Abstract

The West Nile flavovirus has been a cause for concern in the United States since the first
reported cases surfaced in 1999. Infection has been documented in over twenty-seven
mammalian species and veterinary vaccines have been developed to curb viral spread
among equine species. To date, no viable vaccine exists for human use, though strides
have been made in human clinical and in-vivo trials with mice and human subjects, alike.
Attenuated vaccines resulting from mutations in the non-structural components of
West Nile Virus have exhibited immunogenic promise, yet still pose the risk of non-
structural component interaction and post-infective fusion with WNV. Recombinant unit
vaccines pose similar risks yet show great promise in producing and maintaining cellular
and humoral response, whereas DNA vaccines have produce an accurate virus-antibody
targeting scheme. Lastly, the most pertinent and tested of vaccines is the vectored form,
which utilizes viral structural proteins as vessels to target cells of the immune system
directly.
This review documents the aforementioned trends in vaccine development and
their respective efficacies, as well as the history, pathophysiology and clinical treatment
of West Nile Virus.


























Introduction

History: Remote Discovery to Regional Epidemic

West Nile Virus (WNV) is as a mosquito-borne flavivirus and human
neuropathogen from the encephalitic Flaviviridae family. The Centers for Disease
Control and Prevention reported 2,374 cases of WNV in the United States in 2013. Of
those, approximately 4.8% (114) were lethal (Centers for Disease Control and
Prevention, 2014). The first reported cases of human West Nile Virus emerged in the
United States in 1999 (Guharoy et al., 2004). The virus remains widespread in
developing nations, particularly in the Middle east, West Asia and Africa, where it was
first documented in an infirmed Ugandan woman in 1936 (Smithburn et al., 1936).
Although verifiable data is available for developed nations, much off the developing
world in which West Nile is widespread, has failed to produce quantifiable figures on
infection rates. It stands to reasons that due to the lack of resources and health advocacy
in these nations, West Nile infection is likely more prevalent and lethal.

Virology: Anatomy of a Virus. Physiology of a Pathogen.

The viral genome is approximately 10.8 kb of single-stranded, positive-sense
RNA transcribed into a singular glycoprotein. A combination of host and viral proteases
cleave the glycoprotein into ten functional proteins (figure 1.1).

Figure 1.1a: Structural Components of West Nile Virus
Structural Components Function
Capsid Protein (C) Binds viral RNA
Envelope Protein (E) Receptor binding
Pre-membrane Protein (prM) Prevents premature fusion
Figure 1.1a: Structural components of WNV.









Figure 1.1b: Non-Structural Components of West Nile Virus
Non-Structural Components Function
NS1 Complement inhibition (Chung et al.,
2006)
NS2A Interferon A/B inhibition and virus induced
apoptosis (Melian et al., 2013)
NS2B NS3 co-factor (Wichapong et al, 2010)
NS3 Serine protease involved in viral replication
(Aleshin et al., 2007)
NS4A Induced autophagy in host cell (Mclean et
al., 2011)
NS4B NS3 agonist (Umareddy et al., 2006)
NS5 Interferon A/B antagonist (Mazzon et al.,
2009)
Figure 1.1b: Non-structural components of WNV.

The flavivirus enters the host cell via E-protein mediated endocytosis. The
nucleocapsid is released into the cytoplasm subsequent to fusion, the RNA dissociated
and viral transcription is initiated. Formation of structural components occurs in the
endoplasmic reticulum. Maturation occurs following cleavage of prM in the Golgi
apparatus, producing full functional, infectious vectors (Mukhopadhyay et al., 2005).
Interferon-A (IFN-!) production palys a role in mitigating WNV proliferation
(Lobigs et al., 2004). Non-structural components, less NS1, have exhibited IFN-!
inhibiting activity (Liu et al., 2005). IFN-! INDUCES GTP-binding protein MxA (also
known as interferon inducible protein p78 in mice), which interacts with WNV capsid
protein C, sequestering the structural component in tubular bundles in the cells
cytoplasm (Hoenen et al., 2014).

