DNA vaccines encoding the envelope protein of West Nile virus lineages 1 or 2 administered intramuscularly, via electroporation and with recombinant virus protein induce partial protection in large falcons (Falco spp.)

The efficacy of two different WNV DNA vaccines in falcons was determined by a) assessing
their tolerance, safety and potency to elicit specific antibody responses and b) by
challenging the vaccinated and control birds with a semi-lethal WNV NY99 dose.

Vaccine tolerance and safety

Following vaccination and sampling, behavior, food uptake, movement or general health
of the falcons were not affected at any time. The body weight of the vaccinated and
control animals varied only slightly between +3.5% and ?6.6%. In group B, two birds
(F32, F33) developed an edematous/gelatinous texture of pectoral muscle 1 wpv. One
falcon in group D demonstrated 0.4 cm?×?0.5 cm sized, yellowish, dense skin texture
at the vaccination site 1 and 2 weeks after boost vaccination (F56, 5-6 wpv) with
recombinant WNV protein. The vaccination sites of all other birds were macroscopically
unsuspicious. In none of the oral and fecal swabs from the falcons was WNV plasmid
DNA detected at any time.

Vaccination induced antibody responses

Antibody responses are shown in Figures 2 and 3. All control birds (group E) remained negative by ELISA and VNT. In group A, one
bird (F25) was ELISA-positive from 5 wpv onwards. At the same time, two other falcons
(F29, F30) had low antibody titers of 1:10 by VNT, which lasted for 3 weeks in one
bird. All other individuals of this group remained negative in both tests throughout
the complete trial.

Figure 2. Antibodies detected by ID Screen© WN competition ELISA during the vaccination period
in falcons. Antibodies of all groups detected by the ID Screen© WN competition ELISA (IDVet,
Grabels, France) during the vaccination period are shown. Optical density at 450 nm
is converted to signal/noise% (S/N%) ratio (S/N%?=?ODsample/ODnegative control * 100),
with values ?40% considered positive, 40% and ?50% being equivocal and 50% negative.
The threshold for positive results is indicated as a solid red line. In box-and-whisker
plots, the ends of the whiskers represent the minimum and maximum values, respectively.
Outliers are represented as black dots instead of whisker-ends and the box includes
50% of the values for each group. Vaccination groups with a statistically significant
difference in development of antibody production detected by ELISA and micro-virus
neutralization test in comparison to group E are labeled with an asterisk (Fisher’s
exact test, p-values???-levels of 0.05).

Figure 3. Antibodies detected by the micro-virus neutralization test (VNT) against homologous
challenge WNV. Antibody titers of all groups determined by VNT against the homologous challenge
virus (neutralization titer) during the vaccination period are shown. In box-and-whisker
plots, the ends of the whiskers represent the minimum and maximum values, respectively.
Outliers are represented as black dots instead of whisker-ends and the box includes
50% of the values for each group. Vaccination groups with statistical significant
difference in development of antibody production detected by ELISA and micro-virus
neutralization test in comparison to group E are labeled with an asterisk (Fisher’s
exact test, p-values???-levels of 0.05).

In group B, one falcon (F31) was ELISA-positive 4 wpv and another falcon (F33) had
a low antibody titer of 1:20 5 wpv by VNT. Both individuals were negative at all other
time points examined and all of the other birds of this group remained negative throughout
the entire vaccination period.

Three falcons in group C (F49, F52, F53) were positive by ELISA from 4 wpv onwards.
One of these (F52) and one other falcon (F50) had antibody titers of 1:15 and 1:10
by VNT, respectively, lasting for 1-2 weeks.

None of the birds in group D were positive by ELISA and only two birds (F57, F58)
had an antibody titer of 1:15 by VNT 6 wpv.

In groups A and C, but not in groups B and D, Fisher’s exact test demonstrated a significantly
increased presence of WNV antibodies compared to group E.

