In vivo neutralization of hepatitis B virus infection by an anti-preS1 humanized antibody in chimpanzees

Previously, we generated a murine monoclonal antibody (mAb), KR127, that recognizes amino acids (aa) 37-45 of the preS1 of hepatitis B virus (HBV). In this study, we have constructed a humanized version of KR127 and evaluated its HBV-neutralizing activity in chimpanzees. A study chimpanzee was given a single intravenous dose of the humanized antibody, followed by intravenous challenge with adr subtype of wild type HBV, while a control chimpanzee was only challenged with the virus. The result showed that the study chimpanzee did not develop HBV infection during 1 year, while the control chimpanzee was infected, indicating that the humanized antibody exhibited in vivo virus-neutralizing activity and thus protected the chimpanzee from HBV infection. In addition, the humanized antibody bound to the preS1 of all subtypes of HBV. We first demonstrate that an anti-preS1 mAb can neutralize HBV infection in vivo. This humanized antibody will be useful for the immunoprophylaxis of HBV infection.     

Absence of detectable hepatitis B virus DNA in sera and liver of chimpanzees with non-A, non-B hepatitis

The risk of hepatitis B infections has been reduced by screening of blood donors for hepatitis B surface antigen (HBsAg). However, recipients remain at significant risk of developing post-transfusion hepatitis. Studies have shown that non-A, non-B hepatitis virus(es) are responsible for the majority of post-transfusion hepatitis infections. In spite of many efforts, these non-A, non-B hepatitis viruses have not yet been identified. Epidemiological studies, however, suggest that non-A, non-B hepatitis shares many features with hepatitis B. Recently, Wands et al [1982] showed, in chimpanzees infected with non-A, non-B hepatitis agents, the presence of antigenemia or viremia by radioimmunoassay with monoclonal antibodies directed toward distinct determinants of HBsAg and by molecular hybridization analysis. They suggested that non-A, non-B hepatitis agents may be related, but distinct variant(s) of hepatitis B virus (HBV). In this study, five chimpanzees were inoculated with three different agents that have been shown to transmit non-A, non-B hepatitis. The following inocula were used (I) a factor VIII preparation kindly provided by D.W. Bradley, (II) acute phase serum from a chimpanzee infected with the F strain kindly provided by A.J. Zuckerman, and (III) a DS-antigen serum previously shown by us to transmit non-A, non-B hepatitis [Duermeyer et al, 1983]. All chimpanzees developed a rise in transaminase levels between 8 and 10 weeks after inoculation. None of the chimpanzees was positive for any markers of HBV infection. No evidence was obtained of infection with hepatitis A, cytomegalovirus, or Epstein-Barr virus. One chimpanzee developed chronic liver disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunogenicity in chimpanzees of experimental hepatitis B vaccines prepared from intact hepatitis B virus,purified polypeptides,or polypeptide micelles

The immunogenicity of three experimental hepatitis B vaccines was evaluated in chimpanzees. Although no antibody to hepatitis B surface antigen (anti-HBs) was detected in two chimpanzees that received an aqueous polypeptide vaccine subcutaneously, a strong anti-HBs response was observed two and ten weeks, respectively, following challenge with hepatitis B virus. Inoculation of two additional chimpanzees with a micellar preparation of these polypeptides by the intravenous route resulted in anti-HBs production in one of the chimpanzees. Two chimpanzees inoculated subcutaneously with an aqueous vaccine of formalin-inactivated intact hepatitis B virus developed anti-HBs in low titers, but the development of antibody to the hepatitis B core antigen following challenge inoculations suggested that subclinical HBV infections may have occurred despite prior vaccination.     

Immunization of chimpanzees with hepatitis B virus-derived polypeptides.

Previous studies established that the purified polypeptides derived from the 22-nm particles associated with hepatitis B surface antigen (HBsAg) produce both humoral and cellular immunity against HBsAg in guinea pigs. Therefore, the two major polypeptides with molecular weights of 22,000 and 25,000 (P22 and P25, respectively) were isolated, adsorbed to an alum adjuvant, and used to immunize four nonimmune chimpanzees. A vigorous anti-HBs response was observed in all four animals after one inoculation of an alum-adsorbed polypeptide vaccine containing 40 micrograms of protein. After one to two booster inoculations, anti-HBs switched from being predominantly immunoglobulin M to the immunoglobin G class, indicating the establishment of immunological memory. Challenge of the vaccinated chimpanzees with 30,000 chimpanzee infectious doses of hepatitis B virus provided evidence for the efficacy of this vaccine. None of the four animals developed serological markers associated with an active hepatitis B infection, and no biochemical or histopathological changes of hepatitis were observed. A nonvaccinated control chimpanzee that was inoculated with the same hepatitis B virus material developed hepatitis B infection, confirming infectivity of the challenge inoculum.     

