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Molecular mechanisms of arenavirus cell entry and antibody-mediated neutralization

Periodic Reporting for period 1 - ARENAVIRUS (Molecular mechanisms of arenavirus cell entry and antibody-mediated neutralization)

Reporting period: 2015-05-01 to 2017-04-30

Emerging infectious disease events are dominated by zoonoses: infections that are naturally transmissible from animals to humans or vice versa. A diverse and deadly group of exotic emerging viruses are the viral hemorrhagic fever (VHF) agents, including the rodent-borne mammalian arenaviruses (genus Mammarenavirus). The research carried out in the scope of the ‘ARENAVIRUS’ project focused on New World (NW) clade B arenaviruses responsible for hemorrhagic fever (HF) outbreaks in South America with high case fatality rates. These include Junin (JUNV), Machupo (MACV) and Guanarito (GTOV) viruses, which are responsible for HF outbreaks in rural areas of Argentina, Bolivia and Venezuela, respectively. Despite the significant threat to the public health and economy in the affected areas, there are no internationally approved drugs for preventing or treating NW arenavirus HF. To aid in rational, structure-based antiviral and vaccine design, the ‘ARENAVIRUS’ project is aimed at demonstrating the molecular mechanisms by which arenaviruses attach to human cells and are neutralized by the humoral immune response.
A primary determinant of zoonotic spread of clade B arenaviruses is their ability to recognize the human transferrin receptor 1 (hTfR1) in addition to the TfR1 of their natural rodent hosts. Cellular targeting, attachment, and membrane fusion is directed by the trimeric multi-functional glycoprotein (GPC) spikes, which decorate arenaviral envelope surface. Each protomer in the trimer consists of: a myristoylated stable signal peptide (SSP), a receptor attachment subunit (GP1), and a transmembrane fusion subunit (GP2). Remarkably, despite conserved utilization of TfR1 during viral attachment, the sequence similarity at the receptor binding site across the NW arenavirus GP1s is low. Before the start date of the project, the Host Laboratory and others had solved the GP1 structure of the MACV alone (1) and in complex with TfR1 (2). However, due to the substantial residue-sequence variability, molecular requirements for arenavirus zoonosis remained unknown.
The GPC spike also comprises the primary target for the host neutralizing immune response. As a preliminary work, the Researcher had demonstrated that the publicly available JUNV glycoprotein-specific neutralizing antibodies (raised by Sanchez et al. (3) and obtained from BEI Resources (Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH)) specifically target the GP1 subunit. Therefore, NW arenavirus GP1s were selected as immunogens for raising novel (cross)neutralizing antibodies against pathogenic NW arenaviruses.

1. Bowden TA et al. (2009) Unusual molecular architecture of the machupo virus attachment glycoprotein, Journal of virology. 83, 8259-65.
2. Abraham J et al. (2010) Structural basis for receptor recognition by New World hemorrhagic fever arenaviruses, Nature structural & molecular biology. 17, 438-44.
3. Sanchez A, et al. (1989) Junin virus monoclonal antibodies: characterization and cross-reactivity with other arenaviruses. J Gen Virol 70 ( Pt 5):1125-1132.
NW arenavirus attachment glycoproteins were used as immunogens to generate glycoprotein-specific neutralizing antibodies (nAbs) in mice. As a result, seven MACV GP1-reactive monoclonal antibodies (mAbs) were successfully isolated using antigen-specific single B cell sorting technique. No mAbs against the Bolivian haemorrhagic fever virus have been reported in the literature to date. Thus, the obtained anti-MACV GP1 mAbs might be of diagnostic and/or therapeutic interest. Even though a heterologous prime-boost immunization strategy, including both MACV GP1 and JUNV GP1 as immunogens, was used to raise the murine antibodies, no cross-reactivity of the isolated MACV GP1-reactive mAbs with JUNV GP1 or other NW arenavirus GP1s was observed. Neutralization tests for these antibodies, as well as functional and structural studies are ongoing.

In parallel, publicly available nAbs, raised against radiation-inactivated JUNV (3), were obtained from BEI Resources, tested for their reactivity with NW arenavirus GP1s and used to produce Fab fragments, suitable for co-crystallization experiments with JUNV GP1. Resulting from this approach, a high-resolution structure of the JUNV GP1–nAb OD01 complex was solved (PBD ID: 5NUZ). Our JUNV GP1–nAb co-crystal structure reveals the molecular basis for how a mouse-derived nAb (OD01) interferes with the host-cell attachment of JUNV by targeting the receptor-binding surface of the GP1. Comparison of our structure with another JUNV GP1–nAb complex reported during the project period (JUNV GP1–GD01 (4)) reveals largely overlapping epitopes but highly distinct antibody binding modes. Despite differences in GP1 recognition, we find that both antibodies present a key tyrosine residue, albeit on different chain, that inserts into a central pocket on JUNV GP1 and effectively mimics the contacts made by the host TfR1. These data provide a molecular-level description of how antibodies derived from different germline origins arrive at equivalent immunological solutions to virus neutralization. Immuno-focusing of the antibody response to mimic natural host-receptor interactions reveals a key point of vulnerability on the JUNV surface.These data provide valuable information for rational development of small-molecule antivirals targeting the receptor binding site and for design of synergetic, non-competing combinations of therapeutic anti-JUNV mAbs. The publication presenting the JUNV GP1-nAb OD01 complex structure is in press: 'Zeltina A, Krumm SA, Sahin M, Struwe WB, Harlos K, Nunberg JH, Crispin M, Pinschewer DD, Doores KJ, Bowden TA. 2017. Convergent immunological solutions to Argentine hemorrhagic fever virus neutralization. Proc Natl Acad Sci U S A.'

Furthermore, crystallization trials of the JUNV attachment glycoprotein alone and in complex with the human TfR1 were performed. Different N-linked-glycan handling and complex formation strategies were explored. ~3000 crystallization conditions for the glycoprotein alone and ~2500 conditions for the JUNV GP1–TfR1 complex were screened. Unfortunately, no crystal hits were obtained. The possible crystallization hurdles include the endoglycosidase-resistant glycosylation on the JUNV GP1 as well as the low affinity of the JUNV GP1 – TfR1 binding. Currently, further NW arenavirus attachment glycoproteins and TfR1 orthologues from their native rodent host species are being tested for expression efficiency and binding affinity.

3. Sanchez A, et al. (1989) Junin virus monoclonal antibodies: characterization and cross-reactivity with other arenaviruses. J Gen Virol 70 ( Pt 5):1125-1132.
4. Mahmutovic S et al. (2015) Molecular Basis for Antibody-Mediated Neutralization of New World Hemorrhagic Fever Mammarenaviruses, Cell Host Microbe. 18(6), 705-13.
Our data demonstrates that the TfR1 binding site is a major neutralization epitope on the surface of the JUNV, thus facilitating knowledge-based development of novel antiviral therapeutics to lessen the threat of the New World arenaviruses in the affected communities.