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RESEARCH ARTICLE
INFECTIOUS DISEASE
Efficacy of a virus-vectored vaccine against human and

bovine respiratory syncytial virus infections
Human respiratory syncytial virus (HRSV) is a major cause of lower respiratory tract disease in children and the elderly for
which there is still no effective vaccine. We have previously shown that PanAd3-RSV, which is a chimpanzee adenovirus
vectored vaccine candidate that expresses a secreted form of the HRSV F protein together with the N and M2-1 proteins of
HRSV, is immunogenic in rodents and nonhuman primates, and protects mice and cotton rats from HRSV challenge.
Because the extent to which protection demonstrated in rodent models will translate to humans is unclear, we have
exploited the calf model of bovine RSV (BRSV) infection, which mimics HRSV disease in children more closely than do
experimental models of unnatural laboratory hosts, to evaluate the safety and efficacy of the PanAd3-RSV vaccine. We
show that PanAd3-RSV alone and in combination with a modified vaccinia Ankara expressing the same HRSV antigens
(MVA-RSV) induced neutralizing antibodies and cellular immunity in young seronegative calves and protected against
upper and lower respiratory tract infection and pulmonary disease induced by heterologous BRSV challenge. There
was no evidence either of enhanced pulmonary pathology or of enhanced respiratory disease in vaccinated calves
after BRSV challenge. These findings support the continued evaluation of the vectored RSV vaccines in man.
INTRODUCTION
Human respiratory syncytial virus (HRSV) is a leading cause of bronchiolitis and severe respiratory disease in infants, young children, immunocompromised individuals, and the elderly throughout the world,
resulting in up to 200,000 deaths per year in children under the age of
5 years worldwide and ~10,000 deaths in adults >65 years of age in the
United States each year (1, 2). The major barriers to vaccine development are the young age of the main target population and the history
of vaccine-exacerbated disease in infants who received a formalininactivated HRSV (FI-HRSV) vaccine, which failed to protect against
infection and primed for enhanced respiratory disease (ERD) after
natural HRSV infection (3, 4). Several HRSV vaccine candidates based
on live attenuated virus, purified proteins, or recombinant vectors
expressing HRSV proteins have shown efficacy in preclinical studies,
but few have progressed to clinical trials or they have had only limited
success, and none have progressed to licensure (5). One promising vaccine approach that has shown efficacy in mice and cotton rats is based
on replication-defective adenovirus (Ad) vectors. Human and chimpanzee Ad-vectored vaccines have been shown to be safe in humans
and induce potent immune responses (6). A single intranasal (IN) or
intramuscular (IM) dose of human Ad serotype 5 (Ad5) or a nonhuman
primate Ad expressing the HRSV F protein, which is relatively well conserved between different HRSV subtype A and B strains, induces protective immunity against HRSV in mice and cotton rats without
enhancing lung pathology (79). However, mice and cotton rats are only
semipermissive for HRSV replication, and the pathogenesis of HRSV
infection in these small-animal models does not fully recapitulate that
in infants. The failure of small-animal models to predict HRSV vaccine
1
The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK. 2ReiThera Srl,
Rome 00144, Italy. 3Istituto Superiore di Sanit, Rome 00161, Italy. 4Keires AG, Basel CH-4051,
Switzerland. 5CEINGE, Naples 80131, Italy. 6Department of Molecular Medicine and Medical
Biotechnology, University Federico II, Naples 80138, Italy.
*Corresponding author. E-mail: geraldine.taylor@pirbright.ac.uk
Present address: Quintiles Ltd., Quintiles Drug Research Unit at Guys Hospital, 6 Newcomen
Street, London SE1 1YR, UK.
efficacy and safety in humans highlights the need for an animal model
that more closely resembles HRSV infection and disease in infants for
evaluation of HRSV vaccine candidates.
HRSV is antigenically and genetically closely related to bovine RSV
(BRSV), which is a major cause of respiratory disease in young calves,
and the epidemiology and pathogenesis of infection with these viruses
in their respective hosts are similar (10). The development of BRSV vaccines faces similar challenges to that of HRSV, such as the need to be
effective in very young individuals in the presence of maternally derived
serum antibodies (MDAs) and the history of ERD in vaccinated calves
undergoing a natural BRSV infection (11). Exacerbated respiratory disease, similar to that seen in FI-HRSVvaccinated infants, has also been
reproduced experimentally in FI-BRSVvaccinated calves (12, 13).
These similarities between HRSV and BRSV infections suggest that
the calf may be a valuable preclinical animal model to evaluate the safety
and efficacy of vaccines containing HRSV proteins with a high degree of
homology to BRSV.
We have generated a replication-incompetent, chimpanzee-derived
AdV (ChAd)vectored vaccine encoding a secreted form of the HRSV
F, together with N and M2-1 proteins (PanAd3-RSV), to induce neutralizing antibodies and prime a T helper 1 (TH1)biased immune response and showed that it was immunogenic and protected mice and
cotton rats against HRSV challenge, without exacerbating pulmonary
pathology (14). Because studies with other virus-vectored vaccines have
shown that heterologous prime/boost with ChAd and modified vaccinia
Ankara (MVA) is more immunogenic than homologous prime/boost
regimens in phase 1 and 2a human clinical trials (15, 16), we constructed
an MVA encoding the same HRSV antigen, consisting of secreted F, N,
and M2-1 (MVA-RSV), and showed that in nonhuman primates, it
could effectively boost humoral and cellular responses primed by PanAd3RSV, and induce mucosal antibodies when the priming dose was
delivered IN (14). However, a pediatric vaccine based on heterologous
prime/boost can pose serious challenges to clinical development in
terms of large-scale manufacturing and, most importantly, safety. The
safety of each individual product should be carefully assessed in case of a
www.ScienceTranslationalMedicine.org
12 August 2015
Vol 7 Issue 300 300ra127
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Geraldine Taylor,1* Michelle Thom,1 Stefania Capone,2 Angiolo Pierantoni,2 Efrain Guzman,1
Rebecca Herbert,1 Elisa Scarselli,2 Federico Napolitano,2 Alessandro Giuliani,3
Antonella Folgori,2 Stefano Colloca,2 Riccardo Cortese,4 Alfredo Nicosia,2,5,6 Alessandra Vitelli2
RESEARCH ARTICLE
RESULTS
receiving FI-HRSV, developed neutralizing antibody (nAb) titers well

above the protective threshold of 1:100 (log2 6.64) described for this
model (Fig. 1A) (17), and all animals were fully protected against HRSV
replication in the lung (Fig. 1B and table S1). As seen previously (14),
only animals primed by the IN route were completely protected against
viral replication in the nose. Although IM vaccination in either homologous or heterologous prime/boost regimens significantly reduced viral
replication in the URT (more than two logs), they did not completely
abrogate it (Fig. 1B). All regimens were safe and did not prime for
enhanced pulmonary pathology after HRSV challenge (Fig. 1C). Thus, although there was a low score for alveolitis (A = 3.4%) and interstitial pneumonia (IP = 4%) in animals vaccinated with PanAd3-RSV IN/MVA-RSV
IM, these scores were markedly less than those seen in animals vaccinated
with FI-HRSV (A = 35%, IP = 25%) after HRSV infection. Furthermore,
histopathology scores in animals vaccinated by other prime/boost regimens
were similar to those seen in unvaccinated, HRSV-infected controls.
Prime/boost vaccine regimens protect cotton rats against