Effect of ethanol during hepatitis B virus infection in chimpanzees

To determine whether the use of ethyl alcohol (ethanol, C₂H₅ OH) may increase the liver damage caused by hepatitis B virus infection, ethanol was infused into four chimpanzees on one or two occasions during the course of natural or experimentally induced hepatitis B virus infections. A fifth chimpanzee, without active hepatitis B virus infection, served as a control. Moderate elevations of serum aspartate or alanine aminotransferases occurred in four of the five chimpanzees, including the control chimpanzee, in direct association with ethanol infusion; pre-existing enzyme elevations persisted in a fifth chimpanzee. No alteration occurred in the titers of hepatitis B surface antigen or of antibody to hepatitis B core antigen in three of the four infected chimpanzees. There was no significant alteration in the course of hepatitis B virus infection by ethanol infusion in these chimpanzees.     

Successful postexposure vaccination against hepatitis B in chimpanzees

To study the effect of postexposure vaccination, four chimpanzees were vaccinated with hepatitis B (HB) vaccine 4, 8, 48, and 72 hr, respectively, after intravenous injection of an infectious hepatitis B virus (HBV) inoculum. The second and third vaccine inoculations were given 2 and 6 weeks later, i.e., at considerably shorter intervals than recommended either for ordinary prophylactic vaccination or for postexposure vaccination in combination with hepatitis B immune globulin (HBIG). The chimpanzees were followed for 1 year. None showed HBs-antigenemia, liver enzyme elevation (ALT), or histopathological alterations in liver biopsies. Late appearance of anti-HBc was observed only in the serum of the animal whose series of vaccination started 72 hr after HBV inoculation. An unvaccinated control chimpanzee, which received the HBV inoculum only, developed clinical hepatitis B with ALT-elevations and HBs-antigenemia within 2 months of the experimental HBV inoculation. These results indicate that postexposure vaccination against hepatitis B begun within 48 hr after HBV exposure, with short intervals between the vaccine injections, can protect against hepatitis B infection also when concomitant HBIG-prophylaxis is not given.     

Hepatitis b e antigen and antibody: detection by radioimmunoassay in chimpanzees during experimental hepatitis b

Hepatitis B e antigen (HBeAg) and its antibody (anti-HBe) were evaluated using a sensitive radioimmunoassay (RIA) in weekly serum samples obtained from nine chimpanzees experimentally infected with hepatitis B virus (HBV). In two chimpanzees with HBV infection with detectable hepatitis B surface antigen (HBsAg) for less than five weeks, and in one chimpanzee with documented HBV infection with no detectable HBsAg, HBeAg was not detected; in all three, anti-HBe became detectable early in the infection. In six chimpanzees in which HBsAg was detected for 16 weeks or longer, HBeAg was detected early in the infection; in five, anti-HBe became detectable and HBeAg unde-tectable prior to the clearance of HBsAg. The sixth remained HBsAg-positive and HBeAg-positive for more than two years and never developed anti-HBe. These results confirm the sensitivity of this RIA and its value in predicting the course of HBV infections.     

Passively acquired antibody to hepatitis B surface antigen. Pitfall in evaluating immunity to hepatitis B viral infections

Antibody to hepatitis B surface antigen (anti-HBs) has been used clinically to indicate an immune response to hepatitis B virus (HBV) and a protection against reinfection with the virus. We describe a child with hemophilia who had high-titer IgG anti-HBs in his serum and who subsequently developed viral B hepatitis. The child had received a unit of fresh frozen plasma 17 days prior to the determination of anti-HBs. The fresh frozen plasma donor was later found to be anti-HBs positive. The patient's anti-HBs was most likely passively acquired and therefore did not signify immunity to HBV. Various tests, including hepatitis B surface antigen group-specific and subtype determinants, ratio units of anti-HBs, and antibody class, have been used to determine whether or not anti-HBs will confer immunity. Although these tests have been thought to accurately predict immune status against infection with HBV, our case shows this may not be true, especially in patients who have been recently transfused. Anti-HBs testing may be predictive of immunity to HBV in the absence of a source of passively acquired anti-HBs.     

