Objective 3. Develop new and improved diagnostic tools, vaccines, and novel management approaches

Development of novel nanoparticle-base vaccines for infectious bronchitis

PI, Mazhar I. Khan; CoPI, Peter Burkhard, University of Connecticut, Storrs, CT 06269

IBVInfectious bronchitis virus (IBV) causes respiratory disease in poultry as

well as affecting avian renal and reproductive systems.

Controlling of IBV is mainly based on vaccination program. Current available lived attenuated or killed vaccines have been challenged by their effectiveness due to IBV variants and lack of cross-protection.

Here, we are designing novel IBV vaccines using self-assembled peptide nanoparticle (SAPN) which called a highly innovative platform. One of the major immuonogenic genome encodes IBV is a spike (S) protein.

Self-assembled Peptide Nanoparticle (SAPN):

A excellent platform for universal vaccine design for avian influenza virus: defined size and shape, epitope strings, flexibility, easily scale up, etc.

Coiled-coil motifs dictate self-assemble of aligned-epitope monomers into a SAPN

Hydrophobic and ionic interaction

(modified image courtesy of Jaime Castillo-Leon, Book: Self-Assembled Peptide Nanostructure.)

(Kaba et al., 2009, J Immunol 183:7268; Zhao et al., 2014 Vaccine, 32: 327–337)

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Design of IBV Nanoparticle based vaccine prototypes

Sequence analysis of the IBV surface protein in order to identify the best B cell epitope to be displayed on the self-assembling protein nanoparticles (SAPN)

AF006624 (Keeler,C.L. Jr., Reed,K.L., Nix,W.A. and Gelb,J. Jr. Serotype Identification of Avian Infectious Bronchitis Virus (IBV)),

L10384(Jia,W., Karaca,K., Parrish,C.R. and Naqi,S.A. A novel variant of avian infectious bronchitis virus resulting from recombination among three different strains. Arch. Virol. 140 (2), 259-271 (1995))

L18990 Wang,L., Junker,D., Hock,L., Ebiary,E. and Collisson,E.W. Evolutionary implications of genetic variations in the S1 gene of infectious bronchitis virus. Virus Res. 34 (3), 327-338 (1994) and

M21883 (Niesters,H.G., Lenstra,J.A., Spaan,W.J., Zijderveld,A.J., Bleumink-Pluym,N.M., Hong,F., van Scharrenburg,G.J., Horzinek,M.C. and van der Zeijst,B.A. The peplomer protein sequence of the M41 strain of coronavirus IBV and its comparison with Beaudette strains. Virus Res. 5 (2-3), 253-263 (1986).

Research plan

The S protein of IBV contains two coiled-coil sequences as the S protein of SARS.

The second sequence (residues 1056-1083: ILDIDSEIDRIQGVIQGLNDSLIDLEKL) corresponds to the HRC sequence in SARS’ S protein (residues 1158–1185: VVNIQKEIDRLNEVAKNL NESLIDLQEL) and shares a high sequence homology.

We have engineered this coiled-coil sequence onto the trimeric coiled-coil of several versions of our current SAPNs.

Thus, these nanoparticles will present this epitope in a conformation-specific manner to the immune system, hence inducing conformation-specific antibodies with the potential to neutralize the virus in a viral infectivity assay.

Research planA sequence alignment of these proteins revealed the coiled-coil heptad repeat regions of the proteins and the relevant portions of the head domain of the glycoproteinA sufficient sequences similarity between the different structures revealed that the optimal B cell determinant within IBV to be used in a design of an IBV prototype vaccine are the coiled-coil sequences of the stalk domain.

Also, after the heptad repeat pattern, a significant portion of the proteins are highly conserved between the different strains/viruses.

These highly conserved regions are likely very important for the function of the glycoprotein of the virus and hence it was decided to keep those sequences at least in some designs of a prototype IBV nanoparticle vaccine.

Figure 2. Arrangement of two epitomic protein sequences in four different peptidemonomers that are building blocks for four different nanoparticle constructs. Black: His-tag; Green: pentameric coiled coil; Blue: trimeric coiled coil; Magenta: CD4 T cell epitope;Red: B cell epitope; Underscore: Restriction site for sub-cloning; Heptad sequences: The repeatpattern is indicated above the sequences (a d a d …)

IBV SAPN constructs

Figure 1. (left) 3D monomeric building block composed of a pentameric coiled-coil domain (green) andtrimeric de novo designed coiled-coil domain (blue) which is extended by the coiled-coil sequence ofthe S protein of IBV (red); (right) Computer model of the protein nanoparticle icosahedral symmetry(not drawn to size with monomer).

Self-assembly

Approaches:

Bioproduction of SAPN constructs:

Expression of coiled-coil SAPN monoer in E.coli Purification of SAPN by His-Column

Structural and biophysical analysis:

Transmission electron microscopy Dynamic light scattering

Results

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Figure 3. Bio-production and characterization of IBV nanoparticle. A:SDS-PAGE; B: Dynamic light scattering; C: Transmission electronmicroscopy

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Animal trial design:

Two groups of chickens were intramuscularly received 3 doses of 100μg IBV nanoparticle or buffer. Chickens were bled four times (2wks post 1st dose, 2 wks post 2nd dose and 2 wks post 3rd

dose). Sera were evaluated by ELISA and virus neutralization test in embryonated eggs.ELISA plates were coated with whole inactivated virus M41 (Biochek kit) or plates coated with 1 μg/ml IBV nanoparticle. Virus neutralization assay, sera diluted at 1/10 and incubated with IBV M41 1 EID50 1x105.4

for 1 hour at 37°C. Inoculum of virus+sera 100μL mixture were inoculated in SPF eggs and incubated for 8 days at 37°C. Weight of embryos were evaluated to asses neutralization effects. RNA was extracted from allantoic fluid from inoculated eggs and was evaluated by real time RT-PCR for relative gene copy numbers.

Fig 1. ELISA for antibodies induced by IBV-Long-long construct in chickens. Commercial kit (Biochek) was used. Plates were coated with M41 IBV. Sera were diluted at 1/500.

Figure 2. Evaluation of antibody ELISA titer to IBV-Long-long construct in chickens. A, two weekspost first dose of immunization; B, two weeks post second dose of immunization; C, two weekspost third dose of immunization.

Figure 2. Evaluation of ELISA antibody titer to IBV-Long-long construct in chickens. Comparison ofboosters

Figure 3. Virus neutralization in embryonated eggs.

Figure 3. Virus neutralization in embryonated eggs.

Conclusions

1. ELISA antibody titer were gradually elevated began at 2 weeks post 2nd dose and reached peak at 3rd dose of immunization. 2. IBV long-long Nanoparticles construct slightly neutralized viral infection in embryos. Embryos were slightly protected by anti-IBV long sera showing slightly gain of weight3. lower viral load and less lesion of disease while comparing to embryos inoculated with mixture of negative sera and M41 virus were observed.

Future PlanOptimize IBV long-long nanoparticle and test the other IBVnanoparticles (short-short; short-long combinations) andoptimization

Vaccine efficacy will be evaluated by challenging with IBVM41 in SPF chickens

AcknowledgementPathobiology, UConn

Jianping Li, Zeinab Helal

and Qing Fan

Molecular Cell Biology, UConn

Peter Burkhard, Christopher Karch

and Anmin Tan

Supported by PRD-CAP USDA-NIFA