Lamivudine treatment can overcome cytotoxic T-cell hyporesponsiveness in chronic hepatitis B: new perspectives for immune therapy.

The hepatitis B virus (HBV) cytotoxic T lymphocyte (CTL) response in patients with chronic HBV infection is generally weak or totally undetectable. This inability to mount protective CTL responses is believed to be a crucial determinant of viral persistence, and its correction represents an important objective of immune therapies for chronic hepatitis B. However, amplification of CTL responses in vivo may be ineffective if HBV-specific CD8 cells are either absent or nonresponsive to exogenous stimulation. In this study, we asked whether antiviral treatments able to inhibit viral replication and to reduce viral and antigen load can successfully reconstitute CTL responses creating the appropriate conditions for their therapeutic stimulation. For this purpose, the HBV-specific CTL response before and during lamivudine therapy was studied longitudinally in 6 HLA-A2-positive patients with HBeAg+ chronic hepatitis B. Both HBV-specific cytotoxic T cell activity measured by chromium release assay on peptide stimulation in vitro and CD8+ T cell frequency measured ex vivo by HLA-A2/peptide tetramer staining were significantly augmented by lamivudine therapy. This enhancement followed the reconstitution of CD4 reactivity and the decline of viral load induced by therapy. Our study shows that lamivudine treatment in chronic hepatitis B can restore CTL reactivity, making CTL susceptible to exogenous stimulation. This effect may enhance the probability that T cell-based immune therapies delivered after lamivudine treatment can successfully reconstitute a protective CTL response able to cure chronic HBV infection.

Lamivudine for the treatment of chronic hepatitis B.

Lamivudine (Zeffix) is the first of a new class of antiviral agents to become available for the treatment of chronic hepatitis B. The results of controlled clinical trials indicate that in most patients, lamivudine improves necro-inflammatory liver disease, reduces the progression of hepatic fibrosis, normalises serum alanine aminotransferase, and enhances hepatitis B e antigen (HBeAg) seroconversion. For patients with HBeAg-positive chronic hepatitis B, one year of lamivudine therapy results in HBeAg seroconversion rates similar to those obtained with a standard course of interferon-alpha. Moreover, results from two and three years of lamivudine therapy show that the cumulative HBeAg seroconversion rate continues to increase with extended lamivudine therapy. Even in the absence of HBeAg seroconversion, lamivudine therapy leads to improvements in liver disease in many patients. HBV strains (YMDD variants) with reduced in vitro sensitivity to lamivudine were detected in some patients after at least 9 months therapy. Although the clinical benefits to lamivudine were greatest for those patients who remained free of YMDD variants, one year of lamivudine therapy led to improvements in most response parameters compared with placebo, regardless of whether YMDD variants were detected. Controlled and open-label studies show that lamivudine may provide similar benefits to other important groups of patients with chronic hepatitis B, including those with pre-core mutant disease and those with hepatic decompensation. Lamivudine was well tolerated in all patient groups studied. The incidence of adverse events was consistently similar in patients who received lamivudine compared with those given placebo. In conclusion, extensive clinical data provide evidence that lamivudine is a well-tolerated, effective, and convenient medicine for patients with chronic hepatitis B.

Lamivudine treatment can restore T cell responsiveness in chronic hepatitis B.

High viral and/or antigen load may be an important cause of the T cell hyporesponsiveness to hepatitis B virus (HBV) antigens that is often observed in patients with chronic HBV infection. Reduction of viral and antigen load by lamivudine treatment represents an ideal model for investigating this hypothesis. HLA class II restricted T cell responses and serum levels of HBV-DNA, HBsAg, and HBeAg were studied before and during lamivudine treatment in 12 patients with hepatitis B e antigen positive chronic active hepatitis B to assess possible correlations between viral and/or antigen load and vigor of the T cell response. Cell proliferation to HBV nucleocapsid antigens and peptides and frequency of circulating HBV nucleocapsid-specific T cells were assessed to characterize CD4-mediated responses. A highly significant enhancement of the CD4-mediated response to HBV nucleocapsid antigens was already detectable in most patients 7-14 d after the start of lamivudine treatment. This effect was dramatic and persistent in 10 patients but undetectable in 2. It occurred concomitant with a rapid and marked reduction of viremia. Interestingly, lamivudine also enhanced the responses to mitogens and recall antigens, showing that its effect was not limited to HBV-specific T cells. In conclusion, an efficient antiviral T cell response can be restored by lamivudine treatment in patients with chronic hepatitis B concurrently with reduction of viremia, indicating the importance of viral load in the pathogenesis of T cell hyporesponsiveness in these patients. Since lamivudine treatment can overcome T cell hyporeactivity, combining lamivudine with treatments directed to stimulate the T cell response may represent an effective strategy to induce eradication of chronic HBV infection.

