Generation of a Gold Nanorod Vaccine against Ebolavirus and Marburgvirus
Statement of the Problem
Ebolavirus (EBOV) and Marburgvirus (MARV) are members of the Filoviridae family, along with the recently proposed Cuevavirus. EBOV and MARV infect both humans and non- human primates causing severe hemorrhagic fever. Five distinct species make up the EBOV family including Zaire (EBOV), Sudan (SUDV), Tai Forest (TAFV), Reston (RESTV), and Bundibugyo (BDBV) while the MARV family consists of two species; MARV and RAVN (RAVV) (1). Wild-type filovirus infection has been associated with severe case fatality rates in humans, as high as 90% (2). In humans, filovirus infection is characterized by an acute onset of flu-like illness, after an initial incubation period of 2-21 days. Following the initial illness, signs and symptoms of the disease include nausea, vomiting, anorexia, chest pain, cough, edema, postural hypotension, neurologic complications, petechial, and mucosal hemorrhage (3). Natural outbreaks occur sporadically and are typically restricted to the African continent with the most recent being a SUDV outbreak in Uganda in 2012. There have also been several observed wild-type filovirus outbreaks among great apes in Africa that have demonstrated similarly high mortality rates as humans (4). Both EBOV and MARV are categorized as class A pathogens by the CDC and NIH, and both pathogens present a recognized biological weapon threat. Class A pathogens are designated such due to their high mortality rates, potential for major public health impact, potential to cause public panic and social disruption, and can be easily disseminated or transmitted from person to person. Presently, there are no licensed vaccines, prophylactics, or treatments for EBOV or MARV infections in humans. As a
consequence, there is an immediate need for vaccines capable of eliciting protective immunity against both EBOV and MARV.
Background and Relevance to Previous Work Recent progress has been made in the field of nanoparticle research, and it is being realized gold nanorods could be used successfully as vaccine delivery systems. As a vaccine delivery vehicle, gold nanorods are extremely attractive candidates. While vaccine research using this construct is still in its infancy, multiple successful studies have been published including JW Stone et al. which demonstrates the capability of gold nanorods loaded with fusion (F) protein of respiratory syncytial virus (RSV) to efficiently induce an immune response in human T lymphocytes; L Xu et al. which sheds light on the rational design of low- toxic nanomaterials as a versatile platform for HIV-1 vaccine nanoadjuvants/delivery systems; and PA Jorquera et al. which demonstrates the vaccination of mice with the RSV G protein nanoparticle vaccine induces a potent neutralizing antibody response, increased G protein- and M2- specific T cell responses, and a reduction in RSV disease pathogenesis (5-7). A single gold nanorod of approximately 21 nm x 57 nm in size is stabilized by protein ligands on its surface. This is a relatively large amount of surface area, which creates the potential for a high level of antigen presentation (5). Many naturally occurring viral particles are of a similar size, and many viruses such as Ebola and Marburg exhibit a filamentous or roughly filamentous shape (9). This similarity of nanorods to viral particles shape and size will be beneficial, as it will mimic wild-type antigen uptake by host immune cells leading to a more broad and robust immune response. Under suitable conditions, surface-stabilizing ligands on gold nanorods may be exchanged, potentially resulting in the formulation of a vaccine candidate incorporating multiple epitopes into a single vaccine construct (9). Also of note, colloidal gold is
phagocytosed by antigen presenting cells e.g. macrophages and dendritic cells (DCs) which play a key role in the induction of a protective immune response (10, 11). In this proposal we will focus on EBOV and MARV epitopes which will be discussed in the Aims section. Several vaccination strategies for EBOV and MARV (including DNA, adenovirus, bivalent rabies virus, recombinant vesicular stomatitis virus (rVSV), virus-like particles (VLPs) and recombinant parainfluenza virus vectored vaccines) have been developed and have been shown to confer varying degrees of protection in animal models (1). The glycoprotein (GP), with variable contribution from the nucleoprotein (NP), appears to be the key immunogenic protein in vaccine protection (along with VP24 and VP40). Numerous reports document the ability of GP to act as a potent immunogen, and GP has been used in many filovirus vaccine trials (3). While each vaccine strategy has shown promising results and a relative degree of protection in animal models, concerns such as vaccine safety, preexisting vector immunity, manufacturing, or lack of commercial interest have slowed progress. I believe that a nanoparticle approach could mimic wild type antigen presentation and result in a more robust, long-term, and safe immune response, unlike previous filovirus vaccine attempts that either require multiple injections, or do not result in cross-protection (3).
