Surface Of Infected Hepatocytes Research Articles

IMRAS-A clinical trial of mosquito-bite immunization with live, radiation-attenuated P. falciparum sporozoites: Impact of immunization parameters on protective efficacy and generation of a repository of immunologic reagents.

BackgroundImmunization with radiation-attenuated sporozoites (RAS) by mosquito bite provides >90% sterile protection against Plasmodium falciparum (Pf) malaria in humans. RAS invade hepatocytes but do not replicate. CD8+ T cells recognizing parasite-derived peptides on the surface of infected hepatocytes are likely the primary protective mechanism. We conducted a randomized clinical trial of RAS immunization to assess safety, to achieve 50% vaccine efficacy (VE) against controlled human malaria infection (CHMI), and to generate reagents from protected and non-protected subjects for future identification of protective immune mechanisms and antigens.MethodsTwo cohorts (Cohort 1 and Cohort 2) of healthy, malaria-naïve, non-pregnant adults age 18–50 received five monthly immunizations with infected (true-immunized, n = 21) or non-infected (mock-immunized, n = 5) mosquito bites and underwent homologous CHMI at 3 weeks. Immunization parameters were selected for 50% protection based on prior clinical data. Leukapheresis was done to collect plasma and peripheral blood mononuclear cells.ResultsAdverse event rates were similar in true- and mock-immunized subjects. Two true- and two mock-immunized subjects developed large local reactions likely caused by mosquito salivary gland antigens. In Cohort 1, 11 subjects received 810–1235 infected bites; 6/11 (55%) were protected against CHMI vs. 0/3 mock-immunized and 0/6 infectivity controls (VE 55%). In Cohort 2, 10 subjects received 839–1131 infected bites with a higher first dose and a reduced fifth dose; 9/10 (90%) were protected vs. 0/2 mock-immunized and 0/6 controls (VE 90%). Three/3 (100%) protected subjects administered three booster immunizations were protected against repeat CHMI vs. 0/6 controls (VE 100%). Cohort 2 uniquely showed a significant rise in IFN-γ responses after the third and fifth immunizations and higher antibody responses to CSP.ConclusionsPfRAS were generally safe and well tolerated. Cohort 2 had a higher first dose, reduced final dose, higher antibody responses to CSP and significant rise of IFN-γ responses after the third and fifth immunizations. Whether any of these factors contributed to increased protection in Cohort 2 requires further investigation. A cryobank of sera and cells from protected and non-protected individuals was generated for future immunological studies and antigen discovery.Trial registrationClinicalTrials.gov NCT01994525.

Plasmodium berghei-Infected Primary Hepatocytes Process and Present the Circumsporozoite Protein to Specific CD8+ T Cells In Vitro

A substantial and protective response against malaria liver stages is directed against the circumsporozoite protein (CSP) and involves induction of CD8(+) T cells and production of IFN-gamma. CSP-derived peptides have been shown to be presented on the surface of infected hepatocytes in the context of MHC class I molecules. However, little is known about how the CSP and other sporozoite Ags are processed and presented to CD8(+) T cells. We investigated how primary hepatocytes from BALB/c mice process the CSP of Plasmodium berghei after live sporozoite infection and present CSP-derived peptides to specific H-2K(d)-restricted CD8(+) T cells in vitro. Using both wild-type and spect(-/-) P. berghei sporozoites, we show that both infected and traversed primary hepatocytes process and present the CSP. The processing and presentation pathway was found to involve the proteasome, Ag transport through a postendoplasmic reticulum compartment, and aspartic proteases. Thus, it can be hypothesized that infected hepatocytes can contribute in vivo to the elicitation and expansion of a T cell response.

Laser capture microdissection and molecular analysis of Plasmodium yoelii liver-stage parasites

