**Abstract:** We have created candidate live West Nile virus (WNV)
vaccines by site-directed mutagenesis of a WN infectious DNA, targeted
to a flavivirus-conserved stem-and-loop secondary structure at the
3'terminus of the 3'noncoding region (NCR) in West Nile virus RNA,
referred to as the "3'SL". Preliminary data from studies in the mouse
model of the neurovirulence and neuroinvasiveness of 3'SL mutant
viruses, selected for study based on impaired replication in tissue
culture, suggests that they are attenuated compared to the strain of WNV
currently circulating in the US, strain NY/99. Specifically, all four
3'SL mutant WNVs exhibited at least a 10,000-fold higher MLD50 (50%
Mouse Lethal Dose) than strain NY/99, and all mutants elicited
WN-specific neutralizing antibodies. However, these viruses retained
some mouse lethality at very high challenge doses (\~10E4 to 10E5 pfu).
A suitable candidate live WN vaccine ought ideally to exhibit no lethal
effect in the mouse model at any practically attainable dose level (e.g.
up to \~10E7 pfu). Therefore we propose here to introduce additional
mutations *into the 3'SL mutant West Nile genomes* via site-directed
mutagenesis, in order to produce 3'SL mutant viruses containing novel
second-site mutations, on the premise that this strategy will enhance
the attenuation phenotype in mice compared to that of existing 3'SL
mutants. We plan to target the NS2B/NS3 protease genes and the envelope
(E) gene in constructing the new mutant viruses, using data already
available that suggests specific mutations in these two targeted sites
likely to result in further attenuation of virus virulence. Viruses that
are suitably attenuated in mice will then be tested in hamsters for
protective efficacy and immunogenicity.

**Specific Aims:** (1) Development of vaccine candidate live attenuated
West Nile viruses that are avirulent and immunogenic in mouse and
hamster models for West Nile neuroinvasiveness. (2) Use of mouse and
hamster models to evaluate safety of live, attenuated West Nile virus
vaccines.

**Background:** There are currently no licensed vaccines to prevent West
Nile encephalitis in humans. Evidence arising from projects to develop
vaccines to prevent other diseases caused by flaviviruses suggests that
a live virus vaccine is to be preferred, because such vaccines have the
potential to afford lifelong protection after a single dose. Two live
attenuated West Nile vaccines are under development. Both of these
candidate viruses are genetic chimeras consisting of the West Nile virus
surface antigen genes (pre-membrane \[prM\] and envelope \[E\]) inserted
into the genetic "backbone" of known attenuated flavivirus genomes
(yellow fever \[YF\] strain 17D and dengue serotype 4 \[DEN4\],
respectively) from which the native prM and E gene segments have been
deleted. Thus these vaccine viruses expose the vaccinee to only two of
at least ten West Nile genes and to none of the seven WN-specific
non-structural (NS) gene products. While preliminary results arising
from testing of these two chimeric vaccines in animals are encouraging,
there are other data which suggest that exposure to the WN NS proteins
may be essential for eliciting a durable protective WN-specific immune
response in humans. Thus there is the possibiity that YF17D or DEN4
chimeric WN vaccines will not prove satisfactory. For this reason, we
have embarked on a program to develop candidate live, attenuated WN
viruses that deliver all of the WN-specific gene products to the immune
system of the recipient. To achieve this goal, we sought to attenuate a
laboratory strain of WNV. We first targeted the flavivirus-conserved
stem-and-loop secondary structure that is present at the 3'terminus of
the WN genome (the "3'SL"), by site-directed mutagenesis of a WNV
infectious DNA. The rationale behind this approach was based on our
earlier work to develop live, attenuated dengue virus vaccines. In those
efforts, we were successful in generating a candidate 3'SL-mutant DEN1
vaccine virus, DEN1mutF virus, that is attenuated and highly immunogenic
in monkeys. DEN1mutF virus is currently the subject of an NIH patent
application. Our studies of the dengue 3'SL also revealed that large
portions of the dengue-specific nucleotide sequence in this domain could
be replaced by analogous nucleotides of the WN 3'SL without major
alteration in viability of the resulting mutant viruses, provided the
3'SL secondary structure was not disrupted. Thus the work with dengue
suggested that mutations in the 3'SL of any flavivirus genome could
result in attenuation of virulence without a lethal effect on virus
replication.

**Study Design:** The availability of mouse and hamster models for
neurotropism (neuroinvasiveness and neurovirulence) forms a basis for
evaluation and selection of candidate WN vaccine viruses for eventual
further studies in monkeys and humans. In the mouse assay, neonatal
outbreds are infected by the intraperitoneal (IP) route with graded
doses of WN virus. Thus the assay measures the ability of the virus to
pass the blood-brain barrier and then to cause a fatal or disabling
encephalitis. Either endpoint typically occurs by day 8 post-infection.
Immunogenicity is measured by determining the titer of WN neutralizing
antibodies in serum of mice that survive virus challenge, on day 30. The
prototype WN virus, strain NY/99, has an MLD50 of about 0.1 to 0.5 pfu
(plaque-forming unit) in this assay. In the hamster assay, adult animals
are infected by the IP route, also, but the NY/99 strain has a much
higher LD50 of 10E4 pfu (Tesh et al. 2002. Emerg. Inf. Dis.
8:1392-1397). Advantages of the hamster model are that the animals can
be immunized by the intramuscular route and then challenged IP and that
animals will survive challenge with low doses of strain NY/99, making it
possible to assess antibody responses after challenge. Moreover,
hamsters do not vary in their sensitivity to WN infection according to
age, as mice do. This latter property of the mouse model makes it
difficult to do challenge studies that can be done in hamsters. LVVD has
available the use of the BL3 Animal Care facility at WRAIR, under the
auspices of Dr. Robert Putnak, for the purposes of the proposed new
investigation. (See attached letter.)

