Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies

Abstract

Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases and has given moderate success. Direct inhibition of parechovirus infection using monoclonal antibodies is a potential treatment. We have developed two neutralizing monoclonal antibodies against HPeV1 and HPeV2, namely, AM18 and AM28, which also cross-neutralize other viruses. Here, we present the mapping of their epitopes using peptide scanning, surface plasmon resonance, fluorescence-based thermal shift assays, electron cryomicroscopy, and image reconstruction. We determined by peptide scanning and surface plasmon resonance that AM18 recognizes a linear epitope motif including the arginine-glycine-aspartic acid on the C terminus of capsid protein VP1. This epitope is normally used by the virus to attach to host cell surface integrins during entry and is found in 3 other viruses that AM18 neutralizes. Therefore, AM18 is likely to cause virus neutralization by aggregation and by blocking integrin binding to the capsid. Further, we show by electron cryomicroscopy, three-dimensional reconstruction, and pseudoatomic model fitting that ordered RNA interacts with HPeV1 VP1 and VP3. AM28 recognizes quaternary epitopes on the capsid composed of VP0 and VP3 loops from neighboring pentamers, thereby increasing the RNA accessibility temperature for the virus-AM28 complex compared to the virus alone. Thus, inhibition of RNA uncoating probably contributes to neutralization by AM28. IMPORTANCEHuman parechoviruses can cause mild infections to severe diseases in young children, such as neonatal sepsis, encephalitis, and cardiomyopathy. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases. In order to develop more targeted treatment, we have searched for human monoclonal antibodies that would neutralize human parechoviruses 1 and 2, associated with mild infections such as gastroenteritis and severe infections of the central nervous system, and thus allow safe treatment. In the current study, we show how two such promising antibodies interact with the virus, modeling the atomic interactions between the virus and the antibody to propose how neutralization occurs. Both antibodies can cause aggregation; in addition, one antibody interferes with the virus recognizing its target cell, while the other, recognizing only the whole virus, inhibits the genome uncoating and replication in the cell. Human parechoviruses (HPeV) are single-stranded, positivesense RNA viruses in the Parechovirus genus within the Picornaviridae family (1). HPeV infections mainly cause mild gastrointestinal symptoms, although HPeV are also associated with more severe central nervous system symptoms, such as meningitis and neonatal sepsis (2-5). The HPeV genome is about 7,300 bases in length, enclose...

Potent neutralization of hepatitis A virus reveals a receptor mimic mechanism and the receptor recognition site

“…HAV capsid proteins exhibit no notable conformational changes upon binding to R10 Fab with an rmsd of 0.2 Å between the R10 Fab bound and unbound states of HAV. R10 binds to the viral surface along the edges of the pentameric building block of the virus, between the twofold and threefold axes in a similar position to that observed for AM28 antibody bound to parecho virus and NAb E18 binding to EV71 (13,19) (Fig. 3 C and D and Fig.…”

Stable long‐term cultures of self‐renewing B cells and their applications

“…As mentioned in the following paragraph, we were also able to develop potent broadly reacting antibodies against the influenza stem region of group 2 influenza viruses , which should inform generation of an efficacious influenza virus vaccine similar to that done for group 1 influenza . We have also generated broadly reactive highly neutralizing antibodies to human parechovirus , a virus of the picornaviridea family, hCMV and HCV. It is likely that knowledge of the epitopes recognized by these antibodies will also inform vaccine development.…”

Human picornaviruses associated with neurological diseases and their neutralization by antibodies

“…For the parechoviruses, only the antigenicity of HPeV-1 has been studied [169]. There are three HPeV structural proteins: VP0, VP1 and VP3.…”

“…One is localized to residues 83-97 of VP0 [172], another is found on VP1 and encompasses the receptor-recognizing arginine-glycine-aspartic acid (RGD) motif [173], and the third one is formed by VP0 and VP3 [169,174]. Whereas antisera generated against the HPeV-1 VP0 peptide were not tested for HPeV-3 neutralization, two other monoclonal antibodies did not cross-react with HPeV-3 [169,174]. Only a non-neutralizing epitope has been described for HPeV-3 [170].…”

“…A useful approach for this is identification of an individual's immune response to a given virus followed by respective B-cell cloning [182]. This approach was utilized to generate two broadly neutralizing antibodies against HPeV [169,174]. Unfortunately, these antibodies did not neutralize HPeV-3 in a conventional microneutralization test.…”