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.

Inhibition of herpes simplex virus type 1 DNA polymerase activity by peptides from the UL42 accessory protein is largely nonspecific.

To identify regions in the UL42 protein of herpes simplex virus type 1 which affect viral DNA polymerase activity, a series of 96 overlapping pentadecapeptides spanning the entire 488 amino acids of the UL42 protein were synthesized and tested for their ability to inhibit polymerase activity on a primed single-stranded M13 DNA template. Two assays were used: formation of full-length double-stranded M13 molecules and rate of incorporation of deoxyribonucleoside triphosphates. Peptides from five noncontiguous regions of the UL42 protein were found to inhibit herpes simplex virus type 1 polymerase activity in both the presence and absence of UL42 protein. The most active peptides from each region correspond to amino acids 23 to 38 (peptide 6), 64 to 78 (peptide 14), 89 to 102 (peptide 19), 229 to 243 (peptide 47), and 279 to 293 (peptide 57). By two different methods (DNA mobility shift and DNA precipitation), peptides 14, 19, 47, and 57 were found to bind DNA; they most probably inhibit enzyme activity by this mechanism. Peptide 6 did not bind DNA and must act by some mechanism other than competing for DNA. The inhibitory peptides were also tested for activity against mammalian polymerase alpha and the Klenow fragment of Escherichia coli polymerase. Although some limited specificity was demonstrated (up to 10-fold for peptide 6), all the peptides showed significant activity against both polymerase alpha and E. coli polymerase.

Effects of (-)-2'-deoxy-3'-thiacytidine (3TC) 5'-triphosphate on human immunodeficiency virus reverse transcriptase and mammalian DNA polymerases alpha, beta, and gamma.

(-)-2'-Deoxy-3'-thiacytidine (3TC) is a selective inhibitor of human immunodeficiency virus replication in vitro (J. A. V. Coates, N. Cammack, H. J. Jenkinson, A. J. Jowett, M. I. Jowett, B. A. Pearson, C. R. Penn, P. L. Rouse, K. C. Viner, and J. M. Cameron, Antimicrob. Agents Chemother. 36:733-739, 1992). The effect of 3TC 5'-triphosphate on both the RNA-dependent and DNA-dependent activities of human immunodeficiency virus type 1 reverse transcriptase and DNA polymerases alpha, beta, and gamma from HeLa cells was investigated. 3TC 5'-triphosphate is a competitive inhibitor (with respect to dCTP) of the RNA-dependent DNA polymerase activity (apparent Ki = 10.6 +/- 1.0 to 1.24 +/- 5.1 microM, depending on the template and primer used); the DNA-dependent DNA polymerase activity is 50% inhibited by a 3TC 5'-triphosphate concentration of 23.4 +/- 2.5 microM when dCTP is present at a concentration equal to its Km value. Chain elongation studies show that 3TC 5'-triphosphate is incorporated into newly synthesized DNA and that transcription is terminated in a manner identical to that found for ddCTP. The 50% inhibitory concentrations of 3TC 5'-triphosphate against DNA polymerases alpha, beta, and gamma at concentrations of dCTP equal to the Km were 175 +/- 31, 24.8 +/- 10.9, and 43.8 +/- 16.4 microM, respectively. More detailed kinetic studies with 3TC 5'-triphosphate and DNA polymerases beta and gamma are consistent with the fact that inhibition of these enzymes by 3TC 5'-triphosphate is competitive with respect to dCTP. The values of Ki were determined to be 18.7 microM for DNA polymerase beta and 15.8 +/- 0.8 microM for DNA polymerase gamma.

The effect of the UL42 protein on the DNA polymerase activity of the catalytic subunit of the DNA polymerase encoded by herpes simplex virus type 1.

