DNA-mediated immunization in a transgenic mouse model of the hepatitis B surface antigen chronic carrier state.

Transgenic mice expressing the sequences coding for the envelope proteins of the hepatitis B virus (HBV) in the liver have been used as a model of the HBV chronic carrier state. We evaluated the possibility of inducing a specific immune response to the viral envelope antigens and thus potentially controlling chronic HBV infection. Using HBV-specific DNA-mediated immunization in this transgenic model, we show that the immune response induced after a single intramuscular injection of DNA resulted in the complete clearance of circulating hepatitis B surface antigen and in the long-term control of transgene expression in hepatocytes. This response does not involve a detectable cytopathic effect in the liver. Adoptive transfer of fractionated primed spleen cells from DNA-immunized mice shows that T cells are responsible for the down-regulation of HBV mRNA in the liver of transgenic mice. To our knowledge, this is the first demonstration of a potential immunotherapeutic application of DNA-mediated immunization against an infectious disease and raises the possibility of designing more effective ways of treating HBV chronic carriers.

Viral cross talk: intracellular inactivation of the hepatitis B virus during an unrelated viral infection of the liver.

Hepatitis B virus (HBV) infection is thought to be controlled by virus-specific cytotoxic T lymphocytes (CTL). We have recently shown that HBV-specific CTL can abolish HBV replication noncytopathically in the liver of transgenic mice by secreting tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) after antigen recognition. We now demonstrate that hepatocellular HBV replication is also abolished noncytopathically during lymphocytic choriomeningitis virus (LCMV) infection, and we show that this process is mediated by TNF-alpha and IFN-alpha/beta produced by LCMV-infected hepatic macrophages. These results confirm the ability of these inflammatory cytokines to abolish HBV replication; they elucidate the mechanism likely to be responsible for clearance of HBV in chronically infected patients who become superinfected by other hepatotropic viruses; they suggest that pharmacological activation of intrahepatic macrophages may have therapeutic value in chronic HBV infection; and they raise the possibility that conceptually similar events may be operative in other viral infections as well.

Viral dynamics in hepatitis B virus infection.

Treatment of chronic hepatitis B virus (HBV) infections with the reverse transcriptase inhibitor lamivudine leads to a rapid decline in plasma viremia and provides estimates for crucial kinetic constants of HBV replication. We find that in persistently infected patients, HBV particles are cleared from the plasma with a half-life of approximately 1.0 day, which implies a 50% daily turnover of the free virus population. Total viral release into the periphery is approximately 10(11) virus particles per day. Although we have no direct measurement of the infected cell mass, we can estimate the turnover rate of these cells in two ways: (i) by comparing the rate of viral production before and after therapy or (ii) from the decline of hepatitis B antigen during treatment. These two independent methods give equivalent results: we find a wide distribution of half-lives for virus-producing cells, ranging from 10 to 100 days in different patients, which may reflect differences in rates of lysis of infected cells by immune responses. Our analysis provides a quantitative understanding of HBV replication dynamics in vivo and has implications for the optimal timing of drug treatment and immunotherapy in chronic HBV infection. This study also represents a comparison for recent findings on the dynamics of human immunodeficiency virus (HIV) infection. The total daily production of plasma virus is, on average, higher in chronic HBV carriers than in HIV-infected patients, but the half-life of virus-producing cells is much shorter in HIV. Most strikingly, there is no indication of drug resistance in HBV-infected patients treated for up to 24 weeks.

Role of B cells in antigen presentation of the hepatitis B core

The hepatitis B virus (HBV) nucleocapsid or core antigen (HBcAg) is extremely immunogenic during infection and after immunization. For example, during many chronic infections, HBcAg is the only antigen capable of eliciting an immune response, and nanogram amounts of HBcAg elicit antibody production in mice. Recent structural analysis has revealed a number of characteristics that may help explain this potent immunogenicity. Our analysis of how the HBcAg is presented to the immune system revealed that the HBcAg binds to specific membrane Ig (mIg) antigen receptors on a high frequency of resting, murine B cells sufficiently to induce B7.1 and B7.2 costimulatory molecules. This enables HBcAg-specific B cells from unprimed mice to take up, process, and present HBcAg to naive Th cells in vivo and to T cell hybridomas in vitro approximately 105 times more efficiently than classical macrophage or dendritic antigen-presenting cells (APC). These results reveal a structure–function relation for the HBcAg, confirm that B cells can function as primary APC, explain the enhanced immunogenicity of HBcAg, and may have relevance for the induction and/or maintenance of chronic HBV infection.