Understanding the liver stage development of Plasmodial parasites, and by extension the proteins expressed during that phase of the life cycle, has become increasingly more important as investigators have identified the pre-erythrocytic stage of the parasite as a target for effector T lymphocytes and their products. It has been clearly demonstrated that immunization with radiation-attenuated sporozoites can confer long lasting sterile protective immunity and that the immunity is stage-specific [1–3]. This form of immunization is effective against a sporozoite challenge, but not a blood stage challenge. Therefore, the protective immune response must be directed against the sporozoite and/or liver stage antigens. Experimental evidence, in mice, has clearly demonstrated that this protection requires the cellular immune system [4,5] and specifically CD8 T lymphocytes [6–8]. Malarial antigens expressed during the liver stage are processed and presented, in the context of class I MHC molecules, on the surface of infected hepatocytes [9,10]. Identifying these antigens, that may act as targets for cell mediated immunity, would significantly enhance the understanding of how the irradiated sporozoite vaccine model confers protection and ultimately lead to the identification of candidates for a subunit vaccine. Recent advances in tissue microdissection have provided a methodology to allow the isolation of a pure population of liver stage schizonts from heterogeneous liver tissue. In the past, microdissection was done manually or with the aid of a micromanipulator. The area of interest was scraped off of a tissue section using a needle and subsequent processing was performed with the tissue that was recovered. This was a slow, laborious and unreliable practice that frequently yielded an impure cell population. The development of Laser-capture microdissection (LCM) has turned microdissection into a fast, reliable and precise technique. LCM utilizes an infra red laser and a transparent thermoplastic film to selectively isolate cells or regions of interest [11,12]. Briefly, a slide containing a section of infected liver is placed on the microscope stage. A transport arm, holding the thermoplastic film, is then moved over the region of interest. The cell(s) to be isolated are moved to the center of the optical field and the laser is activated. The laser melts the film directly onto the tissue containing the targeted cell. The film rapidly cools and solidifies such that when the film is moved by the transport arm the film/tissue combination shears away from the surrounding tissue. The process can be repeated many times, utilizing areas of the film that have not been melted with the laser. The film, which is held in a plastic ring, the same dimensions as a miAbbre iations: AMA-1, apical membrane antigen 1; CS, circumsporozoite; LCM, laser capture microdissection; MSP1, merrozoite surface protein 1; NYLS3, navy yoelii liver stage 3; RT-PCR, reverse transcriptase-polymerase chain reaction. * Corresponding author. E-mail address: jsacc001@umaryland.edu (J.B. Sacci, Jr). 1 Present address: Celera Genomics, 45 West Gude Dr., Rockville, MD 20852, USA.

Posttranscriptional inhibition of class I major histocompatibility complex presentation on hepatocytes and lymphoid cells in chronic woodchuck hepatitis virus infection.

Woodchuck hepatitis virus (WHV), similar to human hepatitis B virus, causes acute liver inflammation that can progress to chronic hepatitis and hepatocellular carcinoma. WHV also invades cells of the host lymphatic system, where it persists for life. We report here that acute and chronic hepadnavirus hepatitis is characterized by a profound difference in the expression of class I major histocompatibility complex (MHC) molecules on the surface of infected hepatocytes and, notably, lymphoid cells. While acute WHV infection is accompanied by the enhanced hepatocyte surface presentation of class I MHC antigen and upregulated transcription of the relevant hepatic genes, inhibition of class I antigen display on liver cells is a uniform hallmark of chronic WHV infection. This inhibition in chronic hepatitis occurs despite augmented (as in acute infection) expression of hepatic genes for class I MHC heavy chain, beta(2)-microglobulin, and transporters associated with antigen processing (TAP1 and TAP2). Further, the class I antigen inhibition is not related to the histological severity of hepatocellular injury, the extent of lymphocytic infiltrations, the level of intrahepatic gamma interferon induction, or the hepatic WHV load. Importantly, the antigen expression is also inhibited on organ lymphoid cells of chronically infected hosts. The results obtained in this study demonstrate that the defective presentation of class I MHC molecules on cells supporting persistent WHV replication is due to viral posttranscriptional interference. This event may diminish the susceptibility of infected hepatocytes to virus-specific T-cell-mediated elimination, hinder virus clearance, and deregulate the class I MHC-dependent functions of the host immune system. This multifarious effect could be critical for perpetuation of liver damage and evasion of the antiviral immunological surveillance in chronic infection and therefore could be supportive of hepadnavirus persistence.

Malaria peptides expressed on the surface of infected hepatocytes: isolation, synthesis and functional analysis

It is now clear, from the results of various experiments, that the malarial epitopes expressed on the surface of infected hepatocytes are the target of at least two effector mechanisms: (1) a cytotoxicity, restricted by the major histocompatibility complex (MHC), which involves CD8+ and CD4+ T cells: and (2) antibody-dependent, cellular cytotoxicity. Until now, Plasmodium epitopes have been characterized from known parasite proteins and it appeared important to identify ‘natural’ peptides. Epitopes have been investigated using two procedures for acid extraction, one for pools of livers from infected mice (P. yoelii) or cultures (P. vivax) and one for MHC-class-I molecules present in the plasma membranes of infected livers (P. yoelii), followed by fractionation using reversed-phase, high-performance, liquid chromatography (HPLC). Two new assays to estimate the potential interest of each newly isolated epitope have been developed in parallel: (1) a quantitative, HPLC-based assay of PCR products, to estimate...

