individuals proliferate in response to heat-inactivated bacteria. Second, we cloned H. pylori-specific CD4+ T lymphocytes from the PBMCs of three patients and from both the gastric mucosa and PBMCs of a fourth patient. We have found that CD4+ T-cell clones specific for H. pylori from peripheral blood samples and gastric mucosae of infected patients are major histocompatibility complex class II restricted and discriminate between several cytotoxic and noncytotoxic bacterial strains. Moreover, they are polyclonal in terms of T-cell receptor usage and major histocompatibility complex restriction. Our results demonstrate that the T-cell response to the whole bacterium in PBMCs does not correlate with antibody response, infection, or disease. However, H. pylori-specific CD4+ T cells are detectable, at the clonal level, in both the periphery and gastric mucosa of infected patients. Localization of these cells at the site of disease suggests they are effectors of the immune response to the bacteria.

Colonization of human gastric mucosa with cytotoxic strains of the bacterium Helicobacter pylori is associated with peptic ulcer and with chronic gastritis. Since little is known about the T-cell response to H. pylori, we investigated the CD4+ T-cell response both in peripheral blood mononuclear cells (PBMCs) and at the site of infection. First, we compared the bulk PBMC proliferative response to the bacterium in individuals with and without symptoms of gastroduodenal disease. We found that the PBMCs from virtually all individuals proliferate in response to heat-inactivated bacteria. Second, we cloned H. pylori-specific CD4+ T lymphocytes from the PBMCs of three patients and from both the gastric mucosa and PBMCs of a fourth patient. We have found that CD4+ T-cell clones specific for H. pylori from peripheral blood samples and gastric mucosae of infected patients are major histocompatibility complex class II restricted and discriminate between several cytotoxic and noncytotoxic bacterial strains. Moreover, they are polyclonal in terms of T-cell receptor usage and major histocompatibility complex restriction. Our results demonstrate that the T-cell response to the whole bacterium in PBMCs does not correlate with antibody response, infection, or disease. However, H. pylori-specific CD4+ T cells are detectable, at the clonal level, in both the periphery and gastric mucosa of infected patients. Localization of these cells at the site of disease suggests they are effectors of the immune response to the bacteria.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis. The virus does not replicate efficiently in cell cultures, and it is therefore difficult to assess infection-neutralizing antibodies and to evaluate protective immunity in vitro. To study the binding of the HCV envelope to cell-surface receptors, we developed an assay to assess specific binding of recombinant envelope proteins to human cells and neutralization thereof. HCV recombinant envelope proteins expressed in various systems were incubated with human cells, and binding was assessed by flow cytometry using anti-envelope antibodies. Envelope glycoprotein 2 (E2) expressed in mammalian cells, but not in yeast or insect cells, binds human cells with high affinity (Kd approximately 10(-8) M). We then assessed antibodies able to neutralize E2 binding in the sera of both vaccinated and carrier chimpanzees, as well as in the sera of humans infected with various HCV genotypes. Vaccination with recombinant envelope proteins expressed in mammalian cells elicited high titers of neutralizing antibodies that correlated with protection from HCV challenge. HCV infection does not elicit neutralizing antibodies in most chimpanzees and humans, although low titers of neutralizing antibodies were detectable in a minority of infections. The ability to neutralize binding of E2 derived from the HCV-1 genotype was equally distributed among sera from patients infected with HCV genotypes 1, 2, and 3, demonstrating that binding of E2 is partly independent of E2 hypervariable regions. However, a mouse monoclonal antibody raised against the E2 hypervariable region 1 can partially neutralize binding of E2, indicating that at least two neutralizing epitopes, one of which is hypervariable, should exist on the E2 protein. The neutralization-of-binding assay described will be useful to study protective immunity to HCV infection and for vaccine development.

We investigated whether human resting T cells could be activated to proliferate and display effector function in the absence of T cell receptor occupancy. We report that combination of interleukin 2 (IL-2), tumor necrosis factor alpha, and IL-6 activated highly purified naive (CD45RA+) and memory (CD45RO+) resting CD4+ T cells to proliferate. Under this condition, memory resting T cells could also display effector function as measured by lymphokine synthesis and help for immunoglobulin production by B cells. This novel Ag-independent pathway of T cell activation may play an important role in vivo in recruiting effector T cells at the site of immune response and in maintaining the clonal size of memory T cells in the absence of antigenic stimulation. Moreover, cytokines can induce proliferation of naive T cells without switch to memory phenotype and this may help the maintenance of the peripheral pool of naive T cells.

