Mechanisms of protective immunity against asexual blood stages of Plasmodium falciparum in the experimental host Saimiri

In the Saimiri monkey, an experimental host for human malaria, acquired protection against Plasmodium falciparum blood stages depends on the IgG antibody populations developed. In vivo protective anti-falciparum activity of IgG antibodies is correlated with the in vivo opsonizing activity promoting phagocytosis of parasited red bloood cells. In contrast, non protective antibodies inhibit this mechanism by competing at the target level. A similar phenomenon can be and human infection. Anti-cytoadherent and anti-rosette antibodies developed by Saimiri and humans prevent the development of physiopathological events like cerebral malaria which can also occur in this experimental host. Furthermore, transfer to protective human anti-falciparum IgG antibodies into infected Saimiri monkeys exerts an anti parasite activity as efficient as that observed when it is transfered into acute falciparum malaria patients, making the Saimiri an even more attractive host. Studies on the role of immunocompetent cells in the protective immune reponse are still in their infancy, however the existance of a restricted polymorphism of MHC II class molecules in the Saimiri confers additional theoretical and practical importance to this model.

Imunodiagnóstico

In the last years, the knowledge about immunodiagnosis in schistosomiasis has increased considerably. This was due to a better immunologic understanding of the host-parasite interaction and the evolution of the technical procedures by the introduction of new concepts and techniques. The search of more sensitive, specific, practical and less expensive diagnostic tests has led to the development of a great number of immunological tests that could complement the limitations of the parasitological diagnosis. The antigens of differnt life cycle stages of Schistosoma mansoni may be used as target for immunodiagnostic tests and the anti-schistosome antibodies in sera of infected patients can be detected. The research of circulating schistosome antigens, the production of specific antisera and the application of these antibodies in immunodiagnostic tests have also been discussed.

Modern immunological approaches to assess malaria transmission and immunity and to diagnose plasmodial infection

The present paper reviews our recent data concerning the use of immunological methods employing monoclonal antibodies and synthetic peptides to study malaria transmission and immunity and to diagnose plasmodial infection. As concerns malaria transmission, we studied the main vectors of human malaria and the plasmodial species transmitted in endemic areas of Rondônia state, Brazil. The natural infection on anopheline was evaluated by immunoradiometric assay (IRMA) using monoclonal antibodies to an immunodominant sporozoite surface antigen (CS protein) demonstrated to be species specific. Our results showed that among six species of Anopheles found infected, An. darlingi was the main vector transmitting Plasmodium falciparum and P. vivax malaria in the immediate vicinity of houses. In order to assess the level of anti-CS antibodies we studied, by IRMA using the synthetic peptide corresponding to the repetitive epitope of the sporozoite CS protein, sera of individuals living in the same areas where the entomological survey has been performed. In this assay the prevalence of anti-CS antibodies was very low and did not reflect the malaria transmission rate in the studied areas. In relation to malaria diagnosis, a monoclonal antibody specific to an epitope of a 50 kDa exoantigen, the major component of supernatant collected at the time of schizont rupture, was used as a probe for the detection of P. falciparum antigens. This assay seemed to be more sensitive than parasitological examination for malaria diagnosis since it was able to detect plasmodial antigens in both symptomatic and asymtomatic individuals with negative thick blood smear at different intervals after a last parasitologically confirmed confirmed attack of malaria.

Immunogenicity of multiple antigen peptides containing Plasmodium vivax CS epitopes in BALB/c mice

Multiple antigen peptide systems (MAPs) allow the incorporation of various epitopes in to a single synthetic peptide immunogen. We have characterized the immune response of BALB/c mice to a series of MAPs assembled with different B and T cell epitopes derived from the Plasmodium vivax circumsporozoite (CS) protein. A B-cell epitope from the central repeat domain and two T-cell epitopes from the amino and carboxyl flanking regions were used to assembled eight different MAPs. An additional universal T cell epitope (ptt-30) from tetanus toxin protein was included. Immunogenicity in terms of antibody responses and in vitro T lymphocyte proliferation was evaluated. MAPs containing B and T cell epitopes induced high titers of anti-peptides antibodies, which recognized the native protein on sporozoites as determined by IFAT. The antibody specificity was also determined by a competitive inhibition assay with different MAPs. A MAP containing the B cell epitope (p11) and the universal epitope ptt-30 together with another composed of p11 and the promiscuous T cell epitope (p25) proved to be the most immunogenic. The strong antibody response and specificity for the cognate protein indicates that further studies designed to assess the potential of these proteins as human malaria vaccine candidates are warranted.