Clinical Presentations and Diagnosis

The World Health Organization estimates that 80% of affected individuals are
asymptomatic following infection (World Health Organization, 2011), however immune-
compromised patients, in particular the elderly, are at higher risk to develop neurological
complications. The most commonly occurring complication is encephalitis.
Recorded serological analysis of plasma and cerebrospinal fluid of a 55-year-old
African-American male suffering from WNV induced encephalitis is depicted in figure
1.2.

Plasma
Component
Patient Values Normal Range Condition
Leukocyte Count 12 x 10
3
/ uL 4.5-10 x 10
3
/ uL Elevated
Neutrophil % 84% 54-62% Elevated
Lymphocyte % 16% 25-33% Low
Creatine
Phosphokinase
Concentration
(CPK)

336 u/L

8/150 u/L

Elevated
Figure 1.2a: Plasma Analysis
(Gulharoy et al., 2004)

CSF Component Patient Values Normal Range Condition
White Blood Cell 62 cells/ uL 0-5 cells / uL Elevated
Erythrocyte Count 51 cells / uL 0 cells/uL Elevated
Neutrophil % 67% Less than 2% Elevated
Lymphocyte % 18% 60-70% Low
Monocyte % 12% Up to 30% Normal
Glucose
Concentration
82 mg/dL 50-80 mg/dL Normal
Protein 51 mg/dL 15-60 mg/dL Normal
Figure 1.2b: CSF Analysis

(Gulharoy et al., 2004)
Vaccine Development
Inactivated/Killed Virus

Studies conducted on mice that have been vaccinated with strains of formalin-
inactivated WNV NY99 exhibited a 100% survival rate relative to control subjects
injected with phosphate-buffered saline solution. The vaccinated mice were observed for
physical reactivity and antibody production over a period of 30 days and vaccinated
twice, within a week from onset of study, prior to administration of WNV.
Cross-neutralization tests using vaccinated mouse sera indicated that mice
injected with 830 ng of inactivated WNV vaccine produced the most efficacious FRNT
50

titer results (Guillermo et al, 2008). Previous studies have shown similar efficacy with
sequential vaccinations (Lim et al., 2008).

Attenuated Viruses

Modified strains of WNV obtained by point mutations exhibited amino acid
changes in non-structural components of the viral genome. This suggests that attenuated
strains of WNV utilized for vaccine development presented alterations in coding
sequences responsible for replicative machinery and pathogenesis. These non-structural
attenuated vaccines prove to be more precarious as they are responsible for inducing
virogenesis by increasing the likelihood of combining with structural components
following WNV challenges.
Mice inoculated with modified strains in some instances produced detectable
WNV-specific antibody levels as early as 3 weeks, and exhibited a high survival rate
relative to unvaccinated groups. The virulence of the strain corresponds to a more
substantial immune response, though greater potency often corresponded to a greater
mortality rate post-inoculation, again exhibiting the affinity of non-structural vaccine
components for structural components produced following WNV challenges
(Yamshchinkov et al., 2004).
Induced mutations in structural components of the WNV genome coincided with
a sharp decrease in neuroinvasiveness relative to wild-type WNV strains. Furthermore,
transfected cells analyzed for viral growth kinetics indicated that the mutation inhibited
viral growth in Vero and C6/36 cell cultures, indicating that laboratory-induced mutation
hedged against the risk of virulence produced by non-structural components of WNV (Yu
et al., 2008).

Recombinant Subunit Vaccines

WNV component vaccines have shown promise in conferring immunity against
the virus, Mice tested for humoral and cellular responses following inoculation with
structural and non-structural viral proteins stimulated production cytokines. Mice
vaccinated twice at 4-week intervals showed a dose-dependent increase in humoral IgG
response for non-structural (NS1) subunit vaccinations (Siirin et al., 2008). Vaccines
employ9ing structural subunits (protein E) maintained a constant, dose-independent level
of IgG1 production, while IgG2a titers decreased with increased dosage. Subunit cellular
response for both structural and non-structural components were additive with each
component yielding significant increases in IFN-", IL-4, IL-5 and IL-10 post-inoculation
(Liberman et al., 2007). Recombinant protein E subunit vaccines have continued to show
evidence of strong humoral responses and increased survivability in mice (Bonafe et al.,
2009).
Recombinant subunits from vectors of transmission, such as mosquitos, have thus
far shown little evidence of sustaining a humoral response against WNV. Recombinant
mosquito salivary protein D7 enhances pathogenesis of WNV virus in mice, thusly
contributing to host susceptibility (Reagan et al., 2012).

