In vivo activity of a mixture of two human monoclonal antibodies (anti-HBs) in a chronic hepatitis B virus carrier chimpanzee

A 35-year-old female hepatitis B virus carrier chimpanzee was infused with one dose of a mixture of human monoclonal antibodies 9H9 and 4-7B (antibodies against hepatitis B virus surface antigen; HBsAg). Blood samples were taken before and up to 3 weeks after infusion. HBsAg and antibodies against HBsAg (anti-HBs) were quantified by radioimmunoassay and enzyme immunoassay. Free anti-HBs was never detected. Thirty min after the start of the infusion the HBsAg level was minimal with maximum loading of the chimpanzee HBsAg with human immunoglobulin. HBsAg complexes could be dissociated by acid treatment. The HBsAg level was completely restored on day 7. Similar results were obtained for the preS1-containing particles that may represent the infectious viral particles in the chimpanzee serum. A mouse monoclonal anti-HBs (HBs.OT40) was found to compete with 9H9 in artificial immune complexes with the pre-treatment HBsAg from the chimpanzee. Used as a conjugate, HBs.OT40 yielded a maximum decrease in the signal in the 30 min sample compared to non-competing anti-HBs conjugates. This indicates binding of HBsAg with 9H9 in the circulation of the chimpanzee. Immune-complexed 4-7B could not be detected by its corresponding 4-7B peptide conjugate, probably due to its low concentration in the complexes. It is concluded that human monoclonal anti-HBs can effectively reduce the level of HBsAg in serum from this chronic carrier. Monoclonals 9H9 and 4-7B may complement each other due to their different mechanisms of inactivation, probably with higher efficiency than that monitored by our HBsAg screening assays

Clinical characteristics of women captured by extending the definition of severe postpartum haemorrhage with 'refractoriness to treatment': a cohort study

Background: The absence of a uniform and clinically relevant definition of severe postpartum haemorrhage hampers comparative studies and optimization of clinical management. The concept of persistent postpartum haemorrhage, based on refractoriness to initial first-line treatment, was proposed as an alternative to common definitions that are either based on estimations of blood loss or transfused units of packed red blood cells (RBC). We compared characteristics and outcomes of women with severe postpartum haemorrhage captured by these three types of definitions. Methods: In this large retrospective cohort study in 61 hospitals in the Netherlands we included 1391 consecutive women with postpartum haemorrhage who received either ≥4 units of RBC or a multicomponent transfusion. Clinical characteristics and outcomes of women with severe postpartum haemorrhage defined as persistent postpartum haemorrhage were compared to definitions based on estimated blood loss or transfused units of RBC within 24 h following birth. Adverse maternal outcome was a composite of maternal mortality, hysterectomy, arterial embolisation and intensive care unit admission. Results: One thousand two hundred sixty out of 1391 women (90.6%) with postpartum haemorrhage fulfilled the definition of persistent postpartum haemorrhage. The majority, 820/1260 (65.1%), fulfilled this definition within 1 h following birth, compared to 819/1391 (58.7%) applying the definition of ≥1 L blood loss and 37/845 (4.4%) applying the definition of ≥4 units of RBC. The definition persistent postpartum haemorrhage captured 430/471 adverse maternal outcomes (91.3%), compared to 471/471 (100%) for ≥1 L blood loss and 383/471 (81.3%) for ≥4 units of RBC. Persistent postpartum haemorrhage did not capture all adverse outcomes because of missing data on timing of initial, first-line treatment. Conclusion: The definition persistent postpartum haemo

Hepatitis B from diagnosis towards prophylactic single domain antibodies

Diagnosis of the hepatitis B Virus (HBV) is important for treatment and prevention of further spread of the virus. Today, detection of hepatitis B virus surface antigen (HBsAg) is the method of choice for the screening and initial diagnosis of HBV. Genetic diversity and discovery of HBV variants with mutations in the immunological dominant region of HBsAg caused none reactivity in some diagnostic assays. Characterization of anti-HBs monoclonal antibodies lead to discovery of a unique monoclonal antibody revealing the true topology of the small s-protein. The knowledge of the antibody recognition spectra, lead to the development of a diagnostics assay showing for the first time detection of all HBsAg mutants based on three different epitope regions. The three epitope regions should in theory enable detection of all known HBsAg mutants, which was proven after testing HBsAg mutants in direct comparison to other diagnostic assay lacking complete mutant detection. The improved HBsAg screening assay permitted better and earlier diagnosis of HBV infections, leaving the diagnosed HBV patient for treatment. Today the main motivation for intervening in an on-going HBV infection is in case the liver is compromised. Depending on the status of the patient, antiviral therapy is started or in worse case when the liver stops to function, a liver transplantation is the final choice. Transplantation of the liver is preceded by antiviral therapy in combination with Hepatitis B immunoglobulin (HBIg) additions. In a quest for an alternative source for HBig we isolated and selected single-domain antibodies (VHHs) that recognize the major small s-protein of HBV. Testing of five VHH in an in-vitro neutralization experiment identified one VHH that could neutralize HBV comparable with good neutralizing anti-HBs reference antibodies. Pepscan analysis and amino acid substitution experiments suggested mutual recognition of the VHH of both the “a”-determinant and c-terminus of the s-protein, fixating the structure and preventing conformational changes needed during viral entry. Alternatively binding to a yet to be discovered HBV (co-) receptor is blocked by the VHH. Ab initio modeling of the dimer “a”-determinant sequence (aa100-155) with VHH S5 revealed a tempting structure that could explain our and published observations. Mainly the location of the cysteine residues in the newly found structure was striking, showing possible formation of inter and intra chain disulfide bridges. Separate analysed c-terminal hydrophobic region (155-226) showed structural homology with Arfaptin 2, a BAR domain protein. The homologous structure predicted importance of the region giving curvature to the aggregated s-proteins in the HBsAg particles. We conclude from the modelling data that the protruding spikes are dimers of the “a”-determinant with peripheral stabilized c-terminal s-protein fragments on the 3- and 5-fold axis

