http://orcid.org/0000-0002-4410-865X
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ABSTRACT
Immunoinformatics tools were used to predict human leukocyte antigen (HLA) class II-restricted T cell epitopes within the envelope glycoproteins and nucleocapsid proteins of Ebola virus (EBOV) and Sudan virus (SUDV) and the structural proteins of Venezuelan equine encephalitis virus (VEEV). Selected epitopes were tested for binding to soluble HLA molecules representing 5 class II alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, and DRB1*1501). All but one of the 25 tested peptides bound to at least one of the DRB1 alleles, and 4 of the peptides bound at least moderately or weakly to all 5 DRB1 alleles. Additional algorithms were used to design a single “string-of-beads” expression construct with 44 selected epitopes arranged to avoid creation of spurious junctional epitopes. Seventeen of these 44 predicted epitopes were conserved between the major histocompatibility complex (MHC) of humans and mice, allowing initial testing in mice. BALB/c mice vaccinated with the multi-epitope construct developed statistically significant cellular immune responses to EBOV, SUDV, and VEEV peptides as measured by interferon (IFN)-γ ELISpot assays. Significant levels of antibodies to VEEV, but not EBOV, were also detected in vaccinated BALB/c mice. To assess immunogenicity in the context of a human MHC, HLA-DR3 transgenic mice were vaccinated with the multi-epitope construct and boosted with a mixture of the 25 peptides used in the binding assays. The vaccinated HLA-DR3 mice developed significant cellular immune responses to 4 of the 25 (16%) tested individual class II peptides as measured by IFN-γ ELISpot assays. In addition, these mice developed antibodies against EBOV and VEEV as measured by ELISA. While a low but significant level of protection was observed in vaccinated transgenic mice after aerosol exposure to VEEV, no protection was observed after intraperitoneal challenge with mouse-adapted EBOV. These studies provide proof of concept for the use of an informatics approach to design a multi-agent, multi-epitope immunogen and provide a basis for further testing aimed at focusing immune responses toward desired protective T cell epitopes.
Abbreviations
| EP | = | electroporation |
| ELISpot | = | enzyme-linked immunospot |
| ELISA | = | enzyme-linked immunosorbent assay |
| EBOV | = | Ebola virus |
| HLA | = | human leukocyte antigen |
| IM | = | intramuscular |
| MHC | = | major histocompatibility complex |
| PRNT | = | plaque reduction neutralization test |
| SUDV | = | Sudan virus |
| VEEV | = | Venezuelan equine encephalitis virus |
Disclosure of potential conflicts of interest
Anne S. De Groot and William D. Martin are founders and majority owners of EpiVax, Inc., a bio-technology company that provides access to immunoinformatics tools and designs vaccines for commercial clients. Leonard Moise holds options at EpiVax, Inc., and both he and Frances Terry are employees of EpiVax, Inc. Due to this relationship with EpiVax, these authors acknowledge that there is a potential conflict of interest inherent in the publication of this manuscript and assert that they made an effort to reduce or eliminate that conflict, where possible.
Acknowledgments
We would like to thank Michelle Richards, Daniel Mitchell, Rebecca Grant-Klein, and Nicole Van Deusen for their assistance in performing the animal studies and immunogenicity analyses. This work was performed while Callie Bounds was a National Research Council postdoctoral Associate.
Funding
The studies described herein were supported by Grant R.R.0001_07_RD_B to USAMRIID from the Joint Science and Technology Office for Chemical and Biological Defense of the Defense Threat and Reduction Agency. The opinions, interpretations, conclusions, and recommendations contained herein are those of the authors and are not necessarily endorsed by the US. Army.