Non-A, non-B hepatitis in chimpanzees: interference with acute hepatitis A virus and chronic hepatitis B virus infections

Two chimpanzees with persistent non-A, non-B (NANB) hepatitis were superinfected with marmoset-passaged MS-1 HAV. Two control chimpanzees were also infected with marmoset-passaged HAV. Neither animal with persistent NANB hepatitis developed elevated alanine aminotransferase (ALT) activity, whereas both control chimpanzees exhibited ALT elevations within 3 weeks after inoculation. In addition, both NANB-infected chimpanzees demonstrated a delayed anti-HAV antibody response in which one animal failed to produce detectable IgM anti-HAV. With the exception of one stool, all serial liver biopsy specimens and daily stool suspensions from the superinfected chimpanzees were negative for HAV antigen. One chimpanzee with a chronic HBV infection was superinfected with non-A, non-B hepatitis and was shown to develop elevated ALT activity and hepatocyte ultrastructural alterations accompanied by a marked reduction in the titer of serum HBsAg. Our combined findings indicate that acute and persistent non-A, non-B hepatitis infections are capable of interferring with two distinctly different hepatotropic viruses. These results also suggest that in vitro detection of non-A, non-B hepatitis infection or virus(es) may be achieved by antibody-independent methodologies that employ the basic principle of viral interference.     

Antigenicity and immunogenicity of hepatitis B virus particles produced by mouse cells transfected with cloned viral DNA

Antigenicity and immunogenicity of the hepatitis B surface antigen (HBsAg) 22-nm particles produced by mouse cells transfected with HBV DNA were studied. Both the a group and the y subtype determinants were present on the particles. Injected into mice the particles induced formation of anti-HBs. An antibody titer of 400 IU/mP, 7 weeks after the primary injection was obtained in one experiment. The affinity constant of these antibodies to human HBsAg particles was about 1 × 10^8? mol/liter. The anti-HBs antibodies react specifically with both the a group and the y subtype HBV determinants. These results show that these particles have antigenic and immunogenic properties similar to those of the 22-nm particles present in human serum.     

Dissociated antibody responses to the S and pre-S2 regions of the hepatitis B virus after vaccination in hemophiliacs

The membranes of hepatocytes and the pre-S2 envelope protein of the hepatitis B virus (HBV) contain binding sites for polymerized human albumin, which is thought to act as a link between HBV and hepatocytes. Hence, anti-pre-S2 antibodies should prevent HBV uptake by the liver, and there is indeed preliminary evidence that they protect chimpanzees from HBV infection. To evaluate whether a plasma-derived vaccine containing the pre-S2 sequence induced an anti-pre-S2 response in 105 vaccinated hemophiliacs, anti-pre-S2 was measured in parallel with antibody to hepatitis B surface antigen (anti-HBs). Eighty-five percent of the hemophiliacs had both anti-pre-S2 and anti-HBs when vaccination was completed, 13% had anti-HBs alone, and 2% (two cases) had anti-pre-S2 alone. Eighty-seven percent of anti-pre-S2-positive hemophiliacs compared with only 50% of anti-pre-S2-negative hemophiliacs (P less than 0.001) developed high anti-HBs titers (greater than or equal to 1,000 mlU/ml). This study demonstrates, therefore, that the antibody responses to the S and pre-S2 regions of HBV may be dissociated after vaccination in hemophiliacs and that higher anti-HBs titers are attained in anti-pre-S2-positive hemophiliacs.     

Failure of preexisting antibody against hepatitis B surface antigen to prevent subsequent hepatitis B infection.

We studied a patient who developed acute hepatitis B virus (HBV) infection despite the presence of preexisting antibody to the surface antigen of HBV (anti-HBs). Anti-HBs has been reported to consist primarily of antibody against the common a determinant of HBV. Antibody directed against this major determinant appears to confer protection against HBV, regardless of the subtype. Our patient was shown to have had preexisting anti-HBs of anti-d but not anti-a specificity. She subsequently developed non-A, non-B viral hepatitis followed by an episode of acute hepatitis B after exposure to HBV of the ayw subtype.     

Analysis of the antigenic epitopes of hepatitis B surface antigen involved in the induction of a protective antibody response.

Vaccination with hepatitis B surface antigen (HBsAg) has shown that antibody directed against the common 'a' determinant of this antigen is protective against infection with hepatitis B virus (HBV). In this study the antigenic epitopes of the 'a' determinant have been analysed by competitive inhibition assays and by binding studies to synthetic peptides using a panel of monoclonal antibodies prepared against HBsAg, all of which are shown to recognise the common group determinant. One murine monoclonal antibody used in this study, RFHBs1, has been shown previously to block infectivity of HBV in susceptible chimpanzees ((1983) J. Med. Virol. 16, 89-95). This antibody bound to a cyclical synthetic peptide analogue of amino acids 124 to 137 of the major HBsAg polypeptide.     