DNA Vaccines

Equine DNA vaccines rely on co-expression of structural WNV components, such
as E envelope and prM membrane protein. Protein prM facilitates E protein stability
throughout structural assembly and each has been implicated as an epitope in antibody
bonding (Vazquez et al., 2002)(Beasley & Barrett, 2002).
The outer domain of protein E consists of regions DI, DII and DIII. Although all
regions elicit antibody response, DIII, which is responsible for receptor binding, is the
strongest (Beasley & Barrett, 2002). Ectodomain inoculations accompanied by periodic
boosts with DIII showed significantly elevated IFN-" AND IgG levels (Schneeweiss et
al., 2011). DNA vaccinations have been shown to be the most accurate method of
antibody induction for WNV, however the risk of virulence is still substantial due to the
likelihood of vaccine-induced, translated non-structural components associating with
structural components from the WNV challenge.

Vectored Vaccines

Lentiviral vectors, used widely for their long incubation periods and the ability to
transfect both mitotic and non-mitotic cells have shown promise in transducing dendritic
cells and eliciting strong immune responses from cytotoxic CD8+ cells (Esslinger et al.,
2003) (Esslinger et al., 2002). Chimerized lentiviruses infused with WNV protein E
showed strong evidence of a considerably substantial and prolonged humoral response in
mice after a single inoculation (Igelsias et al., 2006).
Vectored Influenza A virus chimerized with the DIII region of WNV protein E
protected mono-inoculated mice by stimulating antibody and helper CD4+ cells (Martina
et al., 2006). Live chimeric viruses have been shown to illicit cytotoxic CD8+ memory
(Smith et al., 2011). Live attenuated WNV has also been shown to exhibit higher
humoral antibody responses compared to inactivated WNV and WNV sera (Tesh et al.,
2002). Human trials for attenuated yellow fever virus infused with WNV prM and E
protein sequences exhibit high seroconversion rates for WNV antibodies (Dayan et al.,
2012).

Pharmacological Intervention

There are currently no known agents that effectively treat WNV infection.
Ribavirin, an antivral medication, has been shown to be efficacious against neurological
complications such as encephalitis (Odelola et al., 1978), and high concentrations of the
drug inhibit WNV in in-vitro observations (Jordan et al., 2000). Interferon alpha-2b
proved protective and therapeutic before and after WNV challenges in primate cells,
whereas Ribavirin has been shown to be protective but not therapeutic (Anderson &
Rahal, 2002).

Conclusion

Development of vaccines to curb infection in humans is guided by the developed
worlds urgency to respond to the possibility of widespread, pandemic risks. As such,
West Nile Virus vaccine development ahs only been targeted towards organisms most
likely to be affected in the western world domesticated animals. The lack of literature
on vaccine formulation, despite the first documented instances of infection arising over
eighty years ago, was broadly augmented following the West Nile Virus outbreak among
the human population in New York in 1999.
In the last 15 years, an upsurge in West Nile Virus experimentation has produced
captivating results. The most promising of these findings is a live attenuated, chimeric
vaccine consisting of a yellow fever vector infused with West Nile Virus structural
components pre-membrane protein prM and envelope protein E. Research shows that
structural components have served as the building blocks of subunit and DNA vaccines
that have showed strong efficacy in mice populations.
Other less prominent yet equally revealing studies found that formalin-inactivated
West Nile Virus has the potential to induce a humoral response in mice, whereas a
peptide-attenuated virus strain has the potential to diminish the neuroinvasiveness of the
pathogen.
Steps to develop therapeutic guidelines for viral encephalitis have also been
discussed. To date, interferon alpha-2b has proven to be both protective and therapeutic
in uninfected and infected mice, respectively.
An important consideration is the lack of health infrastructure in developing
nations that are susceptible to West Nile infection. Due to a lack of preventative measure
in all facets of the West Nile lifecycle, including mosquito nets to ward off carriers and
proper palliative care to prevent on-set encephalitis, West Nile Vaccines are drastically
needed to preclude a growing pandemic in an exponentially globalizing world.