Clinical signs following WNV NY99 challenge

Details on the clinical scores and development of body weights are provided in Figure 4. In the control group E, two of the eight control birds (F55, F27) had to be euthanized
3 dpi and 14 dpi, respectively, due to severe central nervous signs (clinical score
3) such as seizures, torticollis, ataxia and an inability to stand. Two other birds
(F42, F51) were found dead on 5 dpi and 8 dpi, respectively, after showing greenish
discoloration of urates, enophthalmos, inappetence and ruffled feathers on the previous
day. Similar symptoms (clinical scores 2-3) were observed in the four other control
birds between 3 dpi and 11 dpi temporarily, but clinical signs ceased afterwards.
All control birds dropped their weight from 1.56% to 27.01% (median: 5.75%).

Four falcons of group A had clinical scores equal to or above 2 and two falcons (F26,
F29) died on 4 dpi and 8 dpi, respectively. Body weight dropped slightly in the three
survivor birds (0.7-7.1%) and decreased rapidly and severely in the two deceased falcons
(21.9% and 10.5%, respectively). Median weight loss of all birds in group A was 6.8%.

In group B, two falcons (F31, F33) had clinical scores of 2 on one day and one falcon
(F35) had clinical scores of 2 and 3 over several days. No bird died and weight losses
ranged from 0.76% to 10.9% (median: 9.6%).

One falcon (F50) of group C had a clinical score of 2 on one day (7 dpi) and another
bird (F54) had this score after ten days. However, clinical score in the latter increased
to 3 for one day. No falcon died and the weight loss in four birds ranged from 9.1%
to 16% (median: 11.6%), whereas in one bird (F54), it was up to 27.1%.

In group D, one falcon (F60) had a clinical score of 2 and one falcon (F59) had a
clinical score of 3. The latter animal was euthanized on 18 dpi because of ataxia,
hypermetria and weight loss of 30.9%. The other birds in this group dropped weight
from 1.5% to 12% (median: 8.7%).

In groups B, C and D, but not in group A, significantly reduced clinical scores were
demonstrated by Wilcoxon rank sum test compared to the control group E (p-values 0.01154, 0.02391, 0.04592; for group A 0.1703).

Virus shedding, viremia and antibody response after challenge infection

Oral and cloacal virus shedding data are shown in Figures 5 and 6, virus genome detection in the blood in Figure 7 and virus re-isolation data from blood in Figure 8. Serology results are depicted in Figures 9 and 10.

Figure 4. Clinical score: following criteria were classified daily: general condition, posture,
plumage, behavior, excrements, neurological status, hydration status, respiration
and food uptake (alterations: +++?=?3 points, ++?=?2 points, +?=?1 point, +/ -?=?0.5
points and -?=?0 points). Points were summed up and total points of one day were classified in a clinical score
system: 0–0.5 points?=?0?=?healthy; 1–4.5 points?=?1?=?mild affection; 5–10.5 points?=?2?=?moderate
affection; 11 points and more?=?3?=?severe affection; 4?=?dead. Weight presents the
change of body weight (??=?decrease, +?=?increase) in percent as comparison of the
first and the last measurement of each falcon during the trial. Vaccination groups
with statistical significant difference in sum of clinical scores in comparison to
group E are labelled with an asterisk (one-sided Wilcoxon rank sum test with continuity
correction, p-values???-levels of 0.05). Abbreviation: dpi (days post infection).

Figure 5. Oral virus shedding of falcons during WNV lineage 1 challenge. The oral virus shedding of falcons during challenge is displayed. Cycle threshold
values (Ct values) of swabs for all groups are shown. In box-and-whisker plots the
ends of the whiskers represent the minimum and maximum values, respectively. Outliers
are represented as black dots instead of whisker-ends. This includes 50% of the values
of each group, with the median value of each group being represented by a line in
the middle. Vaccination groups with statistical significant difference in the level
of oral viral genome shedding in comparison to group E are labeled with an asterisk
(ANOVA over all sampling days, p-values???-levels of 0.05). There was no group with a statistically significant difference
in duration of oral viral genome shedding (one-sided Wilcoxon rank sum test with continuity
correction, cut-off Ct value 35.0, p-values???-levels of 0.05).