HRSV without exacerbating pulmonary pathology
We previously showed that a single IN vaccination with PanAd3-RSV
protected cotton rats from HRSV infection in both the URT and LRT
for at least 3 months, without enhancement of pulmonary pathology
(14). However, homologous PanAd3-RSV prime/boost and heterologous PanAd3-RSV/MVA-RSV prime/boost elicited higher humoral
and cellular immunity than did a single PanAd3-RSV administration
in nonhuman primates (14). Therefore, before further development
of these regimens, we tested the safety and efficacy of different homologous and heterologous prime/boost vaccine vector combinations in
cotton rats after HRSV challenge.
Groups of cotton rats were primed IN or IM with 5 108 viral particles (vp) of PanAd3-RSV and boosted IM, 4 weeks later, with PanAd3RSV at the same dose or with 1 107 plaque-forming units (pfu) of
MVA-RSV. Animals were challenged IN with 1 105 pfu of HRSV,
3 weeks after boosting. Control groups included unvaccinated animals
and a group vaccinated with FI-HRSV as a control for the induction of
enhanced pulmonary pathology. All vaccinated animals, except those
Prime/boost vaccine regimens protect against BRSV

infection and lung pathology in young,
BRSV-seronegative calves
Six different PanAd3-RSV and MVA-RSV vaccine regimens were evaluated in groups (n = 3 to 5) of 2- to 4-week-old, BRSV-seronegative
calves in four different studies (Table 1). As controls, groups of three
to six calves were vaccinated with PanAd3 and MVA vectors encoding
unrelated antigens. Calves were challenged 4 weeks after vaccination by
combined IN and intratracheal (IT) administration of 104 pfu of BRSV,
Snook strain. Animals were euthanized 6 days after the challenge, when
virus replication peaks in the nose and lung, causing extensive pulmonary pathology.
In study 1, all control animals except one (indicated by * in Fig. 2A
and calf #104955 in table S2) showed high levels of viral replication in
both the LRT (Fig. 2A and table S2) and URT, similar to that seen in
previous studies in calves that were not vaccinated (Fig. 2B and fig.
S1) (18). In contrast, IN administration of PanAd3-RSV almost completely abolished BRSV replication in the lung (Fig. 2A) and resulted in
Fig. 1. Vaccine immunogenicity, efficacy, and safety in cotton rats.

Animals (n = 5 to 10) were primed with 5 108 vp of PanAd3-RSV, IN or
IM, boosted IM 4 weeks later with PanAd3-RSV at the same dose or with
1 107 pfu of MVA-RSV, and challenged IN 3 weeks later with 1 105
pfu of RSV/Long strain. FI-RSV: two doses IM. (A) RSV neutralizing titer in
vaccinated and control animals expressed as the serum dilution (log2)
reducing plaques by 60% compared with controls at study week 4 (gray
bars, post-prime) and week 7, at the time of challenge (black bars, post-
boost). (B) RSV titers in lung homogenates and nasal tissue collected
5 days after RSV challenge. Data are expressed as pfu/g. Dashed lines
show limits of detection of the two assays. (C) Histological analysis of
lung sections 5 days after RSV challenge. Four parameters were evaluated: peribronchiolitis (PB), perivasculitis (PV), interstitial pneumonia (IP),
and alveolitis (A). Slides were scored blind on a 0 to 4 severity scale, and
values were converted to a 0 to 100% histopathology score. Data in (A)
to (C) are shown as means + SD.
12 August 2015
misconducted vaccination course, such as missing prime or boost. In

addition, IN administration in babies could be inefficient due to frequent rhinitis or stuffy nose. Therefore, to provide support for the clinical development of the vectored vaccines, we evaluated the safety and
efficacy of different vaccination regimens and routes against HRSV in
cotton rats and against BRSV in young seronegative calves. All tested
vaccine regimens proved to be safe and protective in cotton rats. All
the tested regimens were safe and protected calves against heterologous
BRSV challenge to various degrees. In particular, we showed that
PanAd3-RSV delivered IN by a spray, followed by IM administration of
MVA-RSV, completely prevented upper (URT) and lower respiratory
tract (LRT) infection and pulmonary pathology.
reduced levels of viral replication in the nasopharynx (Fig. 2B and fig. S1).
Notably, calves vaccinated with PanAd3-RSV IN/MVA-RSV IM were
completely protected against BRSV replication in both the LRT and
URT (Fig. 2, A and B, fig. S1, and table S2). Macroscopic lung lesions were
observed in control animals, with the exception of one individual, which
showed only limited virus replication in the LRT. Although the number
of animals in this study does not allow for a statistical analysis of the data,
it is clear that a single IN dose of PanAd3-RSV strongly reduced the extent of macroscopic lung lesions, whereas the heterologous prime/boost
regimen completely prevented the formation of lung lesions (Fig. 2C).
As seen in infants hospitalized with HRSV infection, BRSV-infected
calves develop a massive infiltration of polymorphonuclear neutrophils
(PMNs) in the lungs as a result of viral replication in the LRT (19, 20).
After BRSV challenge, PMNs in bronchoalveolar lavage (BAL) from control calves ranged from 50 to 65%, and a similar proportion of PMNs
were seen in BAL from calves vaccinated with PanAd3-RSV IN alone,
suggesting that some lung viral replication had occurred. In contrast,
there were few (1 to 1.9%) PMNs in BAL from calves in the heterologous
IN/IM vaccine group, suggesting that vaccination had effectively prevented viral replication in the lungs (Fig. 2D, fig. S2, and table S2). None
of the vaccinated animals had evidence of pulmonary eosinophilia (fig.
S2), which is a feature of vaccine ERD in children and calves (3, 13). Microscopic lung lesions in control calves were characterized by a nonsuppurative exudative alveolitis and bronchiolitis (Fig. 3, A and B). About
50% of the microscope fields from control calves contained alveoli with
extensive inflammation, giving a score of 3, and 60% of the bronchioles
showed extensive peribronchiolar accumulations of mononuclear cells
and neutrophils and a bronchiolar exudate, with a score of 2 to 3 (Fig.
3, B and G, and table S4). In contrast, histopathological lesions in animals
vaccinated IN with PanAd3-RSV were essentially restricted to a few
bronchioles (Fig. 3, C and D) (<5% with a score of 2 to 3), and the extent
of alveolitis was reduced (<5% score of 2 to 3) (Fig. 3, G and H, and table S4).
Calves vaccinated with PanAd3-RSV IN/MVA-RSV IM did not show
any microscopic lung lesions (Fig. 3, E and F).
Because the peak incidence of severe HRSV disease occurs in infants
at 2 to 3 months of age, we next evaluated four prime/boost regimens in
which the interval between the two administrations was reduced to

4 weeks (studies 2 and 3 in Table 1). Although three of the four regimens
resulted in complete protection against BRSV replication in the LRT of
all calves (Fig. 2A and table S2), virus was detected in the LRT of three of
four calves vaccinated IN/IN with PanAd3-RSV (Fig. 2A). Once again, the
heterologous prime/boost regimen showed a higher degree of protective
efficacy in the URT, and virus was not detected at any time after challenge
(Fig. 2B and fig. S1). Although virus was isolated from the nasopharynx of
75% of calves vaccinated by homologous prime/boost regimens (Fig. 2B),
virus had been cleared from all but one of the calves by day 6 after infection (fig. S1). In contrast, high titers of virus were still detected in the
nasopharynx of control calves at this time (Fig. 2B and fig. S1).
Consistent with the virological data, none of the calves receiving the
heterologous prime/boost regimen displayed macroscopic lung lesions
(Fig. 2C and table S2), and macroscopic lesions were minimal in most
animals vaccinated with PanAd3-RSV IM/IM or IN/IM (Fig. 2C). In
contrast, three of four calves vaccinated with PanAd3-RSV IN/IN
had macroscopic lung lesions (Fig. 2C). The proportion of neutrophils
in BAL was reduced by >50% in calves vaccinated by the heterologous
prime/boost or the homologous PanAd3-RSV IN/IM regimens (Fig.
2D). However, there was a high proportion of neutrophils (46 to
61%) in BAL from all but one of the calves vaccinated with PanAd3RSV IM/IM, suggesting that transient virus replication had occurred
in the lungs of these animals (Fig. 2, A and D). The BAL from calves
vaccinated with PanAd3-RSV IN/IN also contained a high proportion
of neutrophils (30 to 49%), which was associated with infectious BRSV
in the lungs (Fig. 2D). Bacteria were not isolated from the BAL of any of
the vaccinated calves, indicating that they did not contribute to the pulmonary inflammatory response. Eosinophils were not detected in BAL
from any of the calves (fig. S2).
Inferential test based on the single measures highlighted some statistically significant results with Kruskal-Wallis test of one-way analysis
of variance (ANOVA). Post hoc analyses revealed that the regimens
showing a statistically significant difference with controls were as
follows: PanAd IN/MVA IM (P < 0.03) and PanAd IM/MVA IM (P <
0.01) for RSV in nasal secretions; PanAd IN/MVA IM (P < 0.01) and
Table 1. Design of calf studies. n.a., not available.