Identification of hepatitis B virus indigenous to chimpanzees

Hepatitis B viruses (HBV) and related viruses, classified in the Hepadnaviridae family, are found in a wide variety of mammals and birds. Although the chimpanzee has been the primary experimental model of HBV infection, this species has not been considered a natural host for the virus. Retrospective analysis of 13 predominantly wild-caught chimpanzees with chronic HBV infection identified a unique chimpanzee HBV strain in 11 animals. Nucleotide and derived amino acid analysis of the complete HBV genome and the gene coding for the hepatitis B surface antigen (S gene) identified sequence patterns that could be used to reliably identify chimpanzee HBV. This analysis indicated that chimpanzee HBV is distinct from known human HBV genotypes and is closely related to HBVs previously isolated from a chimpanzee, gibbons, gorillas, and orangutans.

Inhibition of MHC class I-restricted antigen presentation by γ2-herpesviruses

The γ-herpesviruses, in contrast to the α- and β-herpesviruses, are not known to inhibit antigen presentation to CD8+ cytotoxic T lymphocytes (CTLs) during lytic cycle replication. However, murine γ-herpesvirus 68 causes a chronic lytic infection in CD4+ T cell-deficient mice despite the persistence of a substantial CTL response, suggesting that CTL evasion occurs. Here we show that, distinct from host protein synthesis shutoff, γ-herpesvirus 68 down-regulates surface MHC class I expression on lytically infected fibroblasts and inhibits their recognition by antigen-specific CTLs. The viral K3 gene, encoding a zinc-finger-containing protein, dramatically reduced the half-life of nascent class I molecules and the level of surface MHC class I expression and was by itself sufficient to block antigen presentation. The homologous K3 and K5 genes of the related Kaposi's sarcoma-associated virus also inhibited antigen presentation and decreased cell surface expression of HLA class I antigens. Thus it appears that an immune evasion strategy shared by at least two γ-herpesviruses allows continued lytic infection in the face of strong CTL immunity.

Efficient pyrophosphorolysis by a hepatitis B virus polymerase may be a primer-unblocking mechanism

Effective antiviral agents are thought to inhibit hepatitis B virus (HBV) DNA synthesis irreversibly by chain termination because reverse transcriptases (RT) lack an exonucleolytic activity that can remove incorporated nucleotides. However, since the parameters governing this inhibition are poorly defined, fully delineating the catalytic mechanism of the HBV-RT promises to facilitate the development of antiviral drugs for treating chronic HBV infection. To this end, pyrophosphorolysis and pyrophosphate exchange, two nonhydrolytic RT activities that result in the removal of newly incorporated nucleotides, were characterized by using endogenous avian HBV replication complexes assembled in vivo. Although these activities are presumed to be physiologically irrelevant for every polymerase examined, the efficiency with which they are catalyzed by the avian HBV-RT strongly suggests that it is the first known polymerase to catalyze these reactions under replicative conditions. The ability to remove newly incorporated nucleotides during replication has important biological and clinical implications: these activities may serve a primer-unblocking function in vivo. Analysis of pyrophosphorolysis on chain-terminated DNA revealed that the potent anti-HBV drug β-l-(−)-2′,3′-dideoxy-3′-thiacytidine (3TC) was difficult to remove by pyrophosphorolysis, in contrast to ineffective chain terminators such as ddC. This disparity may account for the strong antiviral efficacy of 3TC versus that of ddC. The HBV-RT pyrophosphorolytic activity may therefore be a novel determinant of antiviral drug efficacy, and could serve as a target for future antiviral drug therapy. The strong inhibitory effect of cytoplasmic pyrophosphate concentrations on viral DNA synthesis may also partly account for the apparent slow rate of HBV genome replication.

Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic Pseudomonas aeruginosa pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing P. aeruginosa from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30–100 times as many P. aeruginosa as cells lacking CFTR or expressing mutant ΔF508 CFTR protein. Purified CFTR inhibited ingestion of P. aeruginosa by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to P. aeruginosa and a synthetic peptide of this region inhibited P. aeruginosa internalization in vivo, leading to increased bacterial lung burdens. CFTR clears P. aeruginosa from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.

Therapeutic passive vaccination against chronic Lyme disease in mice

Passive and active immunization against outer surface protein A (OspA) has been successful in protecting laboratory animals against subsequent infection with Borrelia burgdorferi. Antibodies (Abs) to OspA convey full protection, but only when they are present at the time of infection. Abs inactivate spirochetes within the tick and block their transmission to mammals, but do not affect established infection because of the loss of OspA in the vertebrate host. Our initial finding that the presence of high serum titers of anti-OspC Abs (5 to 10 μg/ml) correlates with spontaneous resolution of disease and infection in experimentally challenged immunocompetent mice suggested that therapeutic vaccination with OspC may be feasible. We now show that polyclonal and monospecific mouse immune sera to recombinant OspC, but not to OspA, of B. burgdorferi resolve chronic arthritis and carditis and clear disseminated spirochetes in experimentally infected C.B.-17 severe combined immunodeficient mice in a dose-dependent manner. This was verified by macroscopical and microscopical examination of affected tissues and recultivation of spirochetes from ear biopsies. Complete resolution of disease and infection was achieved, independent of whether OspC-specific immune sera (10 μg OspC-specific Abs) were repeatedly given (4× in 3- to 4-day intervals) before the onset (day 10 postinfection) or at the time of fully established arthritis and carditis (days 19 or 60 postinfection). The results indicate that in mice spirochetes constitutively express OspC and are readily susceptible to protective OspC-specific Abs throughout the infection. Thus, an OspC-based vaccine appears to be a candidate for therapy of Lyme disease.