Figure 6. Cloacal virus shedding of falcons during WNV lineage 1 challenge. The cloacal virus shedding of falcons during challenge is displayed. Cycle threshold
values (Ct values) of swabs for all groups are shown. In box-and-whisker plots the
ends of the whiskers represent the minimum and maximum values, respectively. Outliers
are represented as black dots instead of whisker-ends. The box includes 50% of the
values of each group and median value of each group is represented by a line in the
middle. Vaccination groups with a statistically significant difference in the level
of cloacal viral genome shedding in comparison to group E are labeled with an asterisk
(ANOVA over all sampling days, p-values???-levels of 0.05). There was no group with a statistically significant difference
in duration of cloacal viral genome shedding (one-sided Wilcoxon rank sum test with
continuity correction, cut-off Ct value 35.0, p-values???-levels of 0.05).

Figure 7. Viremia of falcons during WNV lineage 1 challenge detected by qRT-PCR. The cycle threshold values (Ct values) of whole blood for all groups during the challenge
period are displayed. In box-and-whisker plots the ends of the whiskers represent
the minimum and maximum values, respectively. Outliers are represented as black dots
instead of whisker-ends. The box includes 50% of the values of each group and median
viremia value of each group is represented by a line in the middle. Vaccination groups
with a statistically significant difference in the level of viremia in comparison
to group E are labeled with an asterisk (ANOVA over all sampling days, p-values???-levels of 0.05). There was no group with a statistically significant difference
in duration of viremia (one-sided Wilcoxon rank sum test with continuity correction,
cut-off Ct value 35.0, p-values???-levels of 0.05).

Figure 8. Viremia of falcons during WNV lineage 1 challenge detected by virus titration. The results of blood titration for all groups during the challenge period are given
in log10 tissue culture infection dose 50 per mL (TCID50/mL) whole blood. In box-and-whisker
plots the ends of the whiskers represent the minimum and maximum values, respectively.
Outliers are represented as black dots instead of whisker-ends. The box includes 50%
of the values of each group and the median viremia value of each group is represented
by a line in the middle.

Figure 9. Antibodies detected by ELISA during challenge in falcons. Antibodies of all groups detected by the ID Screen© WN competition ELISA (IDVet,
Grabels, France) during the challenge period are shown. Optical density at 450 nm
is converted to signal/noise% (S/N%) ratio (S/N%?=?ODsample/ODnegative control * 100),
with values???40% considered positive, 40% and ?50% equivocal and 50% negative.
The threshold for positive results is indicated as a solid red line. In box-and-whisker
plots, the ends of the whiskers represent the minimum and maximum values, respectively.
Outliers are represented as black dots instead of whisker-ends and the box includes
50% of the values for each group.

Figure 10. Antibodies detected by micro-virus neutralization test (VNT) during challenge in falcons. Antibody titers of all groups determined by VNT against homologous challenge virus
(neutralization titer) during the challenge period are shown. In box-and-whisker plots
the ends of the whiskers represent the minimum and maximum values, respectively. Outliers
are represented as black dots instead of whisker-ends. The box includes 50% of the
values of each group and median value of each group is represented by a line in the
middle.

All birds of the challenge control group (group E) shed WNV orally and in their feces
from 3 dpi onwards, with Ct values varying from 17.48 to 34.75. However, in the four
surviving control birds (F13, F24, F36, F43), viral genomes were sometimes not detected
(F36 at 20 dpi and F13, F24, F43 after 8-14 dpi). Viral genomes were detected in the
blood of all falcons from 3 dpi onwards, with Ct values ranging from 19.94 to 34.78.
Viremia ceased in most of the survivors on 6-14 dpi (all except F36), while it persisted
in the fatally affected falcons until death. Virus was successfully re-isolated and
titrated from blood taken on 3 and 6 dpi (yielding titers in the range of 10
^1.75
-10
^6.5
TCID 50/mL respectively). First, WNV-specific neutralizing antibodies were detected
on 3 dpi (F36, F55) or 6 dpi (F13, F24, F27, F43), respectively, and ranged from 1:10
to 1:80. However, two of the eight falcons which did not survive the WNV challenge
did not develop a neutralizing antibody response (F42, F51). In the survivors (F13,
F24, F36, F43), the antibody titers increased to a maximum of 1:1280 at the end of
the observation period.