Study
1
3
4
Regimen*
No.
Prime study
day (week)
Boost study
day (week)
BRSV challenge study

day (week)
Sacrifice study
day (week)
Sham (controls)
d0
d56 (wk8)
d84 (wk12)
d90 (wk13)
PanAd3 IN
d56 (wk8)
n.a.
d84 (wk12)
d90 (wk13)
PanAd3 IN/MVA IM
d0
d56 (wk8)
d84 (wk12)
d90 (wk13)
Sham (controls)
d0
d28 (wk4)
d56 (wk8)
d62 (wk9)
PanAd3 IM/PanAd3 IM
d0
d28 (wk4)
d56 (wk8)
d62 (wk9)
PanAd3 IN/PanAd3 IM
d0
d28 (wk4)
d56 (wk8)
d62 (wk9)
PanAd3 IN/PanAd3 IN
d0
d28 (wk4)
d56 (wk8)
d62 (wk9)
Sham (controls)
d0
d28 (wk4)
d56 (wk8)
d62 (wk9)
PanAd3 IM/MVA IM
d0
d28 (wk4)
d56 (wk8)
d62 (wk9)
Sham (controls)
d0
d28 (wk4)
d56 (wk8)
d62 (wk9)
PanAd3 IM/PanAd3 IM
d0
d28 (wk4)
d56 (wk8)
d62 (wk9)
*Calves were primed with 5 1010 vp of PanAd3-RSV in a volume of 2 ml either IN or IM, and boosted IM with the same dose of PanAd3-RSV or with 2 107 pfu of MVA-RSV. As controls, calves were
Calves were
primed with 5 1010 vp of PanAd3 expressing an irrelevant antigen, and boosted IM with the same dose of PanAd3 or with 1 107 pfu of MVA expressing an irrelevant antigen.
challenged IN and IT with 1 104 pfu of BRSV/Snook strain.
12 August 2015
RESEARCH ARTICLE
RESEARCH ARTICLE
PanAd IM/MVA IM (P < 0.002) for % macroscopic lung lesions; and
PanAd IN/MVA IM (P < 0.002), PanAd IM/MVA IM (P < 0.03), and PanAd
IN/PanAd IM (P < 0.03) for % PMN in BAL. Nevertheless, all the vaccine
regimens, apart from homologous PanAd IN/IN, significantly reduced
BRSV replication in the LRT (P < 0.002; P < 0.05 for PanAd IN alone).
Clinical signs of respiratory disease in the calf model of BRSV infection do not usually develop until on or after day 6 of BRSV challenge
(12, 13, 21, 22), which is when these studies were terminated to sample
lung tissue at expected peak viral growth. As expected, there were few
clinical signs of respiratory disease after BRSV challenge in any of the
calves in studies 1 to 3 (table S3).
To confirm and expand the results on safety and efficacy of the homologous vaccine regimen, which may be more appropriate for a pediatric vaccine, we performed another study (study 4 in Table 1) in which
calves were vaccinated with PanAd3-RSV IM/IM and challenged with a
different pool of BRSV to that used in studies 1 to 3. As seen previously,
vaccinated calves were completely protected against BRSV replication

in the lungs. In addition, there was a significant reduction in nasopharyngeal virus replication (P < 0.02), the extent of macroscopic lung
lesions (P < 0.01), and the proportion of neutrophils in BAL (P < 0.005)
compared with controls (Fig. 4 and table S2). Furthermore, in this study,
all the control calves developed clinical signs of respiratory disease, characterized by fever, an increased respiratory rate (60 to 72 breaths/min),
and/or coughing (Fig. 4 and table S3). In contrast, calves vaccinated with
PanAd3-RSV IM/IM developed only minimal signs of disease, none developed fever, and there was only a moderate, transient increase in respiratory rate (48 breaths/min) in two of five calves.
Fig. 2. Vaccine efficacy and safety in calves challenged with BRSV.

Calves (n = 3 to 5) were vaccinated IN and/or IM and challenged with
BRSV as described for studies 1 to 3 in Table 1. (A) BRSV titers, expressed
as log10 pfu/ml, were determined by plaque assay on lysates of different
respiratory tract samples. TrSc, tracheal scrape; LWC, lung wash cells; RA,
right apical lung lobe; RC, right cardiac lung lobe; LC, left cardiac lung
lobe. Each block of bars corresponds to individual animals. (B) Area under the curve (AUC) of BRSV titers in nasal secretions collected on the
day of challenge and for each of the six following days. (C) Area of the
lung showing macroscopic lung lesions expressed as a percentage. (D)
Percentage of PMNs in BAL 6 days after challenge. Individual animal
data and group median are shown in (B) to (D). Control calves are
shown as open square symbols (study 1), open circles (study 2), and
open inverted triangles (study 3). Kruskal-Wallis test of one-way analysis
of variance (ANOVA) showed statistically significant differences compared with controls for RSV in nasal secretions with PanAd IN/MVA IM
(P < 0.03) and PanAd IM/MVA IM (P < 0.01); % lung lesions with PanAd
IN/MVA IM (P < 0.01) and PanAd IM/MVA IM (P < 0.002); and % PMN in
BAL with PanAd IN/MVA IM (P < 0.002), PanAd IM/MVA IM (P < 0.03), and
PanAd IN/PanAd IM (P < 0.03). All regimens, apart from homologous
PanAd IN/IN, significantly reduced BRSV replication in the LRT (P <
0.002; P < 0.05 for PanAd IN alone).
Prime/boost vaccine regimens induced BRSV-specific

antibodies and primed T cells in BRSV-seronegative calves
Immune responses induced by vaccination and challenge were evaluated
in studies 1 to 3 by analysis of BRSV-specific serum immunoglobulin G
12 August 2015
Fig. 3. Histological changes in the lungs of calves 6 days after BRSV challenge. Representative lung
samples from calves in study 1 were formalin-fixed and paraffin-embedded, and sections were stained with
hematoxylin and eosin (H&E). (A and B) Control calf showing bronchioles filled with neutrophils, sloughed
epithelial cells and cell debris, and alveolar collapse. (C and D) Calf vaccinated IN with PanAd3-RSV, showing
bronchial exudates. (E and F) Calf vaccinated IN with PanAd3-RSV and boosted IM with MVA-RSV, showing
normal lung architecture. Arrows indicate bronchioles. Scale bars, 500 mm. (G) Slides from calves in study
1 were scored blind for the extent of bronchiolitis and alveolitis. All bronchioles in each of three lung sections
from each calf were scored 0 to 3 for severity of bronchiolitis, with 0 = peribronchiolar space free from infiltrating cells and no exudate, and 3 = bronchiole completely blocked by cellular exudate and a peribronchiolar accumulation of cells >4 cells thick. Bronchiolitis scores were expressed as the mean % of bronchioles
with a score of 0 to 1 or 2 to 3 SD. Each 25 objective microscope field of each lung section was also scored
0 to 3 for the extent of alveolitis, with 0 = normal architecture, 1 = thickening of alveolar walls with <5
neutrophils per field, 2 = moderate thickening of alveolar walls with >5 neutrophils per field, and 3 =
>75% consolidation. Alveolitis scores were expressed as the mean % of microscope fields with a score of
0 to 1 or 2 to 3 SD (n = 3 to 4). The Kruskal-Wallis test of one-way ANOVA showed that the bronchiolitis
and alveolitis scores in calves vaccinated with PanAd IN/MVA IM were significantly different from controls
(P < 0.0005).
(IgG) and mucosal IgA responses, induction of nAbs, circulating BRSV-specific

