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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
) will be added to a solution of Hexadecyltrimethylammonium
bromide (CTAB) and Chloroauric acid (HAuCl
) 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
, HAuCl
, 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
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)
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.


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