J. Kruining

Inhibitory effect of 9-(2-phosphonylmethoxyethyl)-adenine (PMEA) on human and duck hepatitis B virus infection.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) was evaluated for its inhibitory effect on hepadnavirus replication in three different cell systems, i.e., human hepatoma cell lines HepG2 2.2.15 and HB611 (transfected with human hepatitis B virus (HBV)) and primary cultures of duck hepatocytes infected with duck hepatitis B virus (DHBV). PMEA inhibited HBV release from HepG2 2.2.15 cells and HB611 cells at a 50% inhibitory concentration (IC50) of 0.7 and 1.2 microM, respectively. Intracellular viral DNA synthesis was inhibited at concentrations equivalent to those required to inhibit virus release from the cells. DHBV secretion from duck hepatocytes was inhibited by PMEA at an IC50 of 0.2 microM. HBsAg secretion was inhibited by PMEA in a concentration-dependent manner in HB611 cells and DHBV-infected duck hepatocytes but not HepG2 2.2.15 cells. The 50% cytotoxic concentration, as measured by inhibition of [3H-methyl]deoxythymidine incorporation was 150 microM for the two human hepatoma cell lines and 40 microM for the duck hepatocyte cultures. In a pilot experiment PMEA was found to reduce the amounts of DHBV DNA in the serum of Pekin ducks.

Inhibitory effects of acyclic nucleoside phosphonates on human hepatitis B virus and duck hepatitis B virus infections in tissue culture.

The inhibitory effects of the 9-(2-phosphonylmethoxyethyl)adenine-related compounds (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-adenine, (S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine, (R)-9-(2-phosphonylmethoxypropyl)adenine, (R)-9-(2-phosphonylmethoxypropyl)-2,6-diaminopurine, and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine on human hepatitis B virus replication in the human hepatoma cell line HepG2 2.2.15 and duck hepatitis B virus infection in primary duck hepatocytes were investigated. (R)-9-(2-phosphonylmethoxypropyl-2,6-diaminopurine had the lowest 50% inhibitory concentrations against hepatitis B virus and duck hepatitis B virus, 0.22 and 0.06 microM, respectively, i.e., two- to fivefold lower concentrations than required for (R)-9-(2-phosphonylmethoxypropyl)adenine and 9-(2-phosphonylmethoxyethyl)adenine. All compounds were not toxic in vitro at a concentration of 100 microM.

Conversion of 2′,3′-dideoxyadenosine (ddA) and 2′,3′-didehydro-2′,3′-dideoxyadenosine (d4A) to their corresponding aryloxyphosphoramidate derivatives markedly potentiates their activity against human immunodeficiency virus and hepatitis B virus

2′,3′‐Dideoxyadenosine (ddA), 2′,3′‐didehydro‐2′,3′‐dideoxyadenosine (d4A) and their lipophilic 5′‐monophosphate triester (aryloxyphosphoramidate) prodrugs were evaluated for their anti‐retrovirus and anti‐hepatitis B virus activity in various cell culture models. The aryloxyphosphoramidate derivatives of ddA (Cf 1093) and d4A (Cf 1001) showed markedly superior (100–1000‐fold) efficacies than the parent drugs against human immunodeficiency virus type 1 (HIV‐1), HIV‐2, simian immunodeficiency virus (SIV), Moloney murine sarcoma virus (MSV) and human hepatitis B virus (HBV) replication regardless of the cell type in which the virus replication was studied (i.e., human T‐lymphocyte CEM, MT‐4, Molt/4 and C8166 cells, peripheral blood lymphocytes (PBL), monocyte/macrophages (M/M), murine embryo fibroblasts and human hepatocyte cells). Also the selectivity index (ratio of cytotoxic concentration/antivirally effective concentration) of both aryloxyphosphoramidate prodrugs was markedly increased. In particular the d4A prodrug Cf 1001 showed a selectivity index of 300–3000 as compared with 2–3 for the parental d4A in established laboratory cell lines. Also Cf 1001 had a selectivity index of 400–650 in HIV‐1‐infected PBL and M/M, respectively. Both Cf 1001 and Cf 1093 were equally efficient as 3TC (lamivudine) in inhibiting HBV replication in hepatocytes, and rank among the most potent HIV and HBV inhibitors reported so far in cell culture.

Serum HBeAg quantitation during antiviral therapy for chronic hepatitis B

Hepatitis Be antigen (HBeAg) seroconversion is considered the principal short‐term goal of antiviral therapy in chronic hepatitis B. To test whether the pre‐ and per‐treatment HBeAg quantitation has a higher predictive value than that of hepatitis B virus DNA (HBV‐DNA) quantitation for the outcome of antiviral therapy in chronic hepatitis B. A quantitative measurement of HBV‐DNA and HBeAg (AxSYM HBe 2.0 Quantitative, Abbott Laboratories) was undertaken in serial serum samples from 30 patients with 16‐week interferon‐α (IFN‐α) treatment (follow‐up 36 weeks; 14 responders) and from 15 patients with 24‐week lamivudine treatment (follow‐up 24 weeks; 2 responders).

Immune response after vaccination with recombinant hepatitis B vaccine as compared to that after plasma-derived vaccine

Thirty-one individuals (health care workers) were vaccinated with recombinant hepatitis B vaccine (10 microgram dose) and their immune response (anti-HBs) was compared to that of twenty-five health care workers after vaccination with plasma-derived vaccine (20 microgram dose). Although the seroconversion rate and the percentage of anti-HBs/a antibodies at month 7 were comparable, the geometric mean titre of anti-HBs at month 7 was considerably lower for the recombinant vaccine group (857.4 vs. 6736.5 IU/l). However, vaccinees from the two groups showing seroconversion at month 1 had comparable titres at month 7. Raising the dose of HBsAg in the recombinant vaccine may favourably influence the seroconversion rate at month 1 and thereby the immune response after three injections.

Anti-hepatitis B virus activity of a mixture of two monoclonal antibodies in an “inhibition in solution” assay

Two human monoclonal antibodies with anti‐hepatitis B activity were investigated separately and as a mixture by means of an “inhibition in solution” assay. With this assay the capacity of anti‐HBs antibodies to inhibit the binding of hepatitis B surface antigen (HBsAg) with solid‐phase anti‐HBs (Ausria II, Abbott Laboratories) was studied. Both HBsAg of different subtypes and purified Dane particles were used. One of the monoclonal antibodies (9H9) was directed against a conformational epitope (anti‐“a” like) but induced incomplete inhibition (80% to 90%) of all HBsAg subtypes; the other (4–7B), active against a linear epitope, caused full inhibition of all HBsAg subtypes except one (HBsAg/adw4). In vitro, a mixture of these two monoclonal antibodies was active against HBsAg from liver transplant recipients, including those with suspected variant viruses. (HEPATOLOGY 1995; 22:1078–1083.).

Comparative Anti-Retrovirus and Anti-Hepadnavirus Activity of Three Different Classes of Nucleoside Phosphonate Derivatives

The prototype compounds of three different classes of nucleoside phosphonates [i.e. 9-(2-phosphonomethoxyethyl)adenine (PMEA), 9-(2-phosphonomethoxyethoxy)adenine (PMEoA) and 9-[(2R,5R)-2,5-dihydro-5-(phosphonomethoxy)-2-furanyl]adenine (D4API)] were investigated and compared for their antiviral activities. The three test compounds showed a marked inhibition of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) in CEW and MT-4 cell cultures [50% effective concentration (EC50): 0.8-14μm]. D4API was 2- and 15-fold more inhibitory than PMEA and PMEoA, respectively. In contrast, the activity of PMEA against human hepatitis B virus (HHBV) in human hepatoma Hep G2 2.2.15 cells was 5- and 10-fold more pronounced than the activities of PMEoA and D4API, respectively (EC50 1.2μm versus 10 and 6 μm, respectively). The inhibitory activity of D4API against Moloney murine sarcoma virus (MSV)-induced C3H/3T3 cell transformation was superior to the activities of PMEA and PMEoA by at least one order of magnitude (EC50 for D4API 1.3μM, versus 2.8 and 14 μM for PMEA and PMEoA, respectively). The markedly greater inhibitory effect of D4API on MSV in vitro was in agreement with our In vivo findings that D4API inhibited MSV-induced tumour formation in newborn mice and delayed the MSV-associated animal death at a lower dose than PMEA or PMEoA. Both PMEA and D4API emerged as promising compounds that warrant further investigation for their anti-retrovirus and anti-hepadnavirus activities in vivo.

Comparison of two techniques for detecting antibody to HBsAg during a hepatitis B vaccine study

Two assays for the detection of antibody against hepatitis B surface antigen (anti-HBs) were compared. The first was a direct sandwich radioimmunoassay (RIA) which detects, in principle, antibody against any epitope of hepatitis B surface antigen (HBsAg). The second assay was an inhibition enzyme-linked immunosorbent assay (ELISA). In this assay a fixed amount of HBsAg which can be blocked by anti-HBs is measured in a direct sandwich test. Prevaccination screening sera (n = 191) and follow-up sera obtained from high risk groups (n1 = 85; n2 = 41) during two hepatitis B vaccine studies were compared in RIA and ELISA. In prevaccination sera either HBsAg or anti-HBs were detected by ELISA. Full agreement between the results of RIA and ELISA for anti-HBs was obtained in sera containing more than 10 IU/l anti-HBs. Both tests showed variable results at low titres. Experiments with monoclonal anti-HBs indicated that ELISA is less sensitive for subtype specific antibodies (anti-d, anti-y), which may explain that there were consistent differences between RIA and ELISA in a minority of cases.

Anti-pre-S(2) analysis after hepatitis B vaccination in haemodialysis patients

We investigated the development of anti-pre-S(2) antibodies by enzyme immunoassay and by Western blot analysis in a vaccination study in haemodialysis patients; both the Pasteur plasma vaccine retaining the pre-S(2) epitopes and the Merck, Sharp and Dohme (MSD) plasma vaccine containing only HBsAg were used. By enzyme immunoassay (EIA), one anti-pre-S(2) response at month 7 was registered in 23 patients after 5 injections with the 5 micrograms dose of Pasteur vaccine (PS group), whereas 6 responses were seen in 20 patients after 4 injections with the 10 micrograms dose (PD group). None of the 22 vaccines injected with MSD vaccine (40 micrograms) (4 injections, MD group) showed an anti-pre-S(2) response at month 7. In the Western blot an anti-pre-S(2) response was seen in 12 PS patients, in 8 PD patients and in none of the MD patients. Anti-pre-S(2) responses were predominantly observed in patients with a high anti-HBs response but exceptions occurred. Prevaccination anti-pre-S(2) positivity, in the absence of anti-HBc and anti-HBs, was detected in dialysis patients with EIA as well as Western blot, in 10.8 and 21.6%, respectively; similar findings were made in health care personnel. The possible nature of this phenomenon is discussed. In this study the Western blot technique has been shown to be a suitable test system for qualitative and semi-quantitative analysis of anti-pre-S(2) antibodies after hepatitis B vaccination with a higher sensitivity, but probably also a different specificity than the EIA.