In oral and cloacal swabs and in blood taken from birds of group A, viral genomes
were only detectable between 3 dpi and 8 dpi. A solitary positive result in one bird
(F28, Ct value of 32.82 at 14 dpi), which was negative by qRT-PCR at all other time
points, may be due to an artifact. Virus re-isolations from blood were successful
in all birds on 3 dpi (10
^1.75
-10
⁵
TCID 50/mL) and in two birds (F25, F26) also from samples taken at 6 dpi (10
^2.5
and 10
^3.5
TCID50/mL). Antibody titers from 1:15 to 1:320 were detectable in one bird (F30) from
3 dpi and in three birds from 6 dpi onwards, increasing to a maximum of 1:5120 during
the trial. However, in the two birds from this group which eventually died, antibody
titers remained low (1:40) after challenge (F26) or disappeared (F29) after being
low (1:10) before challenge.

In group B, animal viral genomes were detected in pharyngeal swabs up to 10 dpi and
in cloacal swabs of two birds (F34, F35) even until 19-21 dpi. Birds were viremic
on 3 and 6 dpi, but not at later time points. Virus titers in blood on 3 dpi were
10
^1.75
-10
³
TCID50/mL. Initial antibody titers on 6 dpi ranged from 1:60 to 1:640 and rose to
maximum titers of 1:2560 at the end of the study.

In group C, viral genomes were detected in oral and pharyngeal swabs and in blood
up to 14 dpi. One bird (F54) shed virus orally until 21 dpi. Blood Ct values were
in the range of 21.40 to 34.5. Virus re-isolations were successful from blood samples
taken from three birds on 3 dpi (titers 10
^1.75
-10
^2.75
TCID50/mL), but not from blood samples taken at later time points. Antibody titers
were detected in two falcons (F52 and F53) already by 3 dpi and in all falcons on
6 dpi. One of these falcons (F52) carried already antibodies on 0 dpi as a result
of the vaccination. Initially ranging from 1:10 to 1:480, antibody titers increased
to maximum titers 1:7680 and lasted until to the end of the study.

In group D, viral genomes were detected in pharyngeal swabs beginning at 3 dpi until
the end of the observation period and until 14 dpi in cloacal swabs and blood. Virus
re-isolations were successful in two birds (F60, F59) on 3 dpi with 10
^1.83
TCID50/mL and 10
^2.75
TCID50/mL, respectively, and additionally in one of these birds on 6 dpi (10
²
TCID50/mL). Antibody titers in four birds ranged from 1:10 to 1:20 on 3 dpi; however,
antibodies were present on 0 dpi in two of these birds (F57, F58). The titers increased
in all birds, up to maximum levels of 1:3840. One bird (F59) with a low neutralizing
antibody titer on 6 dpi (1:20), but without a strong humoral response (maximum titers
of 1:320), showed neurological signs and apathy and was eventually euthanized on 18
dpi.

ANOVA demonstrated that oral (p values groups A-D: 0.0006594, 0.000002827, 0.00006856, 0.000122) and cloacal (p values groups A, B, D: 0.0005033, 0.0002035, 0.03737; for group C 0.06771) virus
shedding and virus detection in blood (p values groups A-D: 0.001057, 0.000000268, 0.0006269, 0.0005634) were significantly
reduced in all groups compared to the control group. However, the duration of oral
shedding (p values groups A-D: 0.1013, 0.2041, 0.4702, 0.3014) and viremia (p values groups A-D: 0.1566, 0.09665, 0.4108, 0.4108) was not significantly different,
as determined by the Wilcoxon rank sum test.