antibody-secreting cells (ASCs) (study
1 only), and BRSV-specific interferon g
(IFNg)secreting T cells.
Although the single IN vaccination
with PanAd3-RSV induced little or no detectable serum IgG antibodies until 4 to
8 weeks after vaccination (Fig. 5A, fig. S3,
and table S5), BRSV-specific IgG ASC
could be detected in peripheral blood,
7 days after IN priming (fig. S4), indicating
that PanAd3-RSV IN had primed a B cell
response. This conclusion was confirmed
by the presence of high titers of anti-BRSV
IgG, 6 days after challenge of PanAd3RSV IN immunized calves (>3 log10), as
compared to undetectable antibodies in
infected, control animals (Fig. 5A and
fig. S3). In calves primed with PanAd3RSV by the IN route, a homologous IN
boost had little or no effect on serum
BRSV IgG titers (1 log10 in two of four
calves), whereas both homologous and
heterologous IM boosting increased
BRSV IgG titers to over 3 log10, with higher peak levels and faster kinetics in the latter group (Fig. 5A and fig. S3). This was
consistent with the increased numbers of
circulating BRSV-specific IgG ASC detected in the blood, 5 days after the IM
MVA-RSV boost (fig. S4). IM priming
with PanAd3-RSV was more effective in
inducing serum anti-BRSV IgG than IN
administration (>2 log10; Fig. 5A and fig.
S3), and titers increased further after IM
boosting with either PanAd3-RSV or MVARSV. Levels of BRSV-specific serum antibodies induced in calves vaccinated with
PanAd3-RSV IM/IM in study 4 were similar to those in study 3. There was little or no
increase in serum anti-BRSV IgG in any of
the calves vaccinated by either homologous
or heterologous prime/boost regimens after
BRSV challenge (Fig. 5A and fig. S3).
Mucosal IgA antibodies developed
slowly and were detected in low levels only
in calves that had been primed IN with
PanAd3-RSV (Fig. 5B, fig. S3, and table S5).
However, with the exception of calves vaccinated IN/IN with PanAd3-RSV, mucosal
IgA antibodies increased rapidly after BRSV
challenge and appeared to be greatest in
calves that had been primed IN with
PanAd3-RSV (Fig. 5A and fig. S3).
On the day of challenge, all prime/boost
vaccine groups, with the exception of those
primed IN with PanAd3-RSV only and
12 August 2015
RESEARCH ARTICLE
those in the PanAd3-RSV IN/IN group, had mean titers of HRSVspecific serum nAbs in the range of log2 5.8 to 7.6, which are equal to
or above the previously reported threshold for protection of infants
against HRSV-associated hospitalization (Fig. 5C and table S5) (23).
Fig. 4. Vaccination protects against clinical signs of BRSV respiratory

disease. Calves (n = 5 to 6) were vaccinated IM with PanAd-RSV on two
occasions as indicated in study 4 of Table 1. (A) Severity of clinical signs of
disease expressed as the mean clinical scores per day SD. (B) BRSV titers
expressed as log10 pfu/ml in lysates from different LRT samples as described
in Fig. 2. Each block of bars corresponds to individual animals. (C) AUC of
BRSV titer in nasal secretions collected on the day of challenge and for each
of the six following days. (D) Area of the lung showing macroscopic lung
lesions expressed as a percentage of the total lung area. (E) Percentage of
PMNs in BAL 6 days after challenge. Individual animal data and group
median are shown in (C) to (E). Kruskal-Wallis test of one-way ANOVA
showed statistically significant differences compared with controls for nasopharyngeal virus replication (P < 0.02), % lung lesions (P < 0.01), and % PMN
in BAL (P < 0.005).
As seen for BRSV-specific serum IgG, there was little or no increase

in HRSV nAbs after BRSV challenge. However, a strong anamnestic
response after BRSV challenge was seen in calves vaccinated IN with
PanAd3-RSV, but not in those vaccinated with PanAd3-RSV IN/IN
(Fig. 5C). Analysis of BRSV-specific serum nAbs from calves in study
1 showed that levels of nAbs against BRSV were comparable to those
against HRSV (fig. S5). BRSV nAb titers were not detected 6 days after
challenge in control animals, whereas all vaccinated calves displayed an
anamnestic BRSV-specific nAb response, confirming that BRSV nAbs
were primed in both vaccine groups.
BRSV-specific cell-mediated immune responses were analyzed after
vaccination and after challenge by IFNg-ICS and FACS analysis on
peripheral blood mononuclear cells (PBMCs) after stimulation with
BRSV. Before challenge, most BRSV-specific IFNg-producing cells were
CD4+ (mean 4.1% CD4+IFNg+:1.2% CD8+IFNg+) in animals that had
received the heterologous prime/boost regimens (Fig. 5, D and E, and
table S5). In contrast, most BRSV-specific IFNg-producing cells induced by PanAd3-RSV homologous prime/boost vaccination were
CD8+ (mean 0.3% CD4+IFNg+:1.0% CD8+IFNg+) (Fig. 5, D and E). After BRSV challenge, all vaccinated animals showed a strong anamnestic
CD4+IFNg+ T cell response. However, only the calves vaccinated with
PanAd3-RSV homologous prime/boost showed a strong expansion of
CD8+IFNg+ T cells after BRSV challenge (Fig. 5E). The breadth of
vaccine-induced cellular immunity was explored by IFNg-ICS on PBMCs
from the PanAd3-RSV IN/MVA-RSV IM vaccine group using peptide
pools corresponding to the amino acid sequence of the three vaccine
antigens (F, N and M2-1). The results showed that the IFNg+ T cell
responses were distributed among the three HRSV proteins present in
the vaccine (fig. S6).
In conclusion, there were no significant differences in the levels of
serum antibodies induced by the different prime/boost vaccination regimens, apart from those vaccinated with PanAd3-RSV IN/IN, and a rapid mucosal IgA response after BRSV challenge was only seen in calves
that had been primed by the IN route. Heterologous prime/boost
regimens appeared to be most effective at priming BRSV-specific IFNg+producing CD4+ cells, and homologous PanAd3-RSV prime/boost
regimens were most effective at boosting for a strong CD8+IFNg+ response after BRSV challenge.
Heterologous prime/boost vaccine regimens were ranked as
the most immunogenic and protective by principal
components analysis
To rank the immunological potency and the protective efficacy of the
different vaccination regimens, all the data collected for each animal on
immunological and infection readouts were analyzed in an unsupervised manner using principal components analysis (PCA). Two
separate PCAs were computed for immune response (PCimm) and viral load (PCvirus) variables, respectively (fig. S7). The space spanned by
PCimm1 and PCvirus1 constituted a coherent global evaluation space
(Fig. 6A): points (animals) on the bottom right of the graph are characterized by elevated viral infection readouts and low immune responses, whereas points on the top-left quadrant have high immune
responses and low viral infection readouts. Most of the vaccinated
animals reside in the top-left quadrant, showing good protective efficacy. Some of the protected animals were in the bottom-left quadrant,
meaning that low level of immunity was sufficient to protect from infection or that protection was mediated by local immune responses that
were not measured in this study. The mutual relation between the two
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RESEARCH ARTICLE
main principal components PCimm1 and PCvirus1 scored a highly statistical significant correlation (r = 0.83, P < 0.0001), giving a proof of
concept of the strict relation between the entity of immune response and
the decrease in viral load. Both PCimm1 and PCvirus1 were statistically
significant (one-way ANOVA) as for among groups variation (PCimm1:
F = 31.98, P < 0.001; PCvirus1: F = 23.93, P < 0.001), so pointing to a
statistically significant difference in efficacy among treatment groups.
The vaccine regimen PanAd3 IN/MVA IM is the most efficient in terms
of reduction of viral load (mean PCimm1 = 1.08/mean PCvirus1 =
1.20), whereas PanAd3 IM/MVA IM is more immunogenic (mean
PCimm1 = 0.98/mean PCvirus1 = 0.97).
Despite the importance of HRSV as a respiratory pathogen in man,