Inactivation of hepatitis B virus and non-A, non-B hepatitis by chloroform

To determine whether a non-A, non-B hepatitis agent contained essential lipids, we extracted with chloroform a dilution of human plasma that contained approximately 10(4) chimpanzee infectious doses of non-A, non-B hepatitis virus and then tested for infectivity in chimpanzees. In addition, we treated a serum containing hepatitis B virus in the same way. Both of these samples were also sham extracted as controls. Known chloroform-sensitive and chloroform-resistant viruses were added directly to the hepatitis-containing serum or plasma as internal controls or to fetal calf serum as external controls and were assayed for infectivity in vitro after chloroform extraction or sham extraction. All infectivity of the diluted plasma that contained at least 10(4) chimpanzee infective doses of non-A, non-B hepatitis agent and all infectivity of the serum that contained 10(3.5) chimpanzee infective doses of hepatitis B virus were destroyed by chloroform. The chloroform-sensitive control viruses were completely inactivated, but the chloroform-resistant control viruses lost less than 0.5 log10 of infectivity. Sham-extracted non-A, non-B hepatitis agent-containing plasma was shown to maintain its infectivity in chimpanzees that had initially been inoculated with the chloroform-extracted plasma. Thus, both hepatitis type B and non-A, non-B hepatitis appear to be caused by viruses that can be inactivated by a lipid solvent.     

Lack of genetic restriction by a potential anti-idiotype vaccine for type B viral hepatitis

Anti-idiotype (anti-Id) reagents that bear an internal image capable of mimicking hepatitis B surface antigen (HBsAg) were used to induce an antibody to HBsAg (anti-HBs) response in both rabbits and chimpanzees. The anti-idiotype induced antibody response produced in rabbits recognized HBsAg determinants associated with the induction of protective immunity against hepatitis B virus (HBV). Attesting further to the specificity was the binding of the rabbit anti-idiotype to the anti-idiotype induced anti-HBs containing sera. Our findings suggest that genetic restrictions associated with the induction of an interspecies immune response may not be a limitation of anti-idiotype based vaccines. In addition, anti-idiotype immunization also produced an anti-HBs in chimpanzees, a species susceptible to infection with human HBV. These data demonstrate that internal-image-bearing anti-idiotype reagents can induce an immune response across species barriers. Additionally, the reagents represent a viable alternative approach to vaccination against agents such as hepatitis B virus that cause human disease.     

Failure to detect infectious hepatitis b virus using high dose safety test for hepatitis b vaccine

One hundred milliliters of an inactivated hepatitis B vaccine (20 /m?g/ml) were inoculated intravenously into two colony-born infant chimpanzees. Immediately thereafter each received hepatitis B virus from a documented infectious inoculum intravenously at a separate site. Neither chimpanzee developed elevation of aminotransferase levels, hepatitis B surface antigen (HBsAg), or antibody to hepatitis B core antigen during six months of evaluation, the duration of the currently recommended safety test. Both chimpanzees developed antibody to HBsAg beginning 8 and 9 weeks, respectively, after inoculation. The administration of a large intravenous quantity of vaccine antigen thus appeared capable of masking or preventing infection by simultaneously administered hepatitis B virus. This study suggests that a chimpanzee safety test for hepatitis B vaccine should not employ large quantities of vaccine antigen, since such a safety test may fail to detect small amounts of residual infectious hepatitis B virus.     

Occult hepatitis B virus infection in the setting of hepatitis C virus (HCV) and human immunodeficiency virus (HIV) co‐infection: Clinically relevant or a diagnostic problem?

The clinical relevance of occult hepatitis B virus (HBV) infection, defined as detectable HBV DNA serum/liver, in the absence of hepatitis B surface antigen (HBsAg), is unclear. We determined the prevalence of serum occult HBV infection in HIV/HCV co-infected patients enrolled in APRICOT, a randomized multinational trial that investigated the efficacy and safety of peginterferon alfa-2a (40 kDa) plus ribavirin for treatment of HCV. We also examined the effect of prior HBV exposure to liver histology at baseline. Only HBsAg-negative patients were eligible. At screening, serum HBV DNA was assessed by commercial assay (detection limit = 200 copies/mL). Patients were divided into four serological groups: anti-HBs+/anti-HBc+; anti-HBs-/anti-HBc+; anti-HBs+/ anti-HBc-; anti-HBs-/anti-HBc-. Baseline liver biopsy grade and stage were compared among groups. Serum HBV DNA was undetectable in all patients, (n = 866). Results of anti-HBs and anti-HBc was available for 176 patients: 60 (34.1%) anti-HBs+/anti-HBc+; 60 (34.1%) anti-HBs-/anti-HBc+; 11 (6.3%) anti-HBs+/anti-HBc-; 45 (25.6%) anti-HBs-/anti-HBc-. There were no differences among the groups in the histological grade or stage at baseline liver biopsies. Occult HBV infection in serum was not detected in this large immunocompetent cohort. Moreover, prior exposure to HBV did not appear to have any affect on baseline liver histology.     