Experimental Outline The objective of this proposal is to develop a gold nanorod construct that displays the major protective antigens of EBOV and MARV (GP and NP) for use as a vaccine strategy against EBOV and MARV. In order to achieve this objective, I will focus on three major aims.
Aim 1. To formulate and characterize gold nanorods conjugated to GP and NP as a candidate vaccine preparation by covalent attachment of viral proteins using a layer-by- layer approach. Aim 2. To determine the ability of gold nanorods to deliver viral antigens (GP and NP) effectively to human antigen presenting cells in order to initiate the induction of a protective immune response. Aim 3. To evaluate the ability of the gold nanorod vaccine to induce a protective immune response in suitable animal models.
Aim 1. To formulate and characterize gold nanorods conjugated to GP and NP as a candidate vaccine preparation by covalent attachment of viral proteins using a layer-by- layer approach. Recently, gold nanorods as a vaccine delivery vehicle have generated significant interest. JW Stone et al. demonstrates the capability of gold nanorods loaded with fusion (F) protein of respiratory syncytial virus (RSV) to efficiently induce an immune response in human T lymphocytes (5). The viral proteins of EBOV and MARV; GP and NP, are the key immunogenic proteins used in previous vaccine strategies against EBOV and MARV, and will be the antigens of choice in this study (3). I propose to create a vaccine platform made by covalently attaching recombinant viral proteins GP and NP to gold nanorods.
Experimental Design 1.1 Synthesis of gold nanorods
The gold nanorods will be prepared for covalent attachment of GP and NP using a seed- mediated layer-by-layer approach with charged polyelectrolytes polyacrylic acid (PAA) and polyallylamine hydrogen chloride (PAH) (12, 13). The GP and NP protein expression vectors are readily available and will be used not only as the covalently bound antigens on the gold nanorods, but also for creation of ELISAs discussed in Aim 1.3 (14). Briefly, a volume of ice cold sodium borohydride (NaBH 4 ) will be added to a solution of Hexadecyltrimethylammonium bromide (CTAB) and Chloroauric acid (HAuCl 4 ) and stirred vigorously. The resulting solution will contain small (nm) gold spheres that will be used as a seed solution in the preparation of nanorods. Next, CTAB, AgNO 3 , HAuCl 4 , ascorbic acid and the nanorod seed solution will be combined, mixed, and allowed to incubate overnight. Purification via multiple rounds of centrifugation or cross-flow filtration (to remove excess CTAB and metallic ions) will follow the incubation. 1.2 Coating of nanorods Following the nanorod preparation, poly(acrylic acid, sodium salt) (PAA), will be added to multiple aliquots of the gold nanorods simultaneously with a solution of NaCl. The resulting solution will be allowed to mix, centrifuged, and resuspended. Following PAA coating, the nanrods will be further coated with a layer of poly(allylamine hydrochloride) (PAH) simultaneously with a solution of NaCl. Again the resulting solution will be allowed to mix, centrifuged, and resuspended. Aliquots of PAH-coated nanorods will be combined with recombinant filovirus GP and NP, and mixed. Following mixture, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) will be added (to induce amide bond formation between the nanorods and proteins), incubated, and removed via centrifugation.
1.3 Determination of the protein concentration of protein-nanorod conjugations In order to determine the efficacy of protein-nanorod conjugation, protein concentrations will be determined by GP- and NP-specific enzyme linked immunosorbent assay (ELISA) in comparison to known standards. Purified GP and NP protein will be used for both nanorod conjugations and standard curves. GP and NP protein expression vectors have been developed, as well as ELISAs, in other labs. This will greatly reduce the amount of assay development necessary for this aim (14). 1.4 Confirmation of particle integrity and stability In order to determine the integrity and stability of GP and NP conjugated nanorods, transmission electron microscopy (TEM) will be performed. For TEM, grids will be prepared by placing a carbon-coated copper grid on top of the samples, and then drying them. Furthermore, stability tests will be performed by treating as-prepared nanorods, PAH-coated nanorods, or GP/NP-coated nanorods with 1x PBS (pH 7.4) in order to determine particle integrity under physiological pH and osmolarity. Particles will be tested using ultra-violet-visible (UV-vis) absorption. Interpretation of Results The data from these experiments will allow me to determine the success of gold nanorod particle assembly and the efficacy of the antigenic protein-nanorod conjugation. If GP or NP fails at the conjugation step, there are other potential antigens (VP24 and VP40) that can be used in development. The desired outcome of these experiments are stable GP and NP conjugated nanorods with >10% total soluble NP and GP bound to the nanorod surface.