Malaria peptides expressed on the surface of infected hepatocytes: isolation, synthesis and functional analysis

Malaria peptides expressed on the surface of infected hepatocytes: isolation, synthesis and functional analysis

Recombinant pseudorabies virus carrying a plasmodium gene: herpesvirus as a new live viral vector for inducing T- and B-cell immunity

In Balb/c mice, the sterile protective immunity induced by immunization with radiation-attenuated Plasmodium yoelii sporozoites is eliminated by in vivo depletion of CD8+ Tlymphocytes, suggesting that cytotoxic T lymphocytes (CTL) against malaria antigens expressed on infected hepatocytes are required for mediating this protective immunity. To produce a vaccine that would induce CTL against the P. yoelii circumsporozoite protein (CS), we constructed an attenuated pseudorabies virus (PRV) containing a gene encoding this protein. Balb/c mice that received three doses of 10 7 plaque-forming units (p.f.u.) of this vaccine intravenously at 3 week intervals developed high levels of antibodies to sporozoites (indirect fluorescent antibody titre = 4096) and CTL against a 16 amino acid epitope (SYVPSAEQILEFVKQI, amino acids 281–296) from the P. yoelii CS protein designated PYCTLI. The cytotoxic activity of the CTL was antigen-specific, MHC-restricted, and dependent on CD8 + T cells. Furthermore, these CTL eliminated P. yoelii-infected hepatocytes from in vitro culture, indicating that they recognize this peptide on the surface of infected hepatocytes. However, all nine mice that were challenged with 200 sporozoites developed a blood-stage malaria infection. We attribute this lack of protection to the great difficulty of inducing sterile immunity against this highly infectious parasite P. yoelii. We conclude that recombinant pseudorabies virus (PRV) worked successfully as a live vaccine vector to induce both antibodies and CTL, albeit non-protective in vivo, and the herpesviruses should be considered as carriers for subunit vaccines where T- and B-cell immunity is required.

Malaria antigens expressed on the surface of infected hepatocytes: a role in protective immunity?

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Irradiated sporozoite vaccine induces cytotoxic T lymphocytes that recognize malaria antigens on the surface of infected hepatocytes

The observation that protective immunity induced by immunization with radiation attenuated Plasmodium berghei and Plasmodium yoelii sporozoites is dependent on CD8 + T lymphocytes in some strains of mice led us to speculate that immunization with sporozoites induces cytotoxic T lymphocytes (CTL) that recognize malaria antigens on the surface of malaria-infected hepatocytes. In this report we summarize a series of experiments that confirm this hypothesis. We first showed that when immune mice are challenged with live sporozoites they develop malaria-specific, CD8 + T cell-dependent infiltrates in their livers. Next we demonstrated that spleen cells from immune mice eliminate malaria infected hepatocytes from in vitro culture in an antigen specific and genetically restricted manner, indicating that these immune cells recognize malaria antigens on the surface of infected hepatocytes. Finally we defined a CTL epitope of the P. yoelii CS protein, and demonstrated that CTL against this 16-amino-acid peptide (PYCTL1) eliminate infected hepatocytes from culture in an antigenic specific, and MHC restricted manner, indicating that this 16-amino-acid peptide from the CS protein is present on the surface of the infected hepatocytes. We are currently working on constructing vaccines that induce protective CTL against PYCTL1, and identifying additional pre-erythrocytic stage targets of CTL mediated protective immunity.

Cytotoxic T cells recognize a peptide from the circumsporozoite protein on malaria-infected hepatocytes.

Irradiated malaria sporozoites can induce CD8+ T cells that are required for protection against infection. However, the parasite antigens targeted by this immune response are unknown. We have discovered a 16-amino acid epitope from the Plasmodium yoelii circumsporozoite (CS) protein that is recognized by cytotoxic T cells from immune mice. Lymphocytes stimulated with this peptide can kill P. yoelii liver stage parasites in vitro in an MHC-restricted, antigen-specific manner. Thus, epitopes from the CS protein are presented on the surface of infected hepatocytes and can be targets for T cells, even though intact CS protein has not been detected on the surface of the infected hepatocyte. A vaccine that induced CTL to parasite antigens might protect humans against malaria by eliminating liver stage parasites.

Localization of circumsporozoite antigen in exoerythrocytic schizonts of Plasmodium cynomolgi.

We used colloidal gold probes and post-embedding immunoelectron microscopy to localize circumsporozoite (CS) antigen in 5- and 8-day-old in vitro cultures of Plasmodium cynomolgi exoerythrocytic (EE) schizonts. Both small uninucleated and large multinucleated EE schizonts were found in 5-day-old cultures. A mouse monoclonal antibody to the repeat region of the P. cynomolgi CS protein densely labeled the plasma membrane and surface of 5-day-old EE schizonts as well as the surrounding parasitophorous vacuole membrane and space. Density of labeling decreased significantly as EE schizonts increased in size and maturity. Labeling of large, multinucleated 5-day-old schizonts was sparse and limited to the surface of EE schizonts and to small patches of electron dense material which were attached to the inner surface of the parasitophorous vacuole membrane. Mature 8-day-old EE schizonts with developing merozoites had little detectable labeling. CS antigen was not associated with internal structures within developing schizonts. Labeling was not observed in the host cell cytoplasm or on the surface of infected hepatocytes. These findings indicate that epitopes associated with the repeat region of the P. cynomolgi circumsporozoite protein are sequestered within infected host cells during EE development.