Fas-dependent CD4+ cytotoxic T-cell-mediated pathogenesis during virus infection

β2-Microglobulin-deficient (β2m−) mice generate a CD4+ major histocompatibility complex class II-restricted cytotoxic T-lymphocyte (CTL) response following infection with lymphocytic choriomeningitis (LCM) virus (LCMV). We have determined the cytotoxic mechanism used by these CD4+ CTLs and have examined the role of this cytotoxic activity in pathogenesis of LCM disease in β2m− mice. Lysis of LCMV-infected target cells by CTLs from β2m− mice is inhibited by addition of soluble Fas-Ig fusion proteins or by pretreatment of the CTLs with the protein synthesis inhibitor emetine. In addition, LCMV-infected cell lines that are resistant to anti-Fas-induced apoptosis are refractory to lysis by these virus-specific CD4+ CTLs. These data indicate that LCMV-specific CD4+ CTLs from β2m− mice use a Fas-dependent lytic mechanism. Intracranial (i.c.) infection of β2m− mice with LCMV results in loss of body weight. Fas-deficient β2m−.lpr mice develop a similar wasting disease following i.c. infection. This suggests that Fas-dependent cytotoxicity is not required for LCMV-induced weight loss. A potential mediator of this chronic wasting disease is tumor necrosis factor (TNF)-α, which is produced by LCMV-specific CD4+ CTLs. In contrast to LCMV-induced weight loss, lethal LCM disease in β2m− mice is dependent on Fas-mediated cytotoxicity. Transfer of immune splenocytes from LCMV-infected β2m− mice into irradiated infected β2m− mice results in death of recipient animals. In contrast, transfer of these splenocytes into irradiated infected β2m−.lpr mice does not cause death. Thus a role for CD4+ T-cell-mediated cytotoxicity in virus-induced immunopathology has now been demonstrated.

Effects of protein calorie malnutrition on tuberculosis in mice

Infectious diseases and malnutrition represent major burdens afflicting millions of people in developing countries. Both conditions affect individuals in industrialized nations, particularly the aged, the HIV-infected, and people with chronic diseases. While malnutrition is known to induce a state of immunodeficiency, the mechanisms responsible for compromised antimicrobial resistance in malnourished hosts remain obscure. In the present study, mice fed a 2% protein diet and developing protein calorie malnutrition, in contrast to well-nourished controls receiving a 20% protein diet, rapidly succumbed to infection with Mycobacterium tuberculosis. Malnourished mice exhibited a tissue-specific diminution in the expression of interferon γ, tumor necrosis factor α, and the inducible form of nitric oxide synthase in the lungs, but not the liver. The expression of these molecules critical to the production of mycobactericidal nitrogen oxides was depressed in malnourished animals in the lungs specifically at early times (<14 days) after infection. At later times, levels of expression became comparable to those in well-nourished controls, although the bacillary burden in the malnourished animals continued to rise. Nevertheless, urinary and serum nitrate contents, an index of total nitric oxide (NO) production in vivo, were not detectably diminished in malnourished, mycobacteria-infected mice. In contrast to the selective and early reduction of lymphokines and the inducible form of nitric oxide synthase in the lung, a marked diminution of the granulomatous reaction was observed in malnourished mice throughout the entire course of infection in all tissues examined (lungs, liver, and spleen). Remarkably, the progressively fatal course of tuberculosis observed in the malnourished mice could be reversed by restoring a full protein (20%) diet. The results indicate that protein calorie malnutrition selectively compromises several components of the cellular immune response that are important for containing and restricting tuberculous infection, and suggest that malnutrition-induced susceptibility to some infectious diseases can be reversed or ameliorated by nutritional intervention.