Aim 2. To determine the ability of gold nanorods to deliver viral antigens (GP and NP) effectively to human antigen presenting cells in order to initiate the induction of a protective immune response. I will examine the nascent gold nanorod construct and its ability to elicit a protective immune response from human peripheral blood cells. Gold nanorod vaccines have been shown to deliver protective viral antigens effectively to human antigen presenting cells (5). One of the main purposes of this proposal is to test whether gold nanorods can deliver protective filovirus antigens effectively to human antigen presenting cells in order to initiate the induction of a protective immune response. 2.1 Dendritic cell (DC) maturation and T cell activation To that effect, incubating the NP/GP conjugated nanorods with primary cultures of human DCs and T-lymphocytes will be a key experiment in order to determine if an active immune response can be induced. Human peripheral blood will be collected with the approval of the Institutional Review Board (IRB). Peripheral blood will be collected from healthy adult donors. Peripheral blood mononuclear cells (PBMCs) will be isolated using density gradient centrifugation and ficoll. Monocytes will be isolated using CD14+ magnetic beads, and DCs will be derived from the isolated monocytes in the presence of recombinant human GM-CSF and recombinant human IL-4. T cells will be isolated from the sample donor PBMCs using CD3+ magnetic beads. Monocyte-derived DCs (MDDCs) will then be pulsed with PBS, purified GP and NP in PBS, Ebola or Marburg virus GP/NP protein coated nanorods in PBS, or PAH-coated nanorods in PBS. DCs will be pelleted, washed, resuspended and cultured. Following resuspension and culture of DCs, T cells will be stained with carboxyfluorescein succinimidyl ester (CFSE), a
common fluorescent dye used to track proliferation and migration of lymphocytes, and overlaid onto donor-matched nanorod-activated MDDCs and co-cultured for multiple days. 2.2 Flow cytometric analysis of proliferation of human T cells Following co-culture of T cells and nano-rod activated MDDCs, T cell proliferation will be analyzed using a multi-laser flow cytometer. I expect the results to demonstrate a significant increase in T cell proliferation in cells exposed to GP/NP coated nanorods as compared to T cells exposed to PBS, GP/NP alone, or PAH-coated nanorods alone. Interpretation of Results The data from these experiments will allow me to determine if the vaccine construct is capable of inducing an immune response in human immune cells. Testing the proliferation of human T cells in co-culture with DCs treated with GP/NP coated nanorods will determine whether or not a protective response against GP/NP is possible. If there is no T cell proliferation in response to GP/NP, other experiments will have to be performed to rule out potential cytotoxicity. It is possible that other viral antigens, or combinations of antigens could be used.
Aim 3. To evaluate the ability of the gold nanorod vaccine to induce a protective immune response in suitable animal models. 3.1 Evaluation of gold nanorod vaccine safety and efficacy in mouse model Six- to eight-week-old pathogen-free BALB/c mice will be used to initially determine safety and efficacy of the GP/NP nanorod vaccine construct. Mice will be kept in accordance with IRB specifications. Serum IgG titers against filovirus glycoprotein after vaccination are the best correlate of protection against filovirus in non-human primates (15). To assess the induction of serum IgG titers by our vaccine, BALB/c mice will be immunized intramuscularly (I.M.) with
an escalating dose of gold nanorod particles. The humoral immune response against GP and NP will be measured four weeks after immunization using a mouse IgG-specific sandwich ELISA, specific for filovirus GP and NP. It will also be important to determine if the immune response elicited by the gold nanorod vaccine will provide cross reactivity. Serum from each of the immunized mice will be tested for reactivity against each of the filovirus glycoproteins in an ELISA. If this experiment demonstrates no cross-reactivity, the vaccine can be redesigned with multiple viral antigens from different filoviruses in order to elicit a broader, potentially cross reactive response. 3.2 Evaluation of gold nanorod vaccine safety and efficacy in non-human primates EBOV and MARV seronegative cynomolgus macaques will be used to evaluate the vaccine candidate. Non-human primate immunization and challenge will follow the protocol described by J. Blaney et al. (14). Briefly, macaques will be immunized intramuscularly and bled at specific times post-immunization. For each challenge experiment, physical exams and blood draws will also be performed at specific times post-immunization. Complete blood count (CBC) and serum chemistry will be evaluated on an Advia 120 Automated Hematology Analyzer and an Abaxis Piccolo point-of-care lab system respectively. Levels of viral RNA will be determined using quantitative RT-PCR as described previously (14). For determination of virus titers in non-human primate blood and tissue samples, Vero E6 cells were seeded in 48-well plates. Blood samples will be thawed and serial dilutions prepared. Tissues will be homogenized in DMEM and serial dilutions also prepared. Processed blood and tissue samples will be incubated with cells, and cells will be monitored for cytopathic effect.
