Icon Legend

This session is not in your schedule.

This session is in your schedule. Click again to remove it.

Technologies for Emerging Infectious Diseases

Technologies for Emerging Infectious Diseases - Poster Session E

Poster AA14 - Structural design of a recombinant Plasmodium vivax circumsporozoite protein vaccine candidate

Saturday, October 26, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Jacob N. Arras (he/him/his)

BESIP Scholar, (Bioengineering Summer Internship Program)
NIH - NIBIB/NIAID, Texas, United States

Co-Author(s)

KR

Karine Reiter

Biologist
Process Development Unit, Laboratory of Malaria Immunology and Vaccinology, NIAID/NIH, United States
RH

Raul Herrera

Biologist
Process Development Unit, Laboratory of Malaria Immunology and Vaccinology, NIAID/NIH, United States
VN

Vu Nguyen

Research Scientist
Process Development Unit, Laboratory of Malaria Immunology and Vaccinology, NIAID/NIH, United States
FC

Fernando Cabezas

Laboratory Technician
Laboratory of Malaria Immunology and Vaccinology, NIAID/NIH, United States
Lixin Lang, Ph. D.

Staff Scientist
Molecular Tracer and Imaging Core Facility, NIBIB/NIH, United States
DK

Dale Kiesewetter, PhD

Chief, Molecular Tracer and Imaging Core Facility
NIH - NIBIB, United States
JR

Jonathan Renn, PhD

Scientist
Recombinant Antigen and Antibody Development Unit, Laboratory of Malaria Immunology and Vaccinology, United States
NM

Nicholas MacDonald

Head, Molecular Biology Unit
Molecular Biology Unit, Laboratory of Malaria Immunology and Vaccinology, NIH/NIAID, United States
Richard Leapman, PhD

Scientific Director, Intramural Research Program
NIH - NIBIB
Bethesda, Maryland, United States
PD

Patrick Duffy

Chief
Laboratory of Malaria Immunology and Vaccinology, NIH/NIAID, United States

Primary Investigator(s)

Albert J. Jin, Ph.D (he/him/his)

Chief, Nanoinstrumentation and Force Spectroscopy Section
NIH- NIBIB
Bethesda, Maryland, United States

Last Author(s)

DN

David L. Narum, MS, PhD (he/him/his)

Head, Process Development Unit
Process Development Unit, Laboratory of Malaria Immunology and Vaccinology, NIAID/NIH
Bethesda, Maryland, United States

Co-Author(s)

KJ

Kristine Jones

Introduction: Plasmodium vivax is the most prevalent and geographically widespread malaria parasite with 2.5 billion people at risk of infection. The two clinically efficacious vaccines recommended by WHO against Plasmodium falciparum are composed of the carboxyl-terminal half of the circumsporozoite protein (CSP), providing a strong basis to target a vaccine against the orthologous P. vivax CSP. The P. vivax CSP orthologue is comprised of an amino- and carboxyl-termini with a C-terminal thrombospondin repeat-like domain, and a middle repeat region composed of two allelic variants with about 16 nonameric amino acid repeats, identified as VK210 and VK247. Recombinant PfCSP is a flexible rod-like protein (Rh 4.2 nm, 33 kDa) that binds heparin sulfate proteoglycans on the hepatocyte surface due to motifs identified on the amino- and carboxyl-termini. To support preclinical development of a PvCSP vaccine, we engineered various recombinant forms (including homologous alleles and a chimeric form with both allelic repeat units) and produced in the yeast Pichia pastoris, then purified for biochemical, biophysical, and immunological characterizations.

Materials and

Methods: Recombinant PvCSPs were produced as secreted proteins in the methylotrophic yeast P. pastoris and purified by column chromatography using ionic and hydrophobic properties of each recombinant protein. Purified and well-characterized recombinant PvCSPs, with two different truncated N-terminal boundaries were identified as long and short forms and have been evaluated by analytical size exclusion chromatography with inline static and dynamic light scattering, in silico structure prediction using AlphaFold, atomic force microscopy (AFM), protein-crosslinking mass spectrometry, and heparin binding on a heparin affinity column.

Results, Conclusions, and Discussions: All purified PvCSPs were monomeric and observed to have an extended structure with dynamic light scattering (Hydrodynamic radii (Rh) varying between 3.7 – 4.3 nm for recombinant proteins ranging from 23 – 30 kDa); above the expected Rh of 2.6 nm for a 26 kDa globular protein. AlphaFold generated models with elongated helical structures in the central repeats as well as a propensity for N-terminal and C-terminal interactions. To date, AFM studies at ultrahigh resolution on recombinant VK247 and the chimeric (long and short) proteins have shown elongated monomers and small oligomers that are being validated by protein-crosslinking mass spectrometry. Finally, initial analyses for binding to a heparin affinity column showed that only the recombinant VK247 allele bound while the VK210 allele and chimeric VK247/VK210 did not despite the putative binding motifs being conserved. The basis for this difference in heparin binding is unclear. A solid understanding of the biophysical properties of different recombinant PvCSPs will aid in the design of a PvCSP vaccine candidate.

Acknowledgements (Optional):