there is no effective vaccine. Progress in HRSV vaccine development
has been hampered by the failure of small-animal models to fully recapitulate the pathogenesis of HRSV infection and predict vaccine safety
and efficacy. Here, we report the use of the calf model of BRSV infection
to evaluate the safety, immunogenicity, and protective efficacy of viralvectored vaccines expressing HRSV proteins. We show that compared
to controls, heterologous PanAd3-RSV prime/MVA-RSV boost regimens protected calves against BRSV infection and reduced pulmonary
pathology. Vaccines based on replication-incompetent ChAd and
MVA vectors are capable of inducing a full spectrum of adaptive
humoral and cell-mediated immune responses (6). This approach has
been shown to be safe and highly immunogenic in humans as documented by results obtained in phase 1 clinical trials with candidate vaccines against hepatitis C, malaria, and Ebola (15, 24, 25). Proof of
concept for efficacy of the ChAd/MVA approach was also obtained
in human vaccination and challenge studies with a candidate malaria
vaccine (26).
Our previous studies have shown that an HRSV vaccine consisting
of a secreted form of the F, together with the N and M2-1 proteins,
delivered by a ChAd (PanAd3-RSV) protects cotton rats from HRSV
challenge, without inducing ERD (14). We have extended these observations and shown that ERD is not induced in cotton rats vaccinated by
Fig. 5. Vaccine-induced BRSV-specific antibody and cell-mediated

immune responses. Calves (n = 3 to 5) were vaccinated IN and/or IM and challenged with BRSV as described in Table 1. (A and B) Data are BRSV IgG (A) and
IgA (B) titers in sera and nasal secretions, respectively, collected at the time of
boost (white bars, 4 weeks post-prime), at the time of challenge (gray bars,
4 weeks post-boost), and 6 days after challenge (black bars). The asterisk on
the x axis for regimen PanAd3-RSV IN/MVA-RSV IM indicates that postprime and post-boost data refer to weeks 8 and 12, respectively. Titers
are expressed as log10, and dashed lines indicate limit of detection. (C) HRSV
nAb titers in sera collected at the time of challenge and 6 days after. Titers
are expressed as log2, and dashed line indicates limit of detection. (D and
E) Percentage of CD4+ (D) or CD8+ (E) T lymphocytes secreting IFNg in
response to BRSV stimulation detected by intracellular staining (ICS)
and fluorescence-activated cell sorting (FACS) analysis of PBMCs isolated
at the time of challenge and 6 days after. Data from (A) to (E) are shown as
means + SD.
DISCUSSION
12 August 2015
two administrations of PanAd3-RSV or a heterologous prime/boost

regimen with PanAd3-RSV and MVA expressing the same HRSV antigens. Both vaccination regimens protected cotton rats against viral
replication and did not enhance the extent of alveolitis or interstitial
pneumonia, features characteristic of FI-HRSV ERD in the cotton rat
(27). Although FI-HRSVvaccinated cotton rats showed evidence of
enhanced pulmonary pathology, 3 weeks after challenge, further studies
are needed to determine whether the vectored vaccines prime for ERD
when the HRSV challenge is delayed until immunity has waned, as has
been reported in cotton rats immunized with purified F protein or a
chimeric FG protein (28). IN priming with PanAd3-RSV was more effective than IM priming in preventing HRSV replication in the URT of
cotton rats, whereas both routes were equally effective at preventing viral replication in the lungs. These results are consistent with our previous findings obtained with a single administration of PanAd3-RSV (14)
and with data obtained with human Ad5 encoding HRSV F (9). It is
likely that IN priming leads to higher levels of mucosal immunity than
that induced by the IM route, resulting in better protection of the URT.
These observations contrast with those in cotton rats immunized with a
gorilla Ad encoding F (GC46-F), which induced protection in both the
upper and lower airways after a single IM vaccination (8). However, in
this case, a higher vector dose was used than in our studies, which may
have elicited levels of local immunity sufficient to suppress HRSV replication at the portal of viral infection. Alternatively, expression of the N
and M2-1 proteins may have reduced the immunogenicity of the F protein, and/or the secreted F protein may be predominantly in the postfusion conformation, whereas the membrane-anchored F in GC46-F
may be predominantly in the prefusion conformation, which is known

to induce higher levels of neutralizing antibodies (29).
Because the extent to which vaccine efficacy in rodent models of
HRSV can predict efficacy in man is unclear, we used the calf model
of BRSV infection, which is considered to be a more relevant animal
model than rodents for HRSV pediatric vaccine testing, to evaluate different regimens and routes of our vectored HRSV vaccines. Calves are
the natural hosts of BRSV, and the pathogenesis and epidemiology of
BRSV infection in calves are similar to those in children (10). Furthermore, ERD has been observed after a natural BRSV infection in calves
that had been vaccinated with an inactivated BRSV vaccine, and has
been reproduced experimentally in calves vaccinated with FI-BRSV
(1113), although this has not been consistently reproducible (30). Because genetic vaccines are often less effective in large animals than in
rodents, the calf model provides the opportunity for a more stringent
test of the PanAd3- and MVA-vectored RSV vaccine. The amino acid
identity between the vaccine antigens and the corresponding BRSV proteins is high (F = 81%, n = 93%, M2-1 = 80%) (10); serum antibodies
from BRSV-infected calves are able to neutralize HRSV (31); and CD8+
T cells, which are important in clearance of BRSV from the respiratory
tract of calves (32), recognize the F, N, and M2-1 proteins of BRSV (33).
These observations suggest that immunity elicited by the HRSV proteins contained in the vaccine could be effective against BRSV in calves.
Indeed, all the tested vaccine regimens induced protection in calves
against BRSV, to various degrees. Even a single IN administration of
PanAd3-RSV was able to restrict BRSV replication in the lung and
protect against extensive lung pathology. However, the presence of neutrophils in BAL from these animals suggests that some virus replication may
have taken place in the LRT, but was rapidly
controlled. This is consistent with the accelerated clearance of viral replication
from the URT of calves vaccinated with
PanAd3-RSV IN. In contrast to findings
in cotton rats, PanAd3-RSV IN was not sufficient to completely prevent BRSV replication in the URT. This may be due to the
lower efficacy of a single IN administration in large animals like young calves
weighing ~300-fold more than cotton rats.
The failure of PanAd3-RSV IN boosting
of calves primed IN to increase protective
efficacy may have been due to the induction of mucosal immunity to the vaccine
vector, reducing the immunogenicity of
the IN boost, or to poor uptake of the vaccine as a result of nasal discharge seen at
the time of vaccination. The IN route of
vaccination with PanAd3-RSV may face
similar problems in infants with rhinitis
at the time of vaccination.
Only the heterologous prime/boost regimens protected against nasopharyngeal
Fig. 6. Two-dimensional PCA of global vaccine efficacy. (A)The first principal component PCvirus1 (x
axis) was plotted against the first principal component PCimm1 (y axis). Each dot represents an animal, BRSV replication and the development of
colored for vaccine regimen in study groups 1 to 3. Dots on the bottom right of the graph are characterized macroscopic lung lesions, and significantby elevated viral infection readouts and low immune responses, whereas dots on the top-left quadrant have ly reduced the proportion of PMNs in
high immune responses and low viral infection readouts. (B) Single animals are shown collapsed on their BAL, confirming previous findings of betgroup mean (symbol) and SD (bar).
ter efficacy of the heterologous ChAd
12 August 2015
RESEARCH ARTICLE
prime/MVA boost regimens. Notably, heterologous PanAd3-RSV