In order to evaluate T-cell responses in non-human primates, IFN- ELISPOT will be run according to manufacturers protocols. Macaque sera will also be evaluated for the humoral response to EBOV and MARV. Total IgG and isotype ELISAs specific for GP and NP will be performed according to a previously published protocol (14). In addition pathology samples will be evaluated to confirm the effects of nanorods on macaque tissues and organs. Interpretation of Results The data from these experiments will demonstrate the ability of a gold nanorod vaccine to elicit a safe and protective immune response against Ebola and Marburg in both a small rodent and non-human primate animal model. Both models are important for demonstrating safety and efficacy of the vaccine and will provide critical data for this proposal.
Significance and Application The aims of this study provide an outline of experiments that will evaluate the contributions of humoral immunity and cell-mediated immunity to the protection provided against EBOV and MARV infections via a novel gold nanorod vaccine. In addition, methods which could improve the vaccine generated immune responses will be examined. The goal of these aims is to contribute to the development of a protective gold nanorod vaccine against EBOV and MARV, two highly virulent human pathogens and potential bioweapons. Additionally, these studies could provide the groundwork for vaccine development against other serious threats.
Bibliography
1. Bradfute, S.B., Dye, J.M., Bavari, S. 2011. Filovirus Vaccines. Hum. Vaccin. Jun;7(6):701-11 2. Sanchez A., Geisbert T. W., Felsmann H. (2007). Marburg and Ebola Viruses. Philadelphia, PA: Lippincott Williams and Wilkins 3. Friedrich, B.M., et al. 2012. Potential Vaccines and Post-Exposure Treatments for Filovirus Infections. Viruses. Sep;4(9):1619-50 4. Genton, C., et al. 2012. Recovery Potential of a Western Lowland Gorilla Population Following a Major Ebola Outbreak: Results from a Ten Year Study. PLoS One. May;7(5) 5. Stone, J.W., et al. 2013. Gold Nanorod Vaccine for Respiratory Syncytial Virus. Nanotechnology. Jul;24(29):295102 6. Xu, L., et al. 2012. Surface-engineered Gold Nanorods: Promising DNA Vaccine Adjuvant for HIV-1 Treatment. Nano. Lett. Apr 11;12(4):2003-12 7. Jorquera, P.A., 2013. Nanoparticle Vaccines Encompassing the Respiratory Syncytial Virus (RSV) G Protein CX3C Chemokine Motif Induce Robust Immunity Protecting from Challenge and Disease. PLoS One. Sep 10;8(9):e74905 8. Kassam, A., et al. 2006. Place Exchange Reactions of Alkyl Thiols on Gold Nanoparticles. J. Am. Chem. Soc. Mar 22;128(11):3476-7 9. Chen, S., et al. 1998. Gold Nanoelectrodes of Varied Size: Transition to Molecule-like Charging. Science. Jun 26;280(5372):2098-101 10. Villiers, C. et al. 2010. Analysis of the Toxicity of Gold Nano Particles on the Immune System: Effect on Dendritic Cell Functions. J. Nanopart. Res. Jan 12(1):55-60 11. Shukla, R. et al. 2005. Biocompatibility of Gold Nanoparticles and their Endocytic Fate Inside the Cellular Compartment: A Microscopic Overview. Langmuir. Nov 8;21(23):10644-54 12. Murphy, C.J., et al. 2005. Anisotropic Metal Nanoparticles: Synthesis, assembly, and optical applications. J. Phys. Chem. B. Jul 28;109(29):13857-70 13. Norman, R.S., et al. 2008. Targeted Photothermal Lysis of the Pathogenic Bacteria, Pseudomonas aeruginosa with Gold Nanorods. Nano. Lett. Jan;8(1):302-6 14. Blaney, J.E., et al. 2013. Antibody Quality and Protection from Lethal Ebola Virus Challenge in Nonhuman Primates Immunized with Rabies Virus Based Bivalent Vaccine. PLoS Pathog. May;9(5):e1003389 15. Sullivan, N.J., et al. 2009. Correlates of Protective Immunity for Ebola Vaccines: Implications for Regulatory Approval by the Animal Rule. Nat. Rev. Microbiol. Sep;7(9):684