Endothelial cell integrins and COX-2: mediators and therapeutic targets of tumor angiogenesis.

Vascular integrins are essential regulators and mediators of physiological and pathological angiogenesis, including tumor angiogenesis. Integrins provide the physical interaction with the extracellular matrix (ECM) necessary for cell adhesion, migration and positioning, and induce signaling events essential for cell survival, proliferation and differentiation. Integrins preferentially expressed on neovascular endothelial cells, such as alphaVbeta3 and alpha5beta1, are considered as relevant targets for anti-angiogenic therapies. Anti-integrin antibodies and small molecular integrin inhibitors suppress angiogenesis and tumor progression in many animal models, and are currently tested in clinical trials as anti-angiogenic agents. Cyclooxygense-2 (COX-2), a key enzyme in the synthesis of prostaglandins and thromboxans, is highly up-regulated in tumor cells, stromal cells and angiogenic endothelial cells during tumor progression. Recent experiments have demonstrated that COX-2 promotes tumor angiogenesis. Chronic intake of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors significantly reduces the risk of cancer development, and this effect may be due, at least in part, to the inhibition of tumor angiogenesis. Endothelial cell COX-2 promotes integrin alphaVbeta3-mediated endothelial cell adhesion, spreading, migration and angiogenesis through the prostaglandin-cAMP-PKA-dependent activation of the small GTPase Rac. In this article, we review the role of integrins and COX-2 in angiogenesis, their cross talk, and discuss implications relevant to their targeting to suppress tumor angiogenesis.

An Experimental Approach to the Pathogenesis of "Pipestem" Fibrosis (Symmers' Fibrosis of the Liver)

Pathogenesis of schistosomal hepatic fibrosis ("pipestem" fibrosis of the liver) was investigated by means of the murine model. Although worm load appears as the main pathogenetic factor, alone it is not sufficient to produce that characteristic lesion. By comparing the findings in animals with heavy and prolonged Schistosoma mansoni infection, which developed or not" pipestem" fibrosis, it was observed that the lesion was more frequent in intact animals than in the splenectomized one. However, the size of the spleen, the number of recovered worms, the number of eggs per gram of liver tissue, the level of serum idiotype and anti-idiotype antibodies, the size and volume of periovular granulomas formed in the liver, all that failed to show statistically significant differences between the two groups. After analysing all these data, other factors, that apparently have been hitherto negleted, rested to explain the findings. Among them, the timing and sequence of the egg-induced intrahepatic vascular changes seemed crucial. The sequential development of intrahepatic portal vein obstruction, followed by the opening of periportal collateral veins and the continous arrival of schistosome eggs going to be lodged into the latter, appeared as essential steps in the pathogenesis of "pipestem" fibrosis

Chagas' Disease and HIV Co-infection: Genotypic Characterization of the Trypanosoma cruzi Strain

In the past few years, new aspects of the immunopathology of Chagas' disease have been described in immunosuppressed patients, such as fatal central nervous system lesions related to the reactivation of the parasite. This article is the first description of the genotypic characterization, at the strain level, of Trypanosoma cruzi isolated from a patient with Chagas` disease/AIDS co-infection. The presence of four hypodense lesions was observed in the cranial compute tomographic scan. The diagnosis of AIDS was assessed by the detection of anti-HIV antibodies using enzyme-linked immunosorbent assay (ELISA) and Western blot techniques. The CD4+ lymphocyte counts were maintained under 200 cells/mm3 during one year demonstrating the severity of the state of immunosuppression. Chagas' disease was confirmed by serological and parasitological methods. Trypomastigote forms were visualized in a thick blood smear. The parasite isolated is genotypically similar to the CL strain. The paper reinforces that cerebral Chagas' disease can be considered as another potential opportunistic infection in AIDS resulting from the reactivation of a dormant T. cruzi infection acquired years earlier.

