Betsy Brotman

LONG-INCUBATION POST-TRANSFUSION HEPATITIS WITHOUT SEROLOGICAL EVIDENCE OF EXPOSURE TO HEPATITIS-B VIRUS

Abstract An agent other than hepatitis-B (HB) virus seemed to be the cause of 36 (71%) of 51 cases of post-transfusion hepatitis identified during prospective biweekly serological follow-up of 204 cardiovascular-surgery patients. The sera of the 36 cases showed no evidence of the antigen or antibody response expected to accompany infection by HB virus and to be detectable by the sensitive assays used. Incubation periods and clinical and epidemiological features were inconsistent with hepatitis A. Cytomegalovirus-associated seroconversion was no more common among the HB-negative cases than among HB-positive cases or among patients who did not develop hepatitis. The data suggest that a large proportion of long-incubation post-transfusion hepatitis is unrelated to hepatitis B and that control of post-transfusion hepatitis will require identification of a hepatitis virus(es) type C.

Antibodies to a synthetic peptide from the preS 120–145 region of the hepatitis B virus envelope are virus-neutralizing

Studies with synthetic peptides have provided evidence for the presence of preS coded sequences in the envelope (env) proteins of hepatitis B virus (HBV) and indicated that these sequences are involved in the specific attachment of HBV to liver cells. Scanning of the preS sequence for immunodominant continuous epitopes identifies the sequence within residues preS (120-145) as the most immunogenic in eliciting antibodies recognizing HBV and as the most efficiently binding antibodies from sera of rabbits and humans immunized with HBV env proteins. To assess the potential of preS (120-145) as a synthetic vaccine against hepatitis B, in vitro neutralization of the virus by rabbit antiserum to the peptide was assayed in chimpanzees. The animals, subsequently proven to be susceptible to HBV infection, did not develop hepatitis B as judged by negative serological tests for HBV-associated antigens and antibodies and by normal serum alanine aminotransferase levels and normal liver biopsies. These results establish the role of preS domains in the process of virus neutralization and the potential of synthetic preS analogues for hepatitis B vaccination.

Successful nucleic acid based immunization of newborn chimpanzees against hepatitis B virus.

To determine whether DNA based immunization could protect newborn chimpanzees against a challenge infection with hepatitis B virus, two chimpanzees were immunized on the day of birth with a plasmid coding for hepatitis B surface antigen, and boosted at 6 and 24 weeks. Both animals produced transient antibody to the hepatitis B surface antigen. Following challenge with hepatitis B virus at 33 weeks the two immunized animals developed anamnestic antibody responses, however, neither developed detectable hepatitis B surface antigen or antibody to the core protein, the conventional markers of hepatitis B infection. Both of these markers appeared in an unimmunized control animal. We conclude that DNA based immunization can induce protective immunity in newborn chimpanzees.

Familial clustering of hepatitis B infection.

Abstract In a survey among 449 family contacts of blood donors from 197 households containing carriers of hepatitis B antigen, 6.7 per cent were antigen positive, as compared with 0.8 per cent in control households. The greatest prevalence of B antigen was among siblings (19.7 per cent) and other genetic family contacts (8 per cent). In spouses B antigen was less frequently detected (3.4 per cent). Hepatitis B antibody was detected three times more frequently in the study households than in control households. No differences in prevalence of hepatitis B antibody between specific relatives of antigen carriers were seen. Familial clustering does not appear to be correlated with the presence or absence of liver damage in the asymptomatic donor carrier. Neither venereal nor Maternal-Fetal transmission seems to be of primary importance in the spread of hepatitis B infections in these surveyed families. The evidence supports the hypothesis that hepatitis B virus can be transmitted nonparenterally. (N Engl J Med...

Sterilisation of hepatitis and HTLV-III viruses by exposure to tri(n-butyl)phosphate and sodium cholate.

Blood product sterilisation with 0.3% tri(n-butyl)phosphate (TNBP)/0.2% sodium cholate (CA), a combination known to permit high recovery of factor VIII and factor IX, was evaluated for its effect on hepatitis B (HBV), non-A, non-B (NANB), and human T-lymphotropic type III (HTLV-III) viruses. 2 chimpanzees received factor VIII preparations contaminated with 10(4) chimpanzee infectious doses (CID50) of HBV and treated with TNBP/CA; neither had evidence of HBV infection during 9 months follow-up, but hepatitis B surface antigen (HBsAg) developed 5 and 6 weeks, respectively, after challenge with untreated inoculum. 2 chimpanzees were similarly exposed to 10(4) CID50 of Hutchinson NANB inoculum treated with TNBP/CA; neither became infected during 26 weeks of follow-up but both had characteristic NANB-associated ultrastructural changes 3-5 weeks after exposure to untreated inoculum. 2 chimpanzees inoculated with 80 ml of TNBP/CA-treated factor VIII derived from a pool of thirteen lots obtained from five US manufacturers remained free of any evidence of NANB infection during 32 weeks of follow-up. Subsequently, NANB infection developed in both animals 3-4 weeks after exposure to untreated inoculum. Exposure of HTLV-III diluted into a factor VIII preparation to TNBP/CA inactivated greater than or equal to 10(4.2) tissue culture infective doses within 20 min at 24 degrees C.