IN/MVA-RSV IM boost was the only regimen that completely inhibited
a neutrophil response in the BAL after BRSV challenge, suggesting that
sterilizing immunity had been achieved, which supports the suggestion
that priming at the portal of virus entry affords the greatest protection.
Although virus could not be isolated from the lungs of calves vaccinated
by homologous PanAd3-RSV prime/boost regimens, apart from those
vaccinated IN/IN, the presence of PMNs in BAL suggests that lung virus
replication had occurred and initiated an inflammatory response. However, the IN and IT BRSV challenge does not recapitulate the normal
progression of RSV infection, which starts with initial virus replication
in the nose, followed by spread to the LRT in a proportion of individuals. The rapid clearance of BRSV from the nasopharynx of calves vaccinated by homologous prime/boost regimens suggests that spread of
virus to the lower airways would be restricted in vaccinated individuals.
Prime/boost regimens, with the exception of homologous PanAd3RSV IN/IN, achieved higher levels of immune responses than did a
single PanAd3-RSV administration. On the day of challenge, calves vaccinated by all prime/boost regimens, with the exception of homologous
PanAd3-RSV IN/IN, had developed high and comparable levels of circulating BRSV-specific IgG and HRSV nAbs. However, little or no increase in serum antibody titers was detected in these animals after
challenge. This is unlikely to be due to a lack of cross-reactivity between
HRSV and BRSV as antibodies generated by heterologous prime/boost
neutralize both HRSV and BRSV (fig. S5). Furthermore, the massive
increase in HRSV serum nAbs (more than 10-fold), 6 days after BRSV
challenge, in calves vaccinated with a single PanAd3-RSV dose,
confirms that the vaccine-encoded F is capable of priming B cell responses cross-reacting with BRSV F. The lack of an anamnestic antibody response after BRSV exposure may be due to the high levels of
humoral immunity induced by vaccination. In keeping with this hypothesis, the only prime/boost vaccine group that displayed a slight increase in nAbs after BRSV challenge was that given PanAd3-RSV IN/
PanAd3-RSV IM, where prechallenge titers were, on average, lower
than those in the other three prime/boost groups.
Apart from calves vaccinated with PanAd3-RSV IN/IN, all prime/boost
regimens elicited higher levels of IFNg+ T cell responses than a single
administration of PanAd3-RSV IN. The differences in the phenotype of
T cells induced by homologous PanAd3-RSV prime/boost versus
heterologous regimens in calves are reminiscent of findings in humans vaccinated with vectored hepatitis C virus vaccines, where a heterologous
ChAd/MVA regimen generated more balanced CD8+/CD4+ T cell responses and more sustained memory and effector memory T cell populations than a regimen based on two administrations of Ad vectors, which
preferably induced CD8+ T cells with a higher effector versus memory phenotype (15, 34). Although we did not explore the memory phenotype or the
longevity of the immune responses in our studies, it is possible that the
better protective efficacy afforded by the heterologous prime/boost regimens could be due to the quality of the immune response, as previously
suggested in the case of influenza (35). It should be noted that although only
low levels of BRSV-specific CD4+IFNg+ T cells were induced by the homologous prime/boost regimens, these were rapidly expanded after BRSV challenge and may contribute to the protective efficacy of these regimens. In
contrast, there was little or no expansion of circulating CD8+ IFNg+ T cells
after BRSV challenge in calves vaccinated by heterologous prime/boost
regimens, and yet, these proved to be the most efficacious vaccine regimens. These observations highlight a potential role for CD4+ T cells in
the protective efficacy against RSV infection and associated lung pathol-
ogy, reminiscent of a recent report on protection against influenza infection (36).

The PCA failed to identify specific immune responses that correlated
with protection and highlighted the fact that animals with a low level of
immune responses, such as those that received the first dose of vaccine
in the nose, displayed an equal level of protective efficacy as animals
with high immune responses, such as those primed IM. However, a limitation of our study is that, with the exception of nasal IgA antibodies, all
the immune responses were measured in the blood and therefore do not
reflect the extent of local immunity, which could be very important for a
virus with an ecology restricted to the airways. Further studies are
ongoing to analyze the T cell response in the respiratory tract.
Like HRSV, BRSV vaccineinduced ERD has been reported in
calves vaccinated with an inactivated BRSV vaccine after a natural infection (11). Histological analysis of the lungs from calves that died of
vaccine-induced ERD revealed BRSV antigen in the lungs and histopathological changes consistent with exacerbated infection, with
infiltration of eosinophils, which are not normally present in the natural
infection (11). Similarly, BAL eosinophils were associated with ERD in
FI-BRSVvaccinated calves after experimental BRSV challenge (13, 37).
In contrast, homologous PanAd3-RSV IM/IM vaccination protected
against clinical signs of respiratory disease in study 4, and eosinophils
were not detected in the BAL of any of the vaccinated calves after BRSV
challenge, even in those with virus in the lungs, or with a high proportion (>40%) of PMNs in BAL. In all these studies, calves were challenged
4 weeks after vaccination, and we therefore cannot exclude the possibility that ERD could occur when calves are challenged at a later time
point. However, pulmonary eosinophilia and ERD have been described
in calves vaccinated with FI-BRSV that were not protected against viral
replication when challenged with BRSV 1 month after vaccination (37),
as well as in calves that were protected against viral replication when
challenged 5 months after vaccination (15). Further studies are being
undertaken to determine the duration of immunity induced by the vectored vaccines and the safety when immunity has waned, and to determine vaccine safety and efficacy in calves with MDA at the time of
vaccination.
The principal limitations of the calf studies lie with the open-label
design of the studies and the small group sizes. The nonblinded nature
of the studies means that we cannot exclude or quantify any bias in analysis of the results. However, samples were analyzed without knowledge
of the treatment group, thereby minimizing bias. The small numbers of
calves in some of the studies introduce inherent risks of confounding
chance observations. Nevertheless, group sizes were sufficient to demonstrate that some vaccine regimens induced statistically significant
protection against BRSV infection.
This work provides solid preclinical data on the safety and efficacy of
a viral-vectored HRSV vaccine in a calf model of BRSV infection, which
mimics HRSV disease in children more closely than experimental
models of unnatural laboratory hosts. The BRSV challenge provides a
very stringent test for vaccine-induced cross-protection because there is
90% amino acid identity in the F proteins between different HRSV isolates compared with ~80% identity between the F proteins of HRSV and
BRSV. Nevertheless, the level of protection against BRSV induced in
seronegative, young calves by our vectored vaccine in most prime/boost
vaccine regimens tested is superior to that previously observed with a
replicating vaccinia vector encoding HRSV F and is similar to that of
live attenuated BRSV vaccines (18, 22, 38) or inactivated BRSV immunostimulating complexes (39). We have demonstrated that although the
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MATERIALS AND METHODS