We have already identified four 3'SL mutant WN viruses that are
attenuated in the mouse model system, mutants C2, A6, E+, and A2R. These
mutants were selected for further study after initial screening of about
ten 3'SL mutant viral RNAs, because of the following unique properties:
(i) Mutant C2 displayed a 100-fold *enhanced* replication efficiency in
mosquito cells compared to wt parent virus but grew normally in monkey
kidney cells. (ii) Mutant A6 was retarded in replication in monkey
kidney cells and in mosquito cells and reached 100-fold lower peak
titers in these two cell types, compared to wt parent virus. (iii)
Mutant E+ was similar to A6 in that replication in the two cell types
was retarded at early times after infection, but E+ virus attained peak
titers similar to wt parent virus at late times after infection. (iv)
Mutant A2R virus was completely unable to replicate in mosquito cells
but replicated normally in monkey kidney cells. That is, mutant A2R
virus was a host-range restricted mutant, like our candidate dengue
vaccine, DEN1mutF virus. Preliminary evidence suggests that all of these
candidate viruses have an MLD50 [\>]{.underline}10E4 pfu in mice, and we
have not yet determined whether one or more of these mutants is superior
to the others as a basis for further development. Therefore, step one of
this project will be to continue the study of mutants C2, A6, E+, and
A2R in mice until the MLD50 for each of these viruses is defined with
greater certainty. These results will form the basis for a decision
whether to proceed further with each particular 3'SL mutant virus.
However, we anticipate that all of these viruses will require further
attenuation for serious consideration as candidate live vaccines
suitable for further development. Eventually, very attenuated single or
double mutant WN viruses (see below) will be evaluated in the hamster
model for their ability to protect hamsters against wt WN virus
challenge.

To enhance the attenuation of 3'SL mutant viruses in the mouse (and
hamster) models for WN neurotropism, we plan to introduce "second-site"
mutations targeted to the E gene and/or to domains in the NS2B and NS3
genes known to encode the virus protease activity into 3'SL mutant viral
RNAs. Starting materials for this project will consist of infectious
DNAs encoding the relevant genomes. In all cases, these DNAs have been
prepared by Dr. Li Yu in LVVD and are available in quantity. The general
method for introducing mutations in cloned infectious DNA involves: (1)
Generation of the desired mutation in the context of a PCR product
containing WN nt sequences and overlapping by at least 50 nucleotides
the locations of unique restriction endonuclease (RE) sites in the WN
genome. (2) Cleavage of the WN genome DNA and of the mutant PCR product
by the relevant REs. (3) Insertion of the mutant PCR product into the
cleaved WN DNA by ligation and cloning in bacteria. On occasions when
the WN mutant DNA is unstable in bacteria, the mutant PCR product can be
inserted into cleaved linear WN DNA cloned in a yeast shuttle vector
(pRS424) by co-transformation of yeast, which results in homologous
recombination of the two linear DNAs, yielding stable circular yeast
plasmid DNA. Double- or triple-mutant WN DNAs will be used to generate
RNAs by in vitro transcription, and the resulting RNAs will be used to
transfect both vero and BHK cells by electroporation under conditions
already worked out. Transfected cell monolayers will be followed for
infectivity of RNA by an indirect immune fluorescence assay.

[New mutations in the E gene]{.underline}. These will be based on an
existing live, attenuated Japanese encephalitis (JE) virus vaccine,
strain SA14-14-2. WN and JE viruses are highly related antigenically and
genetically. The E gene sequence of the SA14-14-2 vaccine is known to
differ from that of its virulent parent JE virus, strain SA14, at five
amino acid positions. The WN E gene sequence has complete homology to
the JE strain SA14 E gene sequence in the particular domain affected.
Therefore by analogy we anticipate that introduction into the WN E gene
sequence of mutations that differentiate the JE strain SA14-14-2 E gene
from that of JE strain SA14 will also have an attenuating effect on WN
virus. Actually, this strategy has already been used successfully by
Acambis to enhance the attenuated phenotype of their YF/WN candidate
vaccine (data are in the public domain, published in Journal of
Virology).

[New mutations in NS2B/NS3]{.underline}. Dr. Barry Falgout in LVVD has
previously identified a set of mutations in the viral protease, encoded
within the NS2B and NS3 gene segments, that alter protease activity by
an in vitro study, and he has generated mutant dengue viruses containing
some of these mutations. Thus we already possess data to indicate which
specific mutations of the protease will alter virus replication
efficiency without completely abrogating it. One of these candidate
mutations will be selected for introduction into WN 3'SL mutant viruses
in order to enhance their level of attenuation in the mouse model.

[DNA and RNA sequencing]{.underline}. Novel DNAs will be sequenced to
verify that desired mutations are present and that unwanted occult
mutations are absent. RNAs derived from mutant viruses that replicate
after transfection will be sequenced to prove that mutations introduced
in DNA are present without spontaneous alteration in viral RNA.