Cloning of a Cysteine Protease Required for the Molting of Onchocerca volvulus Third Stage Larvae

We have investigated the involvement of a cysteine protease in the development of Onchocerca volvulus fourth stage larvae (L4) by testing the effect of cysteine protease inhibitors on the survival of third stage larvae (L3), and the molting of L3 to L4 in vitro. When larvae were cultured in the presence of specific inhibitors, the peptidyl monofluoromethylketones, viability of either L3 or L4 was not affected. However, the inhibitors reduced the number of L3 that molted to L4 in vitro in a time- and dose-dependent manner. Molting was completely inhibited in the presence of 50-250 μM inhibitor. Ultrastructural examination of L3 that did not molt in the presence of inhibitors indicated that new L4 cuticle was synthesized, but there was no separation between the L3 and the L4 cuticles. The endogenous cysteine protease was detected in molting larvae after binding to labeled inhibitors, and by antibodies directed against a recombinant O. volvulus L3 cysteine protease that was cloned and expressed. The enzyme was detected in cuticle regions where the separation between the cuticles occurs in molting larvae. These studies suggest that molting and successful development of L4 depends on the expression and release of a cysteine protease.

Exposure to low infective doses of HCV induces cellular immune responses without consistently detectable viremia or seroconversion in chimpanzees

In hepatitis C virus (HCV) infection, there is accumulating data suggesting the presence of cellular immune responses to HCV in exposed but seemingly uninfected populations. Some studies have suggested cross-reactive antigens rather than prior HCV exposure as the main reason for the immune responses. In this study we address this question by analyzing the immune response of chimpanzees that have been sequentially exposed to increasing doses of HCV virions. The level of viremia, as well as the immune responses to HCV at different times after virus inoculation, were examined. Our data indicate that HCV infective doses as low as 1-10 RNA (+) virions induce detectable cellular immune responses in chimpanzees without consistently detectable viremia or persistent seroconversion. However, increasing the infective doses of HCV to 100 RNA (+) virions overcame the low-inoculum-induced immune response and produced high-level viremia followed by seroconversion.

Characterization of an Onchocerca volvulus cDNA clone encoding a genus specific antigen present in infective larvae and adult worms

The isolation and characterization of a recombinant cDNA clone (OV7) expressing an antigen present in Onchocerca volvulus infective larvae and adult stages is described. Using chimpanzee antiserum generated against irradiated infective larvae, we isolated a cDNA clone from a lambda gt11 cDNA expression library derived from adult O. volvulus mRNA. The open reading frame encodes 131 amino acids corresponding to a 15.2-kDa protein. Affinity purified antibodies which bound specifically to OV7 fusion polypeptide recognized a single antigen with an apparent molecular weight of 17,000 in extracts of L3, L4 and adult worms. Immunoelectron microscopy established that the antigen encoded by this clone is present in the hypodermis and the basal layer of the cuticle of L3 and female adult worm, and in the egg shell around developing microfilariae. Since the OV7 fusion polypeptide is onchocerca-specific and is recognized specifically by sera from onchocerciasis patients, and sera from non-patent but infected chimpanzees, and not by sera from patients with other filarial parasites, it may have potential as an antigenic component in a test for detection of non-patent and patent infections of O. volvulus. The OV7 amino acid sequence contains residues that have a probable homology with the cysteine proteinase inhibitor superfamily.

Infectivity of blood from PCR‐positive, HBsAg‐negative, anti‐HBs‐positive cases of resolved hepatitis B infection

BACKGROUND: Numerous reports have noted the existence of sera, particularly from resolving cases of HBV infection, that are positive for HBV DNA by PCR, despite being negative for HBsAg and IgM anti‐HBc. If such blood is infective and detectable by HBV NAT screening, it seems desirable to introduce such screening for transfused blood.

Protection against Chronic Hepatitis C Virus Infection after Rechallenge with Homologous, but Not Heterologous, Genotypes in a Chimpanzee Model

An open question for hepatitis C virus (HCV) vaccine development is whether the various genotypes of this virus protect against the development of chronic infection after heterologous infection with different genotypes. We approached this question by challenging chimpanzees that had recovered from HCV genotype 1a or 1b infection with 6 heterologous genotypes as well as with a homologous genotype (for chimpanzees originally infected with genotype 1a). All 9 chimpanzees rechallenged with a homologous genotype developed self-limited infections. Of 11 chimpanzees challenged with 100 chimpanzee infectious doses of heterologous genotypes, 6 developed self-limited infections, with peak viral loads in acute-phase serum that were ~5-fold lower than those seen during primary infections. One chimpanzee (which had recovered from genotype 1b infection and was rechallenged with genotype 6a) did not develop viremia but did show an anamnestic cell-mediated immune response after rechallenge. Four of the 11 chimpanzees rechallenged with heterologous genotypes developed chronic infections with the genotypes used for rechallenge. These findings suggest that a universally protective HCV vaccine may need to incorporate epitopes from multiple genotypes.