In-vitro cell-mediated cytotoxicity for autologous liver cells in chronic non-A, non-B hepatitis.

To investigate the possible mechanisms of liver cell injury in chronic non-A, non-B (NANB) hepatitis, peripheral blood lymphocytes (PBL) from 16 patients with chronic NANB hepatitis were incubated with autologous hepatocytes in a microcytotoxicity assay. Significant cytotoxicity was demonstrated in 11 patients. T-enriched lymphocytes exhibited significantly greater cytotoxicity than non-T enriched cells. No significant inhibition of cytotoxicity was observed following preincubation of the liver cells with either monoclonal or polyclonal anti-HBc, or monoclonal anti-HBs, or addition of either purified HBsAg or recombinant HBcAg to the culture, indicating that there was no detectable cross-reactivity in this system between hepatitis B virus (HBV) and NANB-associated antigen(s). Preincubation of the patients' hepatocytes with polyclonal IgG purified from a serum of a patient who recovered from an acute NANB hepatitis, did not significantly alter cytotoxicity. Liver cell surface-bound IgG was detected by immunofluorescence in only two of the patients, a finding consistent with existing evidence of poor antibody responses to both liver membrane and NANB-associated antigens. Control experiments using PBL from allogeneic normal donors exhibited normal cytotoxicity for the patients' hepatocytes supporting the hypothesis that antibody-dependent cell-mediated cytotoxicity (ADCC) is unlikely to play a significant role in this clinical setting.     

Requirement of standardizing anti-HBs assay methods in Japan for HBV infection-preventing strategy--discrepancy of anti-HBs measurements among three different kits widely used in Japan

The strategy to eliminate hepatitis B virus (HBV) infection by administrating an HB vaccine is changing worldwide; however, this is not the case in Japan. An important concern about the HBV infection-preventing strategy in Japan may be that the assay methods for the antibody to hepatitis B surface antigen (anti-HBs) are not standardized. The minimum protective anti-HBs titer against HBV infection has been established as 10 mIU/ml by World Health Organization (WHO) -standardized assay methods worldwide, but that is still determined as a "positive" test result by the passive hemagglutination (PHA) method in Japan. We compared anti-HBs measurements in given samples among PHA(Mycell II, Institute of Immunology), chemiluminescent enzyme immunoassay (CLEIA) (Lumipulse, Fujirebio), and chemiluminescent immunoassay (CLIA) (Architect, Abbott), all of which are currently in wide use in Japan. First, anti-HBs measurements in serum from individuals who received a yeast-derived recombinant HB vaccine composed of the major surface protein of either subtype adr or subtype ayw were compared. The results clearly showed that in subtype adr-vaccinees CLIA underestimated the anti-HBs amount compared with CLEIA and PHA, but in ayw-vaccinees, the discordance in the measurements among the three kits was not prominent. Second, anti-HBs measurements in standard or calibration solutions of each assay kit were compared. Surprisingly, CLEIA showed higher measurements in all three kit-associated standard or calibration solutions than CLIA. Thus, the anti-HBs titer of 10 mIU/ml is difficult to introduce in Japan as the minimum protective level against HBV infection. Efforts to standardize anti-HBs assay methods are expected to share international evidence about the HBV infection-preventing strategy.     

The "original" hepatitis B virus of Eastern chimpanzees (Pan trogrodytes schweinfurthii)

Little is known about Hepatitis B Virus (HBV) infections in chimpanzees. Therefore, we investigated the prevalence of chimpanzee HBV (chHBV) infections in captive, wild born chimpanzees in the sanctuary on Ngamba Island, Uganda and one sample from a wild free ranging chimpanzee. In one third of the plasma samples (32.4%; 12/37) we detected antibodies to Hepatitis B (core) antigen. Amongst those individuals HBV DNA was detected in one captive wild born and the wild chimpanzee. In contrast to the only available earlier described HBV sequence from the subspecies Pan troglodytes schweinfurthii, there was no evidence of recombination with human HBV. Our sequences therefore are likely to present the "original" chHBV from P. t. schweinfurthii.