Study design
This study was designed to evaluate the safety and efficacy of different
HRSV vaccination regimens and routes against HRSV in cotton rats
and BRSV in young, seronegative calves. Cotton rats were randomly
allocated and vaccinated with the vectored vaccines or with an FI-RSV
vaccine as a control of vaccine-induced augmented lung pathology. Three
weeks after the last immunization, cotton rats were challenged with
HRSV and sacrificed 5 days later when the virus peaks in the nose and
lungs. Vaccine immunogenicity was determined by analysis of RSVspecific serum nAbs, and protection against HRSV challenge was determined by analysis of nasal and lung virus titers, and analysis of the
severity of histological changes in the lungs at post-mortem examination. The studies were not blinded and the sample sizes varied in the
different groups (from 5 to 10), as indicated in table S1.
Calves were randomly allocated to groups, vaccinated with the vectored vaccines, and challenged with BRSV, as indicated in Table 1. Vaccine immunogenicity was determined by analysis of the development of
BRSV-specific serum and mucosal antibodies, nAbs, and flow cytometric analysis of BRSV-specific IFNg-producing T cells. In some studies,
BRSV-specific antibody-secreting B cells were analyzed by enzymelinked immunospot (ELISPOT). Protective efficacy was determined
by analysis of nasopharyngeal virus replication, lung virus titers, extent
of pulmonary inflammation, and development of clinical signs of respiratory disease after BRSV challenge. A power analysis was used to determine the minimum number of calves required to give a 1.5 log10
difference in lung virus titers between groups with a 95% confidence
and 80% power, assuming an SD of 0.6 log10. This showed that this
power could be achieved with four animals per group. The studies were
not blinded, and the sample sizes varied in the different groups (from
three to six), as indicated in Table 1. Of three groups of four calves in
study 1, one control and one vaccinated calf died with abomasitis and
diarrhea after a change in diet and were excluded from the study; of four
groups of four calves in study 2, one of the control calves had MDAs and
was excluded from the study; of two groups of five calves in study 3, one
of the vaccinated calves underwent a natural BRSV infection during the
course of the study, as determined by an increase in BRSV-specific mucosal IgA antibodies, 4 weeks after the first IM vaccination, and was excluded from the study.
Animal studies were conducted in compliance with the Animal Welfare Act, ARRIVE guidelines, and other federal statutes and regulations relating to animals and experiments involving animals. Calf
experiments were performed under the regulations of the Home Office Scientific Procedures Act (1986) of the UK. The studies had been
approved by The Pirbright Institute Animal Welfare & Ethical Review Body and by the Animal and Plant Health Agency Ethical Review
Committee.
PanAd3-RSV and MVA-RSV vectors

Construction and characterization of PanAd3-RSV and MVA-RSV are
described in detail elsewhere (14). Briefly, the two vectors were generated by inserting in the corresponding viral genomes the HRSV expression cassette, which contained consensus sequences of HRSV proteins
F, N, and M2-1. Protein sequences were chosen among HRSV subgroups A and B, and consensus sequences were derived by alignment
using MUSCLE version 3.6, of all nonidentical sequences and applying
the majority rule. The vaccine F0DTM protein lacked amino acids 525
to 574, which contains the transmembrane and the intracellular regions.
N and M2-1 sequences were spaced by a flexible linker (3GS3G). Consensus F0DTM, N, and M2-1 sequences were optimized for mammalian
expression, including the addition of a Kozak sequence and codon usage
optimization.
Vaccination and challenge of cotton rats
Cotton rat (Sigmodon hispidus) studies were performed at Sigmovir
Biosystems Inc. (Rockville, MD). Prime and boost vaccinations, 4 weeks
apart, were given at the following doses: 5 108 vp for PanAd3-RSV and
1 107 pfu for MVA-RSV. Vaccines were diluted in phosphate-buffered
saline (PBS) to a final 100-ml volume and administered bilaterally (either
50 ml per nostril for IN or 50 ml per quadriceps for IM). One group of
animals received two IM injections, 4 weeks apart, of FI-RSV (lot #100).
Another group of animals was left unvaccinated. Three weeks after the
last vaccination, the animals were challenged IN with 100 ml of RSV/A/Long
at 105 pfu per animal. Five days after challenge, the animals were euthanized and bled, and nasal tissue and lungs were harvested. Lungs were
divided to be processed for viral titration (left lobes) and histopathology
(right lobes). For histopathology, lungs were dissected and fixed with
10% neutral buffered formalin. After fixation, the lungs were embedded
in paraffin, sectioned, and stained with H&E. Four parameters of pulmonary inflammation were evaluated: peribronchiolitis (inflammatory
cell infiltration around the bronchioles), perivasculitis (inflammatory
cell infiltration around the small blood vessels), interstitial pneumonia
(inflammatory cell infiltration and thickening of alveolar walls), and alveolitis (cells within the alveolar spaces). Slides were scored blind on a 0
to 4 severity scale and subsequently converted to a 0 to 100% histopathology score, as described previously (27). Virus titers in lung and
nose homogenates were performed by plaque assay on HEp-2 cells and
expressed as pfu per gram of tissue. RSV nAbs were measured by plaque
reduction assay on HEp-2 cells infected with RSV/A/Long, and the
reciprocal nAb titers were determined at the 60% reduction end point
of the virus control using the statistics program plqrd.manual.entry.
Vaccination and challenge of calves
Calves were obtained from The Pirbright Institute farm and were fed a
limited amount of colostrum containing low levels of BRSV-specific
antibodies, 24 hours after birth, to produce BRSV-seronegative calves.
Different immunization regimens were tested in four separate studies,
which are described in Table 1. Vaccines were diluted in a 2-ml volume
and administered either IN (1 ml per nostril) using a mucosal atomization device (LMA MAD Nasal) or IM at the following doses: 5 1010 vp
for PandAd3-RSV and 2 108 pfu for MVA-RSV. Control animals received prime and boost vaccinations with PanAd3 and MVA vectors
expressing unrelated antigens. Calves were 2 to 4 weeks old at the time
of vaccination, and the interval between prime and boost was 4 weeks,
except for PanAd3 IN/MVA IM regimen, where the two injections were
8 weeks apart (Table 1). Four weeks after the last vaccination, calves
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10
heterologous prime/boost vaccine regimens can completely prevent

BRSV infection in young seronegative calves, efficient protection from
pulmonary disease can be achieved by a less complicated regimen based
on a homologous IM prime/boost. Our studies also suggest that a safe
and protective HRSV vaccine should be based on the ability to activate
both B and T cells in a concerted manner. These preclinical studies of a
viral-vectored HRSV vaccine in calves advance its potential as a human
vaccine for HRSV and support its further evaluation in man.
RESEARCH ARTICLE
BRSV and cells

BRSV used in some immune assays was derived from the full-length
complementary DNA of BRSV strain A51908, variant Atue51908
(GenBank accession no. AF092942) (40), grown in Vero cell monolayers, and infected at a multiplicity of infection (MOI) of 0.1 in Dulbeccos modified Eagles medium (Gibco-BRL) containing 2% heated
fetal calf serum (FCS). Virulent BRSV used to challenge calves consisted
of BAL prepared from a gnotobiotic calf inoculated 6 days previously
with the Snook strain of BRSV, which had been passaged on two previous occasions in gnotobiotic calves (22). The BAL was free from other
viruses, mycoplasmas, and bacteria as assessed by inoculation of tissue
culture cells and mycoplasmal or bacterial media. Virus titers were
determined by plaque assay on fetal calf kidney (FCK) cells.
Serology
Levels of BRSV-specific IgG and IgA were determined as described previously, using BRSV (Snook strain)infected FCK cell lysate as antigen
(32). A lysate of mock-infected FCK cells was used as a control antigen.
BRSV nAb titers were determined by plaque reduction assay on Vero
cells using heat-inactivated serum and BRSV strain A51908, variant
Atue51908, as described previously (41). HRSV nAb titers were determined
by a FACS-based assay using a recombinant HRSV expressing GFP
(HRSV-GFP), a gift from M. E. Peeples (Nationwide Childrens Hospital,
Columbus, OH). The assay was performed as previously described (42).
Briefly, HEp-2 cells were plated (5 104 cells/100 ml each well) in 96well plate(s) and incubated for 2 hours at 37C, 5% CO2. Serial dilutions
of heat-inactivated calf sera were incubated with HRSV-GFP (0.1 MOI)