The effect that the UL42 protein of herpes simplex virus type 1 has on the DNA polymerase activity of the DNA polymerase catalytic subunit (Pol) of the same virus has been investigated. The observed effects are critically dependent on the salt used and its concentration, such that the UL42 protein may inhibit, have little or no effect on, or activate the Pol activity, depending on the condition used. The observed effects are due to the values for Km(app) for activated DNA and Vmaxapp for Pol and the Pol-UL42 protein complex differently varying with salt concentration.

Cellular metabolism of (-) enantiomeric 2'-deoxy-3'-thiacytidine.

The metabolism of (-) enantiomeric 2'-deoxy-3'-thiacytidine (3TC) was examined in human immunodeficiency virus type 1 (HIV-1)-infected and mock-infected human cells. 3TC 5'-triphosphate levels accumulated comparably in HIV-1-infected and mock-infected phytohaemagglutinin-stimulated peripheral blood lymphocytes (PBL) and reached 40% or more of total intracellular 3TC metabolites after 4 hr. The rate of decay of 3TC triphosphate in HIV-1-infected and mock-infected PBL measured as a half-life (T1/2) ranged from 10.5 to 15.5 hr. 3TC did not significantly affect metabolism of deoxynucleotides in the U937 cell line, and was shown to be resistant to the action of human platelet pyrimidine nucleoside phosphorylase.

Kinetics and inhibition of reverse transcriptase from human and simian immunodeficiency viruses.

Reverse transcriptase from the simian immunodeficiency virus (SIV) was found to have kinetic behavior similar to that of enzyme from the human immunodeficiency virus (HIV). Michaelis constants for the substrates TTP and dGTP and inhibition constants for the inhibitors 3'-azido-3'-deoxythymidine 5'-triphosphate, 2',3'-dideoxythymidine 5'-triphosphate, and 2'-3'-dideoxyguanosine 5'-triphosphate were obtained for SIV reverse transcriptase and were found to be similar to the corresponding values for HIV reverse transcriptase. Thus, the interaction of SIV reverse transcriptase with nucleotide analogs appears to be indistinguishable from that of the HIV enzyme, suggesting that SIV/simian acquired immunodeficiency syndrome (SAIDS) is a potentially good model of AIDS.

Synthesis and antiviral evaluation of 6'-substituted aristeromycins: potential mechanism-based inhibitors of S-adenosylhomocysteine hydrolase.

New carbocyclic adenosine analogues substituted at the 6'-position with fluorine, hydroxyl, methylene, or hydroxymethyl have been synthesized as potential mechanism-based inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase. The synthetic routes began with a functionalized (+/-)-azidocyclopentane 2, which was elaborated to the adenosine analogue, or with functionalized cyclopentane epoxides 11, 20, and 27, which were opened directly with adenine in the presence of base. The 6' alpha-fluoro, 6' beta-fluoro, and 6'-methylene carbocyclic adenosine analogues were potent inhibitors of AdoHcy hydrolase. None of the compounds displayed significant activity against herpes simplex virus type 1 or type 2, but several demonstrated potent inhibition of vaccinia virus replication.

New class of antifungal agents: jasplakinolide, a cyclodepsipeptide from the marine sponge, Jaspis species.

Jasplakinolide is a cyclodepsipeptide which represents a new class of antifungal agents with potent activity against Candida albicans. Jasplakinolide is fungicidal against C. albicans with both a MIC and a minimum lethal concentration of 25 micrograms/ml in a broth dilution assay. This activity compares to that of the imidazole miconazole nitrate, which had a MIC of 6.2 micrograms/ml and a minimum lethal concentration of 50 micrograms/ml in the same assay. Topical administration of 2% jasplakinolide cream against a murine vaginal C. albicans infection was equivalent in efficacy to administration of miconazole nitrate at 2%. Subcutaneous administration of jasplakinolide was not effective against a systemic murine C. albicans infection.

Antiviral activity and mechanism of action of ganciclovir.

Ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl]guanine) is a potent inhibitor of viruses of the herpes family, including cytomegalovirus (CMV), that are pathogenic for humans and animals. The primary mechanism of ganciclovir action against CMV is inhibition of the replication of viral DNA by ganciclovir-5'-triphosphate (ganciclovir-TP). This inhibition includes a selective and potent inhibition of the viral DNA polymerase. Ganciclovir is metabolized to the triphosphate form by primarily three cellular enzymes: (1) a deoxyguanosine kinase induced by CMV-infected cells; (2) guanylate kinase; and (3) phosphoglycerate kinase. Other nucleotide-metabolizing enzymes may be involved as well. The selective antiviral response associated with ganciclovir treatment is achieved because of the much weaker inhibition of cellular DNA polymerases by ganciclovir-TP. Activity and selectivity are also amplified by the accumulation of ganciclovir-TP in CMV-infected cells.

Computer-regulated constant pressure ischemia in the rat: the animal model.

A system permitting computer control of partial ischemia in the normotensive rat brain was developed. Right carotid cannulation and bilateral subclavian artery occlusion made the input of blood to the brain dependent solely on left carotid artery flow. Perfusion pressure was controlled by partial compression of this artery with a balloon. The system can produce a range of partial ischemic states maintaining perfusion pressures from 4 to 20 mm Hg. The adequacy of the servo-control system was evaluated in greater detail at requested perfusion pressures of 7 and 12 mm Hg in 14 male Sprague-Dawley rats (300-450 g). Experimentally obtained cerebral perfusion pressures of 6.84 (SD = 0.25, n = 7) and 11.72 (SD = 0.89, n = 7) mm Hg, respectively, demonstrate the efficacy of the system. CBFs were concurrently measured at four separate bilaterally symmetric anatomic sites (cortex, hippocampus, thalamus, and substantia nigra). Significant intra- and interhemispheric differences were found to exist, with regional flows monitored ipsilaterally to the carotid balloon exceeding those of the opposite hemisphere. In summary, this acute model of cerebral ischemia permits control of perfusion pressure over the entire critical partial ischemic range.

In vitro and in vivo activities of phosphate derivatives of 9-(1,3-dihydroxy-2-propoxymethyl)-guanine against cytomegaloviruses.

The anti-cytomegalovirus activities of four phosphate derivatives of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) were evaluated against human, monkey and murine viruses. The 5'-mono-, 3'5'-bis(mono-), and 3',5'-cyclic monophosphate and 5'-homophosphonate forms of DHPG inhibited virus plaque formation at 1-15 microM. The cyclic phosphate and homophosphonate were more active than the other compounds against murine cytomegalovirus (MCMV) in vitro. In an in vivo MCMV infection model, DHPG homophosphonate and DHPG were equally effective at reducing mortality at greater than or equal to 10 mg/kg. The cyclic phosphate was active at 10-20 mg/kg but toxic at greater than or equal to 40 mg/kg. The phosphorylation of DHPG phosphate and DHPG phosphonate, as well as the inhibition of human cytomegalovirus DNA polymerase by their respective triphosphates, were also examined.

Activity of 9-(1,3-dihydroxy-2-propoxymethyl)guanine compared with that of acyclovir against human, monkey, and rodent cytomegaloviruses.

The activities of the purine acyclic nucleoside 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) against two human and five animal strains of cytomegalovirus were compared with those of acyclovir. DHPG was significantly more active than acyclovir against all but one (mouse cytomegalovirus) of the strains tested, with 50% effective doses ranging from 5 to 13 microM, as determined by plaque reduction assays in human embryonic lung (MRC-5) and human embryonic tonsil cells. Both DHPG and acyclovir inhibited virus replication at concentrations considerably lower than those necessary to inhibit cell proliferation. In mode-of-action studies, the triphosphates of DHPG and acyclovir inhibited human cytomegalovirus DNA polymerase. DHPG phosphorylation to the active triphosphate was enhanced in infected cells; however, this enzymatic activity was unrelated to thymidine kinase. In animal studies, DHPG was slightly more effective than acyclovir in reducing mouse cytomegalovirus-induced mortality.

Intracellular metabolism and enzymatic phosphorylation of 9-(1,3-dihydroxy-2-propoxymethyl)guanine and acyclovir in herpes simplex virus-infected and uninfected cells.

The antiherpes agent 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) is a much more potent inhibitor of herpes simplex viruses in vivo than acyclovir, yet both are equally active in vitro against these viruses. To explain this difference, studies were conducted to compare the intracellular metabolism and enzymatic phosphorylation of the two compounds. In herpes type 1 and type 2 infected cells, the levels of DHPG triphosphate were only about 2-fold greater than levels of acyclovir triphosphate at virus-inhibitory concentrations (less than or equal to microM). At concentrations greater than 2.5 microM in herpes type 1 but not in type 2 infected cells, acyclovir phosphorylation was inhibited relative to that of DHPG. When drug was removed after 6 hr from infected cells, acyclovir triphosphate rapidly degraded to acyclovir and was excreted into the culture medium. In contrast, DHPG triphosphate persisted at 60-70% of the original level for 18 hr after drug removal, and DHPG excretion from cells was very slow. This finding could be a key factor to the superior potency of DHPG in animals, despite the fact that blood levels of both compounds fall rapidly after dosing. In uninfected cells, low levels of DHPG and acyclovir triphosphates were produced at 100 microM concentrations. Phosphorylation of DHPG to mono-, di- and triphosphates by purified viral and cell enzymes was more rapid than that of acyclovir. However, acyclovir triphosphate was a much more potent inhibitor of herpes virus and cell DNA polymerases.

Phosphorylation of the antiviral precursor 9-(1,3-dihydroxy-2-propoxymethyl)guanine monophosphate by guanylate kinase isozymes.

Guanylate kinase was purified from human erythrocytes by affinity chromatography using GMP-agarose, and the four isozymes which are present were separated by chromatofocusing. The kinetic properties of each isozyme were analyzed with respect to the natural substrates GMP and dGMP, and the 5'-monophosphate derivatives of the antiviral nucleoside analogs 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) and 9-(2-hydroxyethoxymethyl)guanine (ACV, Acyclovir). The analysis of substrate kinetics yielded Km values for DHPG 5'-monophosphate which were similar with all isozymes (42-54 microM), and about 3-fold higher than the Km values obtained for GMP. Km values obtained with ACV 5'-monophosphate were 10-20-fold higher than the GMP values and varied nearly 4-fold among isozymes (209-753 microM). GMP produced the highest enzyme velocities with all isozymes, followed by dGMP, DHPG 5'-monophosphate, and ACV 5'-monophosphate, in that order. Differences in maximal velocities among isozymes were generally small. DHPG 5'-monophosphate inhibited the isozymes by a simple competitive mechanism with respect to GMP. In contrast, ACV 5'-monophosphate acted as an apparent hyperbolic mixed-type inhibitor. Similar patterns of inhibition were obtained with all isozymes. It is probable that differences is the reactivity of DHPG 5'-monophosphate and ACV 5'-monophosphate with individual guanylate kinase isozymes do not contribute significantly to differences in their antiviral effects.

Narcolepsy: cholinergic receptor changes in an animal model.

An inbred colony of narcoleptic doberman pinschers has been analyzed for muscarinic receptor levels in 19 discrete brain regions. In comparison to age-matched controls, receptors were generally elevated in the brainstem and reduced in forebrain areas. No changes in receptor binding affinity were detected. The increased receptor levels found in the brainstem suggest that cholinoceptive neurons in this region are hypersensitive and may be involved in the initiation of cataplexy and other aspects of the narcolepsy syndrome.

Narcolepsy: biogenic amine deficits in an animal model.

Concentrations of biogenic amine metabolites in discrete brain areas differed significantly between dogs with genetically transmitted narcolepsy and age- and breed-matched controls. Dopamine and 3,4-dihydroxyphenylacetic acid were consistently elevated in the brains of narcoleptic animals, while homovanillic acid was not. Narcoleptic animals consistently exhibited lower utilization of dopamine and higher intraneuronal degradation of dopamine but no uniform decrease in serotonin utilization. Hence neuropathology appears to be associated with genetically transmitted canine narcolepsy. The data indicate a nonglobal depression of dopamine utilization or turnover or both.

Genetic control of dopamine and serotonin receptors in brain regions of inbred mice.

In this report the genetic determinants of dopamine and serotonin receptors are investigated. We have used two types of radioreceptor binding assays to identify and quantify these neurotransmitter receptors in various brain regions of inbred mice. In the first method dopamine and serotonin sites are quantified using [3H]spiperone in the presence of appropriate blanking agents. These results are compared with those obtained by the use of [3H]domperidone and [3H]mianserin to label D2 and S2 sites, respectively. Both methods yield nearly identical results. Strain differences in D2 sites are found in the striatum, olfactory tubercle and pituitary. The density of dopaminergic sites is uncorrelated in the 3 brain regions in all mouse strains studied, suggesting that genetic determination of receptor density is independently regulated in each region. Similar observations have been made for S2 receptors in the striatum, hypothalamus, olfactory tubercle and frontal cortex. Analysis of D3 and D2 binding sites in recombinant inbred lines suggests that each site may be determined monogenically.