Serial study of liver-directed autoantibodies and autoreactive T-lymphocytes in acute viral hepatitis B

To explore the mechanisms underlying liver-directed autoimmune reactions in acute Hepatitis B Virus (HBV) infection, we followed five subjects who were identified in the early incubation phase (30–70 days before the first elevation of transaminases). We assessed serially cellular (using a T-lymphocyte migration inhibitory factor assay) and humoral (RIA) immunity to LSP (a macromolecular, liver-derived lipoprotein complex) and hepatic lectin (HL), the liver-specific receptor for desialylated glycoproteins, which appears to be a major target antigen for autoreactions in autoimmune chronic active hepatitis. Anti-LSP and anti-HL autoantibodies were found, at some stage during acute HBV infection, in 4 5 subjects, whereas cellular immunity to the same antigens was detected in only two patients. Sustained production of anti-HL antibodies was noted only in patients showing cellular immunity to this antigen and was apparently secondary to liver damage, whereas anti-LSP antibodies were first detected at the onset of liver injury when there was no evidence of T-cell immunity to the same antigenic complex. One explanation for this apparent dichotomy between cellular and humoral responses to LSP is that a helper T-cell response to the major envelope component of HBV, HBsAg, which precedes by 10–20 days the development of anti-LSP antibodies, promotes a humoral reaction to autoantigens contained in the LSP preparation, coexpressed with HBsAg, on the surface of infected hepatocytes.

Immunopathological mechanisms of liver cell injury in chronic hepatitis B virus infection

It is generally accepted that the hepatitis B virus is not cytopathic and that liver cell damage in chronic HBV infection is dependent on the host's immune response directed at viral and self-antigens on the surface of infected hepatocytes. In recent years there have been some advances in the understanding of both the viral antigen display on hepatocytes and the resultant host response. Using fluoresceinated polyclonal and monoclonal antibodies to HBV antigens, HBcAg has been identified as the major viral product expressed on the surface of liver cells isolated from patients with chronic HBV infection. In these patients, both T and non-T cells from peripheral blood have been shown to be cytotoxic to autologous hepatocytes. Blocking studies using polyclonal anti-HBs and anti-HBc antibodies indicate that HBcAg, but not HBsAg, is an important target antigen for T-cell attack, and this has recently been confirmed using monoclonal reagents. The non-T cell cytotoxicity appears to be directed at auto-antigens in a liver membrane lipoprotein complex (LSP), probably through an antibody-dependent (ADCC) mechanism. T-cell bypass mechanisms could be responsible for the production of autoantibodies to these normal membrane components. Both these mechanisms of cytolysis are found, most often, in patients with active viral replication in the HBeAg-positive phase of chronic HBV infection. This is presumably because hepatocytes containing free HBV-DNA and expressing HBcAg on their surface will be most susceptible to T-cell attack, while those with integrated HBV-DNA only express HBsAg and are relatively protected.(ABSTRACT TRUNCATED AT 250 WORDS)

T-lymphocyte cytotoxicity to HBsAg-coated target cells in hepatitis B virus infection

The cytotoxic effect of peripheral lymphocytes on chicken red blood cells (ChRBC) coated with purified hepatitis B surface antigen (HBsAg) has been studied as an in vitro parameter of cell-mediated immunity in acute and chronic infection with hepatitis B virus. Using this technique, the mean cytotoxic index of lymphocytes from patients with acute hepatitis B (29.13 +/- 20.88) was significantly higher than that obtained with lymphocytes from control subjects (6.53 +/- 3.75). Only 33.3% of the patients with HBsAg-positive chronic active hepatitis exhibited lymphocyte cytotoxicity to HBsAg-coated target cells and the mean cytotoxic index (11.66 +/- 6.60) was in these cases significantly lower than that found in acute hepatitis B. Healthy chronic carriers of HBsAg failed to show lymphocyte cytotoxicity to target cells. The effector cells detected in acute hepatitis B by this in vitro assay have been demonstrated to be T-lymphocytes, as T-cell depleted subpopulations lacked cytotoxic activity. Target cell lysis could be blocked by addition of HBsAg-coated unlabelled ChRBC as well as of purified HBsAg in the culture tubes. It is suggested that damage to the liver cells in acute hepatitis B is related to the presence of cytotoxic T-lymphocytes reacting with HBsAg on the surface of infected hepatocytes. An inadequate lymphocyte response to the antigen may be responsible for the persistence of the infection in the liver with varied clinical manifestations and associated hepatic lesions.