Hepatitis B from diagnosis towards prophylactic single domain antibodies

Diagnosis of the hepatitis B Virus (HBV) is important for treatment and prevention of further spread of the virus. Today, detection of hepatitis B virus surface antigen (HBsAg) is the method of choice for the screening and initial diagnosis of HBV. Genetic diversity and discovery of HBV variants with mutations in the immunological dominant region of HBsAg caused none reactivity in some diagnostic assays. Characterization of anti-HBs monoclonal antibodies lead to discovery of a unique monoclonal antibody revealing the true topology of the small s-protein. The knowledge of the antibody recognition spectra, lead to the development of a diagnostics assay showing for the first time detection of all HBsAg mutants based on three different epitope regions. The three epitope regions should in theory enable detection of all known HBsAg mutants, which was proven after testing HBsAg mutants in direct comparison to other diagnostic assay lacking complete mutant detection. The improved HBsAg screening assay permitted better and earlier diagnosis of HBV infections, leaving the diagnosed HBV patient for treatment. Today the main motivation for intervening in an on-going HBV infection is in case the liver is compromised. Depending on the status of the patient, antiviral therapy is started or in worse case when the liver stops to function, a liver transplantation is the final choice. Transplantation of the liver is preceded by antiviral therapy in combination with Hepatitis B immunoglobulin (HBIg) additions. In a quest for an alternative source for HBig we isolated and selected single-domain antibodies (VHHs) that recognize the major small s-protein of HBV. Testing of five VHH in an in-vitro neutralization experiment identified one VHH that could neutralize HBV comparable with good neutralizing anti-HBs reference antibodies. Pepscan analysis and amino acid substitution experiments suggested mutual recognition of the VHH of both the “a”-determinant and c-terminus of the s-protein, fixating the structure and preventing conformational changes needed during viral entry. Alternatively binding to a yet to be discovered HBV (co-) receptor is blocked by the VHH. Ab initio modeling of the dimer “a”-determinant sequence (aa100-155) with VHH S5 revealed a tempting structure that could explain our and published observations. Mainly the location of the cysteine residues in the newly found structure was striking, showing possible formation of inter and intra chain disulfide bridges. Separate analysed c-terminal hydrophobic region (155-226) showed structural homology with Arfaptin 2, a BAR domain protein. The homologous structure predicted importance of the region giving curvature to the aggregated s-proteins in the HBsAg particles. We conclude from the modelling data that the protruding spikes are dimers of the “a”-determinant with peripheral stabilized c-terminal s-protein fragments on the 3- and 5-fold axis

Characteristics of de novo structural changes in the human genome.

Small insertions and deletions (indels) and large structural variations (SVs) are major contributors to human genetic diversity and disease. However, mutation rates and characteristics of de novo indels and SVs in the general population have remained largely unexplored. We report 332 validated de novo structural changes identified in whole genomes of 250 families, including complex indels, retrotransposon insertions, and interchromosomal events. These data indicate a mutation rate of 2.94 indels (1-20 bp) and 0.16 SVs (>20 bp) per generation. De novo structural changes affect on average 4.1 kbp of genomic sequence and 29 coding bases per generation, which is 91 and 52 times more nucleotides than de novo substitutions, respectively. This contrasts with the equal genomic footprint of inherited SVs and substitutions. An excess of structural changes originated on paternal haplotypes. Additionally, we observed a nonuniform distribution of de novo SVs across offspring. These results reveal the importance of different mutational mechanisms to changes in human genome structure across generations

Fluid resuscitation during persistent postpartum haemorrhage and maternal outcome: A nationwide cohort study

Clinical epidemiolog