Post mortem examination, histopathology and immunohistochemistry

A summary of the pathological and immunohistochemical findings is provided in Table 1.

Table 1. Histopathological (HE) and immunohistochemical (IHC) results from vaccinated falcons
and non-vaccinated controls infected with WNV lineage 1 NY’99

The gross-pathology, histopathology and IHC of the non-vaccinated positive controls
(group E) were previously described 51]. Briefly, the most consistent finding associated with WNV infection was a non-suppurative
(meningo)-encephalitis (4/8) and an acute or subacute, lymphohistiocytic, necrotizing
myocarditis (5/8). A moderate, non-suppurative, necrotizing arteritis was seen in
the spleen of one bird only (F24). Virus antigens were detected by IHC in brains,
hearts and spleens and at injection sites of the fatally diseased animals (4/8), whereas
challenge survivors displayed only scant (2/8) or no (2/8) WNV antigen staining.

One falcon (F29) of group A died 4 dpi and showed a severe, acute, hemorrhagic enteritis
with positive antigen staining at the injection site, but without other signs typically
associated with WNV infections. In all other birds (4/5) a widely distributed, moderate,
acute, non-suppurative (meningo)-encephalitis was diagnosed. Additional findings included
an acute to subacute, mild, necrotizing myocarditis (2/5), an acute, mild, necrotizing
arteritis in the kidney (2/5), a necrosis of the splenic periarteriolar lymphoid sheaths
(PALS) of different degrees with fibrin deposition (4/5), a mild lymphoplasmacellular
interstitial nephritis (1/5) and a mild lymphohistiocytic neuritis with axonal degeneration
(2/5). The vaccination sites were not altered in any bird of this group. By IHC, scattered
antigen was detected at the virus injection sites in three birds (3/5) and in the
brains of two birds (2/5) (Figure 11), with one of these (F 26) having antigen in all regions of the brain and additionally
in the heart.

Figure 11. Immunohistochemistry of the cerebrum of vaccinated falcons. Single cell necrosis and mild glial reaction with distinct amounts of viral antigen
within neurons and glial cells are demonstrated in the cerebra of two falcons. (A) Falcon in group D, 18 dpi, mab 15R4, bar 50 ?m. (B) Falcon in group A,19 dpi, mab 15R4, bar 20 ?m.

One falcon in group B (F35) demonstrated only a slight lymphohistiocytic pancreatitis.
All other animals (4/5) demonstrated a mild to moderate, widely distributed, acute,
non-suppurative meningoencephalitis. Additionally, a mild, acute, necrotizing myocarditis
(2/5), a mild, necrotizing arteritis in the gut connective tissue (1/5), a mild lymphohistiocytic
hepatitis (1/5) and neuritis (1/5) were seen. The vaccination site of one animal (1/5)
displayed a mild lymphohistiocytic myositis. No WNV antigen was detected by IHC in
any bird of this group.

Four birds (4/5) of group C had acute, non-suppurative encephalitis of different degrees,
varying from slight lesions (1/4) in the cerebellum/cerebrum to moderate and severe
inflammation of all brain areas (1/4) and the meninges (2/4). Additional findings
included an acute necrotizing myocarditis (2/5), a mild to moderate, acute, necrotizing
arteritis (2/5), a mild lymphohistiocytic neuritis (3/5) and nephritis (1/5). At the
vaccination sites, follicular aggregated infiltrations of mononuclear cells with mild
lymphocytolysis (2/5) and an acute lymphohistiocytic myositis (1/5) were seen. IHC
revealed a mild staining reaction in the spleen or brain and heart, respectively,
of two falcons (2/5) (Figure 12), whereas all other birds remained negative.

Figure 12. Immunohistochemistry of the spleen of a vaccinated falcon. The spleen of a falcon in group C, 19 dpi is displayed. Antigen was detected in single
mononuclear cells in follicles, mab 15R4, bar 50 ?m.

