GPRS to residues: let’s un-veil HPV16 to conquer

Structural comparison of four different antibodies interacting with human papillomavirus 16 and mechanisms of neutralization

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Back in 1976, Meisels and Fortin first published evidence to show that HPV was a pre-malignant and malignant agent; and not long after, zur Hausen identified HPV in cervical cancer specimens. It took decades for scientists to develop vaccines for this special virus. However, there is a type-specific limitation to protection in current vaccines and only low-efficiency therapies are available. Thus, a complete understanding of viral latency and natural immunity is essential, which is very important for cancer screening and vaccine usage. Neil Christensen, one of the leading HPV researchers has developed quasi-HPV / (CRPV) infectious particles as well as an extensive library of specific mono-clonal antibodies. Susan Hafenstein brought Cryo-EM technology to PSU in 2009, and established an imaging core for biological research that included a new JEOL 2100 acquired with a successful NIH grant. Thus a productive collaboration was established combining the best of two labs: the viral and immunological reagents of the Christensen Lab with molecular biology and structural approaches from the Hafenstein Lab.

We began our studies with four HPV16 L1 specific neutralizing monoclonal antibodies (N-mAbs), including V5 that is well known and extensively used in HPV research. Preliminary studies by Stephanie and Sarah identified differences between the neutralization behaviors of these monoclonals that posed new questions requiring structural analyses. These questions included: What did the differences mean? What interactions were occurring between capsid and antibodies? Which residues were involved in the interaction? After calculating the optimized binding ratios and conditions, as seen in Methods, through Bob’s golden hands and the efforts from our collaborators James and Alexander, we collected images of HPV16 complexed with V5, 1A, 14J, and 263A2. The structure-function analysis revealed predominantly monovalent binding of each Fab with capsid interactions that involved multiple loops from symmetry related copies of the major capsid protein. The residues identified in Fab-virus interfaces mapped to conformational grooves on the surface of the capsomer. In addition to the known involvement of the FG and HI loops, the DE loop was also found to constitute the core of each epitope. Surprisingly, the epitope mapping also identified minor contributions by EF and BC loops. Complementary immunological assays included N-mAb and Fab neutralization. The specific binding characteristics of the N-mAbs correlated with different neutralizing behaviors in pre- and post-attachment neutralization assays.

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Figure: (A1-A4), reconstruction maps of HPV16-Fab complexes of H16.V5, H16.1A, H16.14J, and H263.A2. (B1-B4), the location of the antibody binding on the surface in corresponding maps. (C), all four epitopes map to a “groove” feature on the capsomer. (D) The roadmap shows the footprint of each antibody mapped to the stereographic projection of a capsomer.

Introducing the authors

Author

Jian Guan, Ph.D, is a Postdoctoral Scientist in Prof. Hafenstein’s Lab in Pennsylvania State University College of Medicine, Hershey Medical Center. Susan Hafenstein is Associate Professor in Department of Medicine, Pennsylvania State College of Medicine. Neil Christensen is Professor of Pathology, in Pennsylvania State College of Medicine.

Structural comparison of four different antibodies interacting with human papillomavirus 16 and mechanisms of neutralization
Virology, Volume 483, September 2015, Pages 253–263
Jian Guan, Stephanie M. Bywaters, Sarah A. Brendle, Hyunwook Lee, Robert E. Ashley, Alexander M. Makhov, James F. Conway, Neil D. Christensen, Susan Hafenstein

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