Vaccines for Emerging and Re-emerging Viruses
I have developed an
extremely simple, innovative, and robust platform technology to present
α-helical epitopes from native proteins to the immune system such that the
resulting conformation-specific antibodies bind to the native protein target. This
procedure involves a process called templating where the helical sequence of
interest is inserted into a parallel two-stranded α-helical coiled-coil and
disulfide-bridged template which maintains the native helical conformation of
the epitope, presents all the surface-exposed residues of the α-helix in the
native protein, and optimizes stability of the immunogen.
Viruses that enter
cells using Class 1 Viral Fusion Proteins such as Ebola, Influenza A,
Respiratory Syncytial Virus and coronaviruses such as SARS-CoV and MERS-CoV are
ideal targets for this technology. The stem domains of the fusion glycoproteins
of these diverse viruses share a common coiled-coil structure that must be
triggered to undergo a massive conformational change in order to induce fusion
of the virus envelope with host cell membranes and initiate virus infections in vitro and in vivo. Our templated, two-stranded immunogens elicited stem
targeted, virus neutralizing antibodies against SARS-CoV and the hemagglutinin
(HA) protein of influenza A.
We have shown that
passive administration of rabbit antibodies to a selected immunogen can protect
mice against death after intranasal challenge with influenza A. This antibody
also recognizes purified HA proteins from different influenza subtypes,
suggesting that this stem-targeted immunogen may serve as a “universal”
influenza vaccine that can protect against multiple subtypes.
This research is the
result of an accumulation of an extensive body of published work on de novo
design of coiled-coil proteins from my laboratory. Selected publications are
Jiang, Z., L. Gera, C.T. Mant and R.S. Hodges. Antibody cross-reactivity
to hemagglutinin protein antigens demonstrates feasibility for development of a
“Universal” Influenza A synthetic peptide vaccine. In Enabling Peptide Research from Basic
Research to Drug Discovery, Proceedings of the 24th American Peptide
Symposium, Orlando, FL (V. Srivastava, A. Yudin and M. Lebl, editors) pp. 36-39
(2015). Published by the American Peptide Society and Propt Scientific
Yan, Z., W.J. Hartsock, Z. Qian, K.V. Holmes and R.S. Hodges. Strategies for designing peptide immunogens
to elicit α-helical conformation specific antibodies reactive with native
proteins. In “Small Wonders: Peptides for Disease Control” (K.
Rajasekaran, J.W. Cary and J. Jaynes, Eds.) ACS Symposium Series, Chapter 6,
pp. 93-136 (2012).
Tripet, B., D. Kao, S. Jeffers, K. Holmes and R.S. Hodges. Template-based Coiled-coil antigens elicit neutralizing
antibodies to the SARS-Coronavirus. J. Structural Biology 155: 176-194 (2006).
Tripet, B., M.W. Howard, M. Jobling, R.K. Holmes, K.V.
Holmes, and R.S. Hodges. Structural
characterization of the SARS-Coronavirus Spike Fusion Protein Core. J. Biol.
Chem. 279: 20836-20849 (2004).
Synthetic Peptide Anti-adhesion Bacterial Vaccine for Prevention of Pseudomonas aeruginosa (PA) Bacterial
I have determined the
key adhesin for the attachment of PA to the epithelial cell surface and
identified the receptor binding domain as a 17-residue region of the pilin
protein. Antibodies to the receptor binding domain (RBD) block adherence of the
organism and provide protection against PA in animal models.
We have determined the
three-dimensional structures of the receptor binding domains from multiple
strains of PA and shown that though sequences vary substantially from one
strain to another, there is a common structural motif.
We have shown that a
synthetic peptide vaccine is superior to a pilin protein-based vaccine. Using a
rational design approach, we have developed a single 17-residue peptide
immunogen that generates antibodies that target the RBD of more than one strain
My present goal is to
design a consensus sequence immunogen that will provide maximum antibody
cross-reactivity and affinity to all known strains of PA.
Hartsock, W.J., C. Hackbarth and R.S. Hodges. Antiadhesion
synthetic consensus sequence peptide-based vaccines for Pseudmonas
aeruginosa. In Handbook of Biologically
Active Peptides, 2nd edition, (A.J. Kastin, Editor in Chief) Chapter
77, pp. 563-570 (2013).
Hackbarth, C. and R.S.
Hodges. Synthetic Peptide Vaccine
Development: Designing Dual Epitopes into a Single Pilin Peptide Immunogen
Generates Antibody Cross-reactivity between Two Strains of Pseudomonas aeruginosa.
Chem. Biol. & Drug Design 76: 293-304 (2010). (Journal Cover Figure) PMC2949483 PMID20807222
Kao, D. and R.S.