Human Immunodeficiency Virus Seroprevalence among Inmates of the Penitentiary Complex of the Region of Campinas, State of São Paulo, Brazil

Six hundred and ninety three male inmates from three penitentiaries, two (A and B) maximum-security systems and one (C) minimum-security facility, located in Campinas, State of São Paulo, Brazil were studied for the presence of human immunodeficiency virus (HIV) antibodies, using a cross-sectional design. The search for anti-HIV antibodies in 693 samples of sera collected was carried out by two serological tests: (a) the Microparticle enzyme immunoassay-HIV-1 and HIV-2 (MEIA) (Abbott Laboratories) and (b) the Western Blot-HIV-1 (WB) (Cambridge Biotech Corporation) to confirm positive results with MEIA. Sera reactivity for HIV antibodies was 14.4%. The highest frequency of anti-HIV antibodies was found in the A and B maximum-security prisons: 17% and 21.5%, respectively. In prison C, the frequency of reagents was 10.9%. Seventy three inmates, initially negative in the MEIA test, were checked again five and seven months later. Three of them, all from the maximum-security facilities, became reactive in the MEIA test, with confirmation in the WB, suggesting that serological conversion had occurred after imprisonment.

Frequency of human toxocariasis in Jos, Plateau State, Nigeria

The enzyme-linked immunosorbent assay (Elisa) was used to examine sera of 104 children and adults in Jos, Plateau State, Nigeria for anti-toxocaral antibodies, out of which 31 (29.8%) were reactive. The seropositive rates were 30.4% for adults, 29.6% for children, 34% for females and 25.9% for males. However, the differences were not significant by age and sex. A highly significant association (p < 0.001) was observed between seropositivity and geophagia but none between seropositivity and dog ownership (p > 0.05).

Lassa virus entry in antigen presenting cells and evaluation of a novel nanoparticle vaccine platform against arena viruses

Arenaviruses are a large and diverse family of viruses that merit significant attention as causative agents of severe hemorrhagic fevers in humans. Lassa virus (LASV) in Africa and the South American hemorrhagic fever viruses Junin (JUNV), Machupo (MACV), and Guanarito (GTOV) have emerged as important human pathogens and represent serious public health problems in their respective endemic areas. A hallmark of fatal arenaviruses hemorrhagic fevers is a marked immunosuppression of the infected patients. Antigen presenting cells (APCs) such as macrophages and in particular dendritic cells (DCs) are early and preferred targets of arenaviruses infection. Instead of being recognized and presented as foreign antigens by DCs, arenaviruses subvert the normal mechanisms of pathogen recognition, invade DCs and establish a productive infection. Viral replication perturbs the DCs' ability to present antigens and to activate T and B cells, contributing to the marked virus-induced immunosuppression observed in fatal disease. Considering their crucial role in the development of an anti-viral immune response, the mechanisms by which arenaviruses, and in particular LASV, invade DCs are of particular interest. The C-type lectin DC-specific Intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) was recently identified as a potential entry receptor for LASV. The first project of my thesis focused therefore on the investigation of the role of DC-SIGN in LASV entry into primary human DCs. My data revealed that DC-SIGN serves as an attachment factor for LASV on human DCs and can facilitate capture of free virus and subsequent cell entry. However, in contrast to other emerging viruses, of the phlebovirus family, I found that DC-SIGN does likely not function as an authentic entry receptor for LASV. Moreover, I was able to show that LASV enters DCs via an unusually slow pathway that depends on actin, but is independent of clathrin and dynamin. Considering the lack of effective treatments and the limited public health infrastructure in endemic regions, the development of protective vaccines against arenaviruses is an urgent need. To address this issue, the second project of my thesis aimed at the development of a novel recombinant arenavirus vaccine based on a nanoparticle (NPs) platform and its evaluation in a small animal model. During the first phase of the project I designed, produced, and characterized suitable vaccine antigens. In the second phase of the project, I generated antigen-conjugated NPs, developed vaccine formulations, and tested the NPs for their ability to elicit anti-viral T cell responses as well as anti-viral antibodies. I demonstrated that the NPs platform is able to activate both cellular and humoral branches of the adaptive anti-viral immunity, providing proof-of-principle. In sum, my first project will allow, in a long term perspective, a better understanding of the viral pathogenesis and contribute to the development of novel antiviral strategies. The second project will expectidly offer a new treatment option against arenaviruses.