The adult liver is an organ without constitutive lymphoid components. Therefore, any intrahepatic T cell found in chronic hepatitis should have migrated to the liver after infection and inflammation. Because of the little information available on the differences between intrahepatic and peripheral T cells, we used recombinant proteins of the hepatitis C virus (HCV) to establish specific T cell lines and clones from liver biopsies of patients with chronic hepatitis C and compared them with those present in peripheral blood mononuclear cells (PBMC). We found that the protein nonstructural 4 (NS4) was able to stimulate CD4+ T cells isolated from liver biopsies, whereas with all the other HCV proteins we consistently failed to establish liver-derived T cell lines from 16 biopsies. We then compared NS4-specific T cell clones obtained on the same day from PBMC and liver of the same patient. We found that the 22 PBMC-derived T cell clones represent, at least, six distinct clonal populations that differ in major histocompatibility complex restriction and response to superantigens, whereas the 27 liver-derived T cell clones appear all identical, as further confirmed by cloning and sequencing of the T cell receptor (TCR) variable and hypervariable regions. Remarkably, none of the PBMC-derived clones has a TCR identical to the liver-derived clone, and even with polymerase chain reaction oligotyping we did not find the liver-derived clonotypic TCR transcript in the PBMC, indicating a preferential intrahepatic localization of these T cells. Functionally, the liver-derived T cells provided help for polyclonal immunoglobulin (Ig)A production by B cells in vitro that is 10-fold more effective than that provided by the PBMC-derived clones, whereas there is no difference in the help provided for IgM and IgG production. Altogether these results demonstrate that the protein NS4 is highly immunogenic for intrahepatic CD4+ T cells primed by HCV in vivo, and that there can be compartmentalization of some NS4-specific CD4+ T cells to the liver of patients with chronic hepatitis C.

Background: Natural hepatitis C virus (HCV) infection elicits poor immunity. Although HCV proteins elicit immune responses in virtually all cases of infection, the great majority of HCV infections become chronic. Currently, no vaccine is available for HCV despite an estimated incidence of approximately 50 000 new cases per year in the USA alone. Objectives: To discuss how the problems associated with developing a vaccine against HCV infection may be overcome and describe recent progress made towards overcoming these problems and developing a vaccine. Study design: A cytofluorimetric assay that can assess the ability of a serum sample to neutralise the binding of the HCV-envelope glycoprotein E2 to human cells (neutralisation of binding or NOB assay) was developed. The assay was used to assess the levels of antibodies capable of neutralising E2 binding in the sera of vaccinated and carrier chimpanzees. Results: Low titres of NOB antibodies were found in the majority of chimpanzees challenged with HCV infection. Chimpanzees immunised with the E1/E2 heterodimer developed NOB antibodies and high levels of neutralising antibodies. These chimpanzees were not protected from challenge with heterologous virus but were protected from subsequent chronic infection. Conclusions: A subunit vaccine composed of recombinant HCV proteins may protect from infection or chronic infection by different HCV genotypes.

Immunization of the female reproductive tract is important for protection against sexually transmitted diseases and other pathogens of the reproductive tract. However, intravaginal immunization with soluble antigens generally does not induce high levels of secretory immunoglobulin A (IgA). We recently developed safe mucosal adjuvants by genetically detoxifying Escherichia coli heat-labile enterotoxin, a molecule with a strong mucosal adjuvant activity, and here we describe the use of the nontoxic mutant LTK63 to induce a response in the mouse vagina against ovalbumin (Ova). We compared intravaginal and intranasal routes of immunization for induction of systemic and vaginal responses against LTK63 and Ova. We found that LTK63 is a potent mucosal immunogen when given by either the intravaginal or intranasal route. It induces a strong systemic antibody response and IgG and long-lasting IgA in the vagina. The appearance of vaginal IgA is delayed in the intranasally immunized mice, but the levels of vaginal anti-LTK63 IgA after repeated immunizations are higher in the intranasally immunized mice than in the intravaginally immunized mice. LTK63 also acts as a mucosal adjuvant, inducing a serum response against Ova, when given by both the intravaginal and intranasal routes. However, vaginal IgA against Ova is stimulated more efficiently when LTK63 and antigen are given intranasally. In conclusion, our results demonstrate that LTK63 can be used as a mucosal adjuvant to induce antigen-specific antibodies in vaginal secretions and show that the intranasal route of immunization is the most effective for this purpose.