for 1 hour at 37C with 5% CO2 in 100 ml of Dulbeccos modified Eagles
medium7% FCS and then added to the cells and left for 20 to 22 hours
at 37C in 5% CO2. Palivizumab (Synagis 100 mg, Abbott, catalog no.
131-026-19) was used as a positive control. Cells were detached by trypsin and collected in PBS containing 1% FCS and 1% formaldehyde, and
then fluorescence was read on a FACSCanto cytometer (BD) using
FACSDiva Software (Becton Dickinson). The percentage of fluorescent
cells for each serum dilution was calculated with respect to the negative
control (100% of infection). The EC50 (median effective concentration) was calculated by curve fitting and nonlinear regression analysis
(GraphPad Prism, GraphPad Software Inc.).
T cell assays
PBMCs were prepared from calves as described previously (32), depleted of WC1+ g/d T cells using an anti-WC1 antibody [CC15 (43)],
anti-mouse IgG microbeads, and MACS columns (Miltenyi Biotec). Cells
were stimulated with BRSV Atue51908 at an MOI of 1, or with uninfected
Vero cell lysates, washed, and aliquoted (2 106 per well) to 96-well
plates, as described previously (33). For peptide stimulation, WC1depleted PBMCs were incubated with HRSV peptide pools covering the
complete sequence of the HRSV vaccine antigens F, N, and M2-1, consisting of 15-mer sequences with 11 amino acids overlap (JPT Peptide
Technologies GmbH). The 269 peptides were dissolved in 100% dimethyl sulfoxide (DMSO) and arranged in four pools as follows: pool
M2-1 (46 peptides), pool N (95 peptides), pool Fa (N-terminal half of F
protein, 64 peptides), and pool Fb (C-terminal half, 64 peptides). Each
peptide pool was used at a final concentration of 3 mg/ml of each peptide. DMSO (Sigma) was used as a negative control. Cells were incubated for ~22 hours, and brefeldin A (1 mg/ml; Calbiochem) was
added for the final 4 hours of the assay. After incubation, all cells were
washed in PBS containing 1% BSA and 0.1% sodium azide, and stained
with Live/Dead Aqua (Life Technologies), fluorescein isothiocyanate
(FITC)anti-bovine CD8 (clone CC63, Serotec), or allophycocyanin
(APC)anti-bovine CD4 [clone CC8, (44), The Pirbright Institute] for
10 min at 20C. After washing, cells were fixed in 1% paraformaldehyde
in PBS for 10 min at 20C, washed, and permeabilized (FACS permeabilizing solution, Becton Dickinson) for 10 min at 20C. Cells were then
washed and stained with phycoerythrin (PE)anti-bovine IFNg (Serotec)
and analyzed using a Fortessa flow cytometer (Becton Dickinson). Data
analysis was performed using FCS Express software (De Novo Software);
gates were set to include live mononuclear cells based on live/dead staining and excluded cell doublet and triplets.
RSV-specific B cell ELISPOT assay
Bovine B cell ELISPOT assays were performed as described previously
(45) using a lysate of BRSV (Snook)infected FCK cells diluted in 0.1 M
carbonate buffer (pH 9.6) to coat wells of MultiScreen-HA ELISPOT
plates (Millipore) for 2 hours at 37C or uninfected FCK cell lysate as
a control. Plates were washed with PBS and blocked with 4% skimmed
milk in PBS for 2 hours at 37C. Twofold dilutions of fresh WC1depleted PBMCs were added to triplicate wells, in 100-ml volumes, starting
from 5 105 cells per well. After 24 hours of incubation, antibodyproducing cells were detected with horseradish peroxidise (HRP)antibovine IgG or HRPanti-bovine IgA (Serotec) for 3 hours at room
temperature. After washing, 100 ml of 3-amino-9-ethylcarbazole substrate (Merck Chemicals) was added for 1 hour at room temperature.
Color development was stopped by rinsing with tap water, and plates
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11
were challenged by IN and IT administration of 104 pfu of BRSV, Snook

strain, in BAL. Calves were bled at defined time points for analysis of
BRSV-specific serum antibody and T cell responses. Nasal secretions
were obtained at weekly intervals using a tampon inserted into the nostril for 15 min. After BRSV challenge, nasopharyngeal swabs were obtained daily and calves were euthanized 6 days after challenge to
determine the extent of gross pneumonic consolidation and lung virus
titers, as described previously (22). Calves were examined daily for clinical signs of disease, and the severity of disease was scored as described
in table S6. Tracheal virus titers were determined by scraping the epithelium from a piece of trachea about 3 cm long into 2 ml of Hanks
balanced salt solution (Sigma) containing 1% bovine serum albumin
(BSA) (Sigma). The apical and cardiac lung lobes were clamped, and
the lungs were lavaged with ~1 liter of PBS to obtain BAL. Cytospin
preparations of BAL cells were fixed and stained with Diff-Quik (Thermo
Fisher Scientific), and differential cell counts were made using oil immersion microscopy. Samples of lung taken from three different apical lung
lobes were homogenized to give a 20% (w/v) suspension. Lung tissue for
histology was also taken from three different apical lobes and fixed in 10%
neutral buffered formalin and paraffin waxembedded, and sections were
stained with H&E. All bronchioles in each lung section were scored 0 to 3
for severity of bronchiolitis, with 3 = bronchiole completely blocked by
cellular exudate and a peribronchiolar accumulation of cells >4 cells thick.
Bronchiolitis scores were expressed as the % of bronchioles with a score of
2 (moderate) to 3 (severe). Each 25 objective microscope field of lung
was also scored 0 to 3 for the extent of alveolitis, with 0 = normal
architecture of alveolar, 1 = thickening of alveolar walls with <5 neutrophils per field, 2 = moderate thickening of alveolar walls with >5 neutrophils per field, and 3 = >75% consolidation. Alveolitis scores were
expressed as the % of microscope fields with a score of 2 (moderate) to
3 (severe).
were allowed to dry before ASCs were counted using the AID ELISPOT
reader. Results were expressed as ASC number per 106 cells and means
of triplicate wells SD.
Principal components analysis
Global vaccine efficacy was estimated by PCA, as a filter for correlated
information (46). PCA is a purely descriptive method that does not depend on specific data distribution. Two separate PCAs were computed
for immune response and viral load variables, respectively; the first was
applied to the data relative to serum IgG, nasal IgA, IFNg+CD8+ lymphocytes, IFNg+CD4+ lymphocytes, and HRSV neutralization titers
before and after challenge. The second analysis was applied to the data
on lung and nasopharyngeal virus titers, macroscopic lung lesions, and
% neutrophils in BAL from 11 control and 19 vaccinated animals in studies
1 to 3 (Table 1). The first principal component of immune response
(PCimm1) accounted for the 47% of total variance, whereas the second
component scored 17% of total variance (fig. S7). PCimm1 was a size
component (47) having near to unity positive loadings with all the variables
and thus constituting a reliable general index of immune response. The
second component (PCimm2) scored 17% of total variance and accounted
mainly for the singular character of nasal IgA (fig. S7). PCA relative to viral
infection gave rise to a global size first component (PCvirus1), accounting
for 64% of total variance and thus representing a reliable global index of
effect (fig. S7). The second component (PCvirus2) accounted for 16% of
total variance and, consistently with the immune space solution, points
to a singularity of local nose response: the variable AUCnose is in fact almost equivalently loaded on the two orthogonal global (PCvirus1) and
nose (PCvirus2) components (0.70 and 0.68, respectively) (fig. S7).
Statistical analysis
Lines present in the scatterplot graphs represent the mean, and bar
graphs depict means SD, as indicated. One-way ANOVA followed
by the Kruskal-Wallis test was performed to analyze multiple groups.