An acute, non-suppurative meningoencephalitis of varying degrees was observed in all
birds from group D (5/5). Additional findings included an acute to subacute, mild
to moderate, necrotizing myocarditis (3/5), a mild, acute, necrotizing arteritis (1/5),
and a mild, acute to subacute, non-suppurative neuritis (4/5) and nephritis (1/5).
The vaccination sites had infiltrations of follicular aggregated mononuclear cells
in the connective tissues (1/5) and a granulomatous dermatitis (2/5). In IHC, one
bird (F59, Figures 11 and 13) was severely affected in cerebrum (15% positive tissue) and mildly affected in cerebellum
and diencephalon. However, IHC results in the spleen and brain of two falcons (F58,
F60) were inconclusive.

Figure 13. Immunohistochemistry of the telencephalon of a vaccinated falcon. The telencephalon of a falcon in group D, 18 dpi, is displayed. Regions close to
ventricles, most notably the area parahippocampalis, were severely affected, mab 15R4,
bar 100 ?m.

In general, the occurrence of scattered antigen in neurons, glial cells and cell processes
of the brain, in cardiocytes and in single monocytes and fibrocytes at the injection
site of vaccinated falcons were similar to findings in the control birds.

Virus loads in organs

The results of qRT-PCR and virology from selected organ samples are summarized in
Table 2. In the organs of all falcons of group E viral genomes were detected with lowest
Ct values of 16.7 (equivalent to 402?895 copies/mg). All organs of the four fatally
affected birds (F27, F42, F51, F55) were qRT-PCR positive, as were 2-5 organs of the
four challenge survivors (F13, F24, F36, F43). Accordingly, virus re-isolations were
possible from organs of the four deceased birds in most cases (10
^1.75
-10
^4.75
TCID50/100 ?L). In the four survivors virus isolations were successful only from the
brain samples of two birds (F13, F43).

Table 2. Viral load of organs in Ct values, copies per mg organ and tissue culture infectious
dose 50 (TCID50) per mg organ

In all birds of group A, viral genomes were detected in the brain and also in the
kidneys in four birds. Heart, spleen, liver and lung were negative in the three survivors,
but positive in two falcons (F26, F29) which died during the study. In the latter
two falcons, viruses could be isolated from all organs (except from heart in F29),
but only from brain (10
^0.75
-10
^5.75
KID50/100 ?L) in two other falcons (F25, F30) and from none of the organs in one falcon
(F28).

Brains of three falcons (F31, F32, F34) belonging to group B carried viral genomes
and the kidney of one falcon (F32) was also qRT-PCR-positive. None of the other birds
and none of the organs were positive by qRT-PCR and virus re-isolation failed in all
birds.

In group C, qRT-PCR was positive in three birds (F49, F50, F54) in the brain, but
not in other organs. Viral genomes were not detected in any other bird and the virology
was negative in all birds for all organs.

Viral genomes were detected in the brain in all birds in group D, in one falcon (F57)
additionally in the kidneys and in another falcon (F59) additionally in all other
organs. In the latter falcon, which had to be euthanized during the trial, virus re-isolation
was successful from the brain with a result of 10
^2.75
TCID50/100 ?L, whereas it failed in all other birds of this group in all organs.

Wilcoxon rank sum test revealed that the viral genome detections was significantly
reduced in all organs of group B animals (p values of brain, spleen, kidney and heart: 0.01767, 0.02228, 0.009749, 0.008443),
in the spleen, kidney and heart of group C animals (p values 0.03582, 0.005709, 0.01536; for brain 0.1286), and in the kidneys of group
D animals (p value 0.04762), but not in the spleen, heart and brain of group D birds (p values 0.1726, 0.1187, 0.7262). No significant reduction of viral genome detection
in organs was present in group A (p values of brain, spleen, kidney, and heart: 0.5476, 0.6532, 0.5476, and 0.5, respectively).