Genetic control of resistance to experimental infection with virulent Mycobacterium tuberculosis

Over 2 billion people are estimated to be infected with virulent Mycobacterium tuberculosis, yet fewer than 10% progress to clinical tuberculosis within their lifetime. Twin studies and variations in the outcome of tuberculosis infection after exposure to similar environmental risks suggest genetic heterogeneity among individuals in their susceptibility to disease. In a mouse model of tuberculosis, we have established that resistance and susceptibility to virulent M. tuberculosis is a complex genetic trait. A new locus with a major effect on tuberculosis susceptibility, designated sst1 (susceptibility to tuberculosis 1), was mapped to a 9-centimorgan (cM) interval on mouse chromosome 1. It is located 10–19 cM distal to a previously identified gene, Nramp1, that controls the innate resistance of mice to the attenuated bacillus Calmette–Guérin vaccine strain. The phenotypic expression of the newly identified locus is distinct from that of Nramp1 in that sst1 controls progression of tuberculosis infection in a lung-specific manner. Mice segregating at the sst1 locus exhibit marked differences in the growth rates of virulent tubercle bacilli in the lungs. Lung lesions in congenic sst1-susceptible mice are characterized by extensive necrosis and unrestricted extracellular multiplication of virulent mycobacteria, whereas sst1-resistant mice develop interstitial granulomas and effectively control multiplication of the bacilli. The resistant allele of sst1, although powerful in controlling infection, is not sufficient to confer full protection against virulent M. tuberculosis, indicating that other genes located outside of the sst1 locus are likely also to be important for controlling tuberculosis infection.

Experimental infection model for Sin Nombre hantavirus in the deer mouse (Peromyscus maniculatus)

The relationship between hantaviruses and their reservoir hosts is not well understood. We successfully passaged a mouse-adapted strain of Sin Nombre virus from deer mice (Peromyscus maniculatus) by i.m. inoculation of 4- to 6-wk-old deer mouse pups. After inoculation with 5 ID50, antibodies to the nucleocapsid (N) antigen first became detectable at 14 d whereas neutralizing antibodies were detectable by 7 d. Viral N antigen first began to appear in heart, lung, liver, spleen, and/or kidney by 7 d, whereas viral RNA was present in those tissues as well as in thymus, salivary gland, intestine, white fat, and brown fat. By 14 d nearly all tissues examined displayed both viral RNA and N antigen. We noted no consistent histopathologic changes associated with infection, even when RNA load was high. Viral RNA titers peaked on 21 d in most tissues, then began to decline by 28 d. Infection persisted for at least 90 d. The RNA titers were highest in heart, lung, and brown fat. Deer mice can be experimentally infected with Sin Nombre virus, which now allows provocative examination of the virus-host relationship. The prominent involvement of heart, lung, and brown fat suggests that these sites may be important tissues for early virus replication or for maintenance of the virus in nature.

Hepatitis C virus lacking the hypervariable region 1 of the second envelope protein is infectious and causes acute resolving or persistent infection in chimpanzees

Persistent infection with hepatitis C virus (HCV) is among the leading causes of chronic liver disease. Previous studies suggested that genetic variation in hypervariable region 1 (HVR1) of the second envelope protein, possibly in response to host immune pressure, influences the outcome of HCV infection. In the present study, a chimpanzee transfected intrahepatically with RNA transcripts of an infectious HCV clone (pCV-H77C) from which HVR1 was deleted became infected; the ΔHVR1 virus was subsequently transmitted to a second chimpanzee. Infection with ΔHVR1 virus resulted in persistent infection in the former chimpanzee and in acute resolving infection in the latter chimpanzee. Both chimpanzees developed hepatitis. The ΔHVR1 virus initially replicated to low titers, but virus titer increased significantly after mutations appeared in the viral genome. Thus, wild-type HCV without HVR1 was apparently attenuated, suggesting a functional role of HVR1. However, our data indicate that HVR1 is not essential for the viability of HCV, the resolution of infection, or the progression to chronicity.

Multiple modes of cellular activation and virus transmission in HIV infection: A role for chronically and latently infected cells in sustaining viral replication

CD4+ T cell activation, required for virus replication in these cells, occurs in local microenvironmental domains in transient bursts. Thus, although most HIV originates from short-lived virus-producing cells, it is unlikely that chronic infection is generally sustained in rapid continuous cycles of productive infection as has been proposed. Such continuity of productive infection cycles would depend on efficient long-range transmission of HIV from one set of domains to another, in turn requiring the maintenance of sufficiently high concentrations of cell-free virus across lymphoid tissues at all times. By contrast, long-lived cellular sources of HIV maintain the capacity to infect newly activated cells at close range despite the temporal and spatial discontinuities of activation events. Such proximal activation and transmission (PAT) involving chronically and latently infected cells may be responsible for sustained infection, particularly when viral loads are low. Once CD4 cells are productively infected through PAT, they can infect other activated cells in their immediate vicinity. Such events propagate locally but generally do not spread systemically, unlike in the acute phase of the infection, because of the early establishment of protective anergy. Importantly, antiretroviral drug treatment is likely to differentially impact long-range transmission and PAT.