Hodges. Advantages of a synthetic
peptide immunogen over a protein immunogen in the development of an anti-pilus
vaccine for Pseudomonas aeruginosa. Chem. Biol. and Drug Design 74: 33-42
(2009). PMC2756486 PMID19519742
Kao, D., M.E. Churchill, R.T. Irvin and R.S. Hodges. Animal protection and
structural studies of a consensus sequence vaccine targeting the receptor
binding domain of the type IV pilus of Pseudomonas
aeruginosa. J. Mol. Biol. 374: 426-442 (2007). PMC3493149 PMID17936788
Antimicrobial peptides (AMPs) as Therapeutics
The dramatic and ever-increasing
emergence of many relevant strains of bacteria resistant to traditional
antibiotics (of which no radically new structural class has been introduced
into medical practice over the past 30 years) is a major issue in human health.
The problem is further complicated by the fact that not only has there been an
explosion of resistance to antibiotics but also a rapid increase in multi-drug
resistance. We have now seen the development of so called “Superbugs” that are
resistant to most or all of the available antibiotics. Vertebrates and other
organisms have developed a defense system to microbial infections composed of
distinct groups of broad spectrum antimicrobial peptides (AMPs).
My group has been studying amphipathic
α-helical AMPs for years and has made six innovative discoveries critical to
the development of AMPs as therapeutics. 1)
Discovery of “specificity determinants,” that is, an amino acid substitution(s)
in the non-polar face of amphipathic α-helical or cyclic β-sheet AMPs that
dramatically reduces toxicity to human red blood cells; 2) The development of a novel method to measure self-association of
small amphipathic molecules; 3) the
discovery that increasing hydrophobicity on the non-polar face beyond an
optimum can lead to inactive AMPs; 4)
the novel discovery that “specificity determinants” when inserted into native
broad-spectrum AMPs directed selectivity to Gram-negative pathogens; 5) Methicillin-resistant S. aureus
(MRSA) strains are more susceptible to AMPs than Methicillin-sensitive S.
aureus (MSSA) strains. This suggested that pathogens in developing antibiotic
resistance are modifying their membrane composition/structure which makes the
membrane more sensitive to AMPs; 6)
advances in our understanding of mechanism of action of amphipathic α-helical
AMPs and the desired properties, for a clinical therapeutic; (i) the importance
of lack of secondary structure in aqueous medium but inducible α-helical
structure in the presence of the hydrophobic environment of the membrane; (ii)
“specificity determinants” reduce or eliminate toxicity by decreasing or
eliminating transmembrane penetration into eukaryotic membranes but allowing
AMP access to the interface region of prokaryotic membranes; (iii) reducing
self-association in aqueous environment is an important property influencing
biological activity; (iv) there is an optimum hydrophobicity window to maintain
high antimicrobial activity; (v) the sole target of the AMP should be the
bacterial membrane and the AMP should not be involved in any stereoselective
interaction with chiral enzymes or lipids or protein receptors; (vi) peptides
should be prepared in the all-D conformation to provide resistance to proteolysis.
Selected publications are listed.
Jiang, Z., L. Gera, C.T. Mant and R.S. Hodges. Design of new antimicrobial peptides (AMPs) with
“specificity determinants” that encode selectivity for gram-negative pathogens
and remove gram-positive and hemolytic activity from broad-spectrum AMPs. In Enabling Peptide Research from Basic
Research to Drug Discovery, Proceedings of the 24th American Peptide
Symposium, Orlando, FL (V. Srivastava, A. Yudin and M. Lebl, editors) pp.
245-248 (2015). Published by the American Peptide Society and Propt Scientific
Jiang, Z., A.J. Vasil, M.L. Vasil and R.S. Hodges. “Specificity
determinants” improve therapeutic indices of two antimicrobial peptides
Piscidin 1 and Dermaseptin S4 against gram-negative pathogens Acinetobacter baumannii and Pseudomonas aeruginosa. Pharmaceuticals 7: 366-391 (2014); DOI
Z. Jiang, A.I. Vasil, L. Gera, M.L. Vasil and R.S. Hodges. Rational design of a-helical
antimicrobial peptides to target gram-negative pathogens, Acinetobacter baumannii and Pseudomonas
aeruginosa: Utilization charge,
“specificity determinants”, total hydrophobicity, hydrophobe type and location
as design parameters to improve the therapeutic
ratio. Chem. Biol & Drug Design 77: 225-240 (2011). (Journal Cover
Figure) PMC3063396 PMID21219588
Hodges, R.S., Z. Jiang, J.
Whitehurst, and C.T. Mant. Devleopment
of antimicrobial peptides as therapeutic agents. In, “Development of Therapeutic Agents
Handbook” (Ed. by Shayne C. Gad), John Wiley and Sons Inc. pp. 285-358 (2011).
