The globoside receptor triggers structural changes in the B19 virus capsid that facilitate virus internalization.

Globoside (Gb4Cer), Ku80 autoantigen, and α5ß1 integrin have been identified as cell receptors/coreceptors for human parvovirus B19 (B19V), but their role and mechanism of interaction with the virus are largely unknown. In UT7/Epo cells, expression of Gb4Cer and CD49e (integrin alpha-5) was high, but expression of Ku80 was insignificant. B19V colocalized with Gb4Cer and, to a lesser extent, with CD49e. However, only anti-Gb4Cer antibodies could disturb virus attachment. Only a small proportion of cell-bound viruses were internalized, while the majority became detached from the receptor. When added to uninfected cells, the receptor-detached virus showed superior cell binding capacity and infectivity. Attachment of B19V to cells triggered conformational changes in the capsid leading to the accessibility of the N terminus of VP1 (VP1u) to antibodies, which was maintained in the receptor-detached virus. VP1u became similarly accessible to antibodies following incubation of B19V particles with increasing concentrations of purified Gb4Cer. The receptor-mediated exposure of VP1u is critical for virus internalization, since capsids lacking VP1 could bind to cells but were not internalized. Moreover, an antibody against the N terminus of VP1u disturbed virus internalization, but only when present during and not after virus attachment, indicating the involvement of this region in binding events required for internalization. These results suggest that Gb4Cer is not only the primary receptor for B19V attachment but also the mediator of capsid rearrangements required for subsequent interactions leading to virus internalization. The capacity of the virus to detach and reattach again would enhance the probability of productive infections.

Local tolerance and stability up to 24 months of a new 20% proline-stabilized polyclonal immunoglobulin for subcutaneous administration.

Subcutaneous administration of human IgG is an alternative to intravenous replacement therapy that is associated with more stable serum IgG levels and fewer systemic adverse events. Highly concentrated IgG solutions are most convenient to minimize infusion volume, but their preparation and stability presents substantial technical difficulties. We report on the stability and local tolerance of IgPro20, an l-proline-stabilized, 20% polyvalent human IgG developed for subcutaneous administration. Stability was tested according to ICH guidelines. Local tolerance and vasoactivity were examined in rabbit and rat models, respectively. The presence of l-proline in IgPro20 reduced viscosity and addition of Polysorbate 80 and inert gassing improved the appearance of the solution. After storage at 25 °C for 24 months, monomer + dimer content, aggregates, and fragments were within specification (≥ 90.0%, ≤ 4.0%, and ≤ 10.0%, respectively), and Fc function and antibody activities were maintained. In rats, intravenous injection of IgPro20 produced mild and transient hypotension comparable to that seen with intravenous IgG products. Local tolerance of IgPro20 in rabbits was comparable to that of a marketed subcutaneous IgG, Beriglobin P. Functionality and quality of IgPro20 are maintained during storage at 25 °C for at least 24 months. The product is well tolerated as assessed in animal models.

Molecular mechanism underlying B19 virus inactivation and comparison to other parvoviruses.

BACKGROUND: B19 virus (B19V) is a human pathogen frequently present in blood specimens. Transmission of the virus occurs mainly via the respiratory route, but it has also been shown to occur through the administration of contaminated plasma-derived products. Parvoviridae are highly resistant to physicochemical treatments; however, B19V is more vulnerable than the rest of parvoviruses. The molecular mechanism governing the inactivation of B19V and the reason for its higher vulnerability remain unknown. STUDY DESIGN AND METHODS: After inactivation of B19V by wet heat and low pH, the integrity of the viral capsid was examined by immunoprecipitation with two monoclonal antibodies directed to the N-terminal of VP1 and to a conformational epitope in VP2. The accessibility of the viral DNA was quantitatively analyzed by a hybridization-extension assay and by nuclease treatment. RESULTS: The integrity of the viral particles was maintained during the inactivation procedure; however, the capsids became totally depleted of viral DNA. The DNA-depleted capsids, although not infectious, were able to attach to target cells. Comparison studies with other members of the Parvoviridae family revealed a remarkable instability of B19V DNA in its encapsidated state. CONCLUSION: Inactivation of B19V by heat or low pH is not mediated by capsid disintegration but by the conversion of the infectious virions into DNA-depleted capsids. The high instability of the viral DNA in its encapsidated state is an exclusive feature of B19V, which explains its lower resistance to inactivation treatments.

Memory B cells producing somatically mutated antiphospholipid antibodies are present in healthy individuals.

Antiphospholipid antibodies (aPLs) are associated with thrombosis and recurrent abortions during autoimmune pathologies, but they are also produced in healthy individuals and during infectious diseases. To analyze the possible links between physiologic and pathologic aPLs, it is of importance to characterize normal aPL production. We took advantage of the known tropism of Epstein-Barr virus (EBV) for B cells in general, and memory B cells in particular, during primary infectious mononucleosis (IMN) in 3 patients to get access to anticardiolipin (aCL)-producing B cells. Flow cytometry analysis of these cells showed that, depending on the patient, 10% to 60% of immunoglobulin M (IgM) aCL-producing B cells express the CD27 marker of memory B cells. Single cell sorting of aCL B cells, followed by single cell reverse transcription-polymerase chain reaction (RT-PCR) amplification of their immunoglobulin variable region genes, showed that some of these cells produce mutated forms of aCL antibodies, confirming their memory B-cell origin. Considering that, during primary IMN, EBV infects and expands already pre-existing memory B cells, we conclude that healthy individuals have a discrete pool of aCL memory cells able to produce mutated forms of antibodies. The implications of this new information are discussed in light of different hypotheses regarding the origin of aCL.

Pathogenic antiphospholipid antibody: an antigen-selected needle in a haystack.

Antiphospholipid antibodies represent a heterogeneous group of autoantibodies directed against anionic phospholipids (PLs) usually linked to protein cofactors. Their presence during the antiphospholipid syndrome is associated with risks of thrombosis and fetal losses. Among 5 randomly selected monoclonal antiphospholipid antibodies, all originating from a single patient suffering from this autoimmune disease, only 1 induced fetal losses when passively injected into pregnant mice. Its antiphospholipid activity was dependent on annexin A5, and its variable regions contained mainly 3 replacement mutations. To clarify the role of these mutations in the pathogenicity of the antibody, they were in vitro reverted to the germ line configuration. The resulting "germ line" antibody reacted with multiple self-antigens and only partially lost its reactivity against PLs, but it was no more dependent on annexin A5 and, more importantly, was no more pathogenic. This study illustrates that the in vivo antigen-driven maturation process of natural autoreactive B cells can be responsible for pathogenicity.