Evaluation of an enzyme immunoassay for hepatitis C virus antibody detection using a recombinant protein derived from the core region of hepatitis C virus genome

This study was undertaken to evaluate an enzyme immunoassay (EIA) for hepatitis C virus antibody detection (anti-HCV), using just one antigen. Anti-HCV EIA was designed to detect anti-HCV IgG using on the solid-phase a recombinant C22 antigen localized at the N-terminal end of the core region of HCV genome, produced by BioMérieux. The serum samples diluted in phosphate buffer saline were added to wells coated with the C22, and incubated. After washings, the wells were loaded with conjugated anti-IgG, and read in a microtiter plate reader (492 nm). Serum samples of 145 patients were divided in two groups: a control group of 39 patients with non-C hepatitis (10 acute hepatitis A, 10 acute hepatitis B, 9 chronic hepatitis B, and 10 autoimmune hepatitis) and a study group consisting of 106 patients with chronic HCV hepatitis. In the study group all patients had anti-HCV detected by a commercially available EIA (Abbott®), specific for HCV structural and nonstructural polypeptides, alanine aminotransferase elevation or positive serum HCV-RNA detected by nested-PCR. They also had a liver biopsy compatible with chronic hepatitis. The test was positive in 101 of the 106 (95%) sera from patients in the study group and negative in 38 of the 39 (97%) sera from those in the control group, showing an accuracy of 96%. According to these results, our EIA could be used to detect anti-HCV in the serum of patients infected with hepatitis C virus.

Macrophage migration inhibitory factor: a counter-regulator of glucocorticoid action and critical mediator of septic shock.

Recent studies have led to the discovery of a mediator that acts as an endogenous counter-regulator of glucocorticoid action within the immune system. Isolated as a product of anterior pituitary cells, this protein was found to have the sequence of macrophage migration inhibitory factor (MIF), one of the first cytokine activities to be described. Macrophages and T cells release MIF in response both to various inflammatory stimuli and upon incubation with low concentrations of glucocorticoids. The glucocorticoid-induced secretion of MIF is tightly regulated and decreases at high, anti-inflammatory steroid concentrations. Once secreted, MIF "overrides" the anti-inflammatory and immunosuppressive effects of steroids on macrophage and T-cell cytokine production. The physiological role of MIF thus appears to be to counter-balance steroid inhibition of the inflammatory response. Anti-MIF antibodies fully protect animals from experimentally induced gram-negative or gram-positive septic shock, an effect that may be the result of the increased anti-inflammatory effects of glucocorticoids after neutralization of endogenous MIF. Anti-MIF therapeutic strategies are presently under development and may prove to be a means to modulate cytokine production in septic shock as well as in other inflammatory disease states.

Immunological and protective properties of novel malaria blood stage peptide antigens

ABSTRACT Malaria is a major worldwide public health problem, with transmission occurring throughout Africa, Asia, Oceania and Latin America. Over two billion people live in malarious areas of the world and it is estimated that 300-500 million cases and 1.5-2.7 million deaths occur annually. The increase in multi-drug resistant parasites and insecticide-resistant vectors has made the development of malaria vaccine a public health priority. The published genome offers tremendous opportunity for the identification of new antigens that can befast-tracked for vaccine development. We identified potential protein antigens present on the surface of asexual malaria blood stages through bioinformatics and published transcriptome and proteorné analysis. Amongst the proteins identified, we selected those that contain predicted a-helical coiled-coil regions, which are generally short and structurally stable as isolated fragments. Peptides were synthesized and used to immunize mice. Most peptides tested were immunogenic as demonstrated in ELISA assays, and induced antibodies of varying titres. In immunofluorescence assays, anti-sera from immunized mice reacted with native proteins expressed at different intraerythrocytic developmental stages of the parasite's cycle. In parallel in vitro ADCI functional studies, human antibodies affinity purified on some of these peptides inhibited parasite growth in association with monocytes in magnitudes similar to that seen in semiimmune African adults. Siudies using human immune sera taken from different malaria endemic regions, demonstrated that majority of peptides were recognized at high prevalence. 73 peptides were next tested in longitudinal studies in two cohorts separated in space and time in coastal Kenya. In these longitudinal analyses, antibody responses to peptides were sequentially examined in two cohorts of children at risk of clinical malaria in order to characterize the level of peptide recognition by age, and the role of anti-peptide antibodies in protection from clinical malaria. Ten peptides were associated ?with a significantly reduced odds ratio for an episode of clinical malaria in the first cohort of children and two of these peptides (LR146 and ÁS202.11) were associated with a significantly reduced odds ratio in both cohorts. This study has identified proteins PFB0145c and MAL6P1.37 among others as likely targets of protective antibodies. Our findings support further studies to systematically assess immunogenicity of peptides of interest in order to establish clear criteria for optimal design of potential vaccine constructs to be tested in clinical trials. RESUME La malaria est un problème de santé publique mondial principalement en Afrique, en Asie, en Océanie et en Amérique latine. Plus de 2 milliards de personnes vivent dans des régions endémiques et le nombre de cas par année est estimé entre 300 et 500 millions. 1.5 à 2.7 millions de décès surviennent annuellement dans ces zones. L'augmentation de la résistance aux médicaments et aux insecticides fait du développement d'un vaccin une priorité. Le séquençage complet du génome du parasite offre l'opportunité d'identifier de nouveaux antigènes qui peuvent rapidement mener au développement d'un vaccin. Des protéines antigéniques potentielles présentes à la surface des globules rouges infectés ont été identifiées par bioinformatique et par l'analyse du protéome et du transcriptome. Nous avons sélectionné, parmi ces protéines, celles contenant des motifs dits "a helical coiled-coil" qui sont généralement courts et structurellement stables. Ces régions ont été obtenues par synthèse peptidique et utilisées pour immuniser des souris. La plupart des peptides testés sont immunogéniques et induisent un titre variable d'anticorps déterminé par ELISA. Les résultats de tests d'immunofluorescence indiquent que les sera produits chez la souris reconnaissent les protéines natives exprimées aux différents stades de développement du parasite. En parallèle, des études d'ADCI in vitro montrent qué des anticorps humains purifiés à partir de ces peptides associés à des monocytes inhibent la croissance du parasite aussi bien que celle observée chez des adultes africains protégés. Des études d'antigénicité utilisant des sera de personnes protégées de différents âges vivant dans des régions endémiques montrent que la majorité des peptides sont reconnus avec une haute prévalence. 73 peptides ont été testés dans une étude longitudinale avec 2 cohortes de la côte du Kenya. Ces 2 groupes viennent de zones bien distinctes et les prélèvements n'ont pas été effectués pendant la même période. Dans cette étude, la réponse anticorps contre les peptides synthétiques a été testée dans les 2 cohortes d'enfants à risque de développer un épisode de malaria afin de caractériser le niveau de reconnaissance des peptides en fonction de l'âge et de déterminer le rôle des anticorps anti-peptides dans la protection contre la malaria. Parmi ces peptides, 10 sont associés à une réduction significative des risques de développer un épisode de malaria dans la première cohorte alors qu'un seul (LR146 et AS202.11) l'est dans les 2 cohortes. Cette étude a identifié, parmi d'autres, les protéines PFB0145c et MAL6P1.37 comme pouvant être la cible d'anticorps. Ces résultats sont en faveur de futures études qui évalueraient systématiquement l'immunogénicité des peptides d'intérêt dans le but d'établir des critères de sélection clairs pour le développement d'un vaccin. Résumé pour un large public La malaria est un problème de santé publique mondial principalement en Afrique, en Asie, en Océanie et en Amérique latine. Plus de 2 milliards de personnes vivent dans des régions endémiques et le nombre de cas par année est estimé entre 300 et 500 millions. 1.5 à 2.7 millions de décès surviennent annuellement dans ces zones. La résistance aux médicaments et aux insecticides augmente de plus en plus d'où la nécessité de développer un vaccin. Le séquençage complet du génome (ensemble des gènes) de P. falciparum a conduit au développement de nouvelles .études à large échelle dans le domaine des protéines du parasite (protéome) ; dans l'utilisation d'algorithmes, de techniques informatiques et statistiques pour l'analyse de données biologiques (bioinformatique) et dans les technologies de transcription et de profiles d'expression (transcriptome). Nous avons identifié, en utilisant les outils ci-dessus, des nouvelles protéines antigéniques qui sont présentes au stade sanguin de la malaria. Nous avons sélectionné, parmi ces protéines, celles contenant un motif dit "a-helical coiled-coil" qui sont des domaines impliqués dans un large éventail de fonctions biologiques. Des peptides représentant ces régions structurellement stables ont été synthétisés et utilisés pour immuniser des souris. La plupart des peptides testés sont immunogéniques et induisent un titre variable d'anticorps déterminé par ELISA. Les résultats de tests d'immunofluorescence indiquent que plusieurs sera de souris immunisées avec ces peptides reconnaissent les protéines natives exprimées à la surface des globules rouges infectés. En parallèle, des études d'ADCI in vitro montrent que des anticorps humains purifiés à partir de ces peptides en présence de monocytes inhibent la croissance du parasite de manière similaire à celle observée chez des adultes africains protégés. Des études d'antigénicité utilisant des sera de personnes immunes de différents âges (adultes et enfants) vivant dans des régions endémiques montrent que la majorité des peptides sont reconnus avec une haute prévalence. 73 peptides ont été testés dans des études épidémiologiques dans 2 villages côtiers du Kenya Ces 2 groupes vivent dans des zones bien distinctes et les prélèvements n'ont pas été effectués pendant la même période. Dans ces études, la réponse anticorps dirigée contre les peptides synthétiques a été testée en utilisant 467 échantillons sanguins d'enfants à risque de développer un épisode de malaria afin de caractériser le niveau de reconnaissance des peptides en fonction de l'âge et de déterminer le rôle des anticorps anti-peptides dans la protection contre la malaria cérébrale. Parmi ces peptides, 10 sont associés à une protection contre un épisode de malaria dans le premier village alors qu'un seul l'est dans les 2 villages. Ces résultats sont en faveur de futures études qui évalueraient systématiquement l'immunogénicité des peptides intéressants dans le but d'établir des critères de sélection clairs pour le développement d'un vaccin.

Variants of the Plasmodium vivax circumsporozoite protein (VK210 and VK247) in Colombian isolates

Phenotypic diversity has been described in the central repeated region of the circumsporozoite protein (CSP) from Plasmodium vivax. Two sequences VK210 (common) and VK247 (variant) have been found widely distributed in P. vivax isolates from several malaria endemic areas around the world. A third protein variant called P. vivax-like showing a sequence similar to the simian parasite P. simio-ovale has also been described. Here, using an immunofluorescent test and specific monoclonal antibodies, we assessed the presence of two of these protein variants (VK210 and VK247) in laboratory produced sporozoite. Both sequences were found in parasite isolates coming from different geographic regions of Colombia. Interestingly, sporozoites carrying the VK247 sequence were more frequently produced in Anopheles albimanus than sporozoites with the VK210 sequence. This difference in sporozoites production was statistically significant (p <0.05, Kruskal-Wallis); not correlation was found with parameters as the total number of parasites or gametocytes in blood from human donors used to feed mosquitoes. Previous studies in the same region have shown a higher prevalence of anti-VK210 antibodies which in theory may suggest their role in blocking the development of sporozoites carrying the CSP VK210 sequence.

Prevalence of hepatitis B and C virus markers among malaria-exposed gold miners in Brazilian Amazon

Hepatitis B and C virus markers were assessed during a survey on malaria in gold mine camps in southern Brazilian Amazon in order to identify risk factors associated to these viral diseases. The study comprised 520 subjects, most of them were gold miners. Missing subjects totaled 49 (8.6%). Among these 520, 82.9% had HBV markers and 7.1% were HBsAg positive. Previous hospitalization, surgery, sexually transmitted diseases and incarceration were quite common among surveyed people, but there is no association between total HBV markers and these factors. On other hand, HBsAg was independently associated to history of sexually transmitted diseases and history of surgery after adjustment. The most frequent HBsAg subtypes identified, adw2 (59%), predominates in populations of Northeast Brazil. The most surveyed people were immigrants coming from that area suggesting that immigrants carried HBV themselves to the study area. Immunoblot (RIBA) confirmed-anti-HCV were found in 2.1%. The only variable associated to anti-HCV in multivariate analysis was illicit intravenous drug. Lack of HCV infection in subjects with such a high HBV markers prevalence reinforces the opinion that HCV is transmitted by restricted routes when compared to HBV. Furthermore, gold miners in Amazon may be considered as a risk group for HBV infection, but not for HCV.