Stability Information Along a Two-stranded α-helical Coiled-Coil
Tropomyosin (Tm) is
most widely known for its role in muscle contraction; its calcium-dependent
cooperation with the troponin complex in regulating the interaction of myosin
and actin required to generate the power stroke within the sarcomeres of muscle
cells. Tm is expressed in all eukaryotic cell types in more than 40 isoforms that
are widely distributed within individual cells. Consequently, Tm plays a
critical role in numerous biologically relevant processes and is associated
with several diseases, including cardiomyopathy and cancer.
Tm is a two-stranded
α-helical coiled-coil of 284-residues per polypeptide chain. How signals are
transmitted along this rod-like molecule of more than 400 Ao remains
a major challenge for protein chemists. We made the first identification of a
stability control region (SCR) residues 97-118 in the 284-residue tropomyosin
sequence that controls overall protein stability but is not required for
folding. We have shown that a single mutation L110A destabilizes the entire Tm
(1-131) molecule, showing that the effect of this mutation is transmitted 165 Ao
along the coiled-coil in the N-terminal direction. The single mutation A109L
prevents the SCR from transmitting stabilizing information and separates the
coiled-coil into two domains, one that is ~9oC more stable than wild
type and one that is ~16oC less stable. Learning how stability information is
transmitted along rod-like molecules is crucial to understanding function and
Kirwan, J.P. and R.S.
Hodges. Transmission of stability
information through the N-domain of tropomyosin is interrupted by a stabilizing
mutation (A109L) in the hydrophobic core of the stability control region
(97-118). J. Biol. Chem. 389: 4356-4366
Kirwan, J.P. and R.S.
Hodges. Critical interactions in the
stability control region of tropomyosin.
J. Structural Biology 170: 294-306 (2010). PMC2856757 PMID20144718
Hodges, R.S., J. Mills, S.
McReynolds, J.P. Kirwan, B. Tripet, and D. Osguthorpe. Identification of a unique “stability control
region” that controls protein stability of tropomyosin: a two-stranded a-helical
coiled-coil. J. Mol. Biol. 392: 747-762 (2009).
Kwok, S.C. and R.S.
Hodges. Stabilizing and
Destabilizing Clusters in the Hydrophobic Core of Long Two-stranded a-Helical
Coiled-Coils. J. Biol. Chem. 279:
Development of New HPLC
and Capillary Electrophoresis Methodology
The development of new
separation methodology is critical to research in academia, biotechnology and pharmaceutical
sectors. A few examples of new methodology developed in our laboratory are
listed below: a new mixed-mode hydrophilic/cation-exchange chromatographic
method for the separation of peptides that rivals reversed-phase chromatography
(RPC) where selectivity is completely different; a new capillary
electrophoresis method called “Ion-interaction capillary zone electrophoresis”
which separates not only by charge/ mass ratio but by subtle differences in
hydrophobicity as well; a novel method to measure self-association of small
amphipathic molecules referred to as temperature profiling in RPC; a one-step
purification of recombinant proteins from a whole cell extract by RPC; and we
have determined the intrinsic hydrophilicity/hydrophobicity of amino acid
side-chains in peptides in the absence of nearest-neighbor or conformational
affects and showed that this scale was the most accurate determination of
side-chain hydrophilicity/hydrophobicity and of general value to all fields of
Mant, C.T. and R.S. Hodges. Separation of peptides on HALO 2-micron
particles. Current Protocols in Protein
Science 85: 11.6.1-11.6.16 (2016). doi:10.1002/cpps.12 (Wileyonlinelibrary.com,
John Wiley & Sons, Inc.).
Mant, C.T., Z. Jiang, B. E. Boyes and R.S. Hodges. An improved approach to hydrophilic interaction
chromatography of peptides: Salt gradients in the presence of high isocratic
acetonitrile concentrations. J.
Chromatography A 1277: 15-25 (2013).
Mant, C.T. and R.S.
Hodges. Design of peptide standards
with the same composition and minimal sequence variation to monitor
performance/selectivity of reversed-phase matrices. J. Chromatography A , 1230: 30-40 (2012).
Mant, C.T., D. Cepeniene and R.S. Hodges. Reversed-phase HPLC of Peptides: Assessing column and
solvent selectivity on standard, polar embedded and polar end-capped
columns. J. Separation Science
33:3005-3021 (2010). No PMC PMID21038458
Mant, C.T., J.M. Kovacs, H.M. Kim, D.D. Pollock and R.S. Hodges. Intrinsic amino acid side-chain
hydrophilicity/hydrophobicity coefficients determined by reversed-phase
high-performance liquid chromatography of model peptides: comparison with other
Biopolymers (Peptide Science) 92: 573-595 (2009). PMC2792893 PMID19795449
Development of New
Broad Spectrum Anticancer Drugs
The goal of the
proposed project is to develop new broad spectrum anticancer drugs using an
innovative and non-traditional approach where we create highly potent small
molecules with a simple 3-component “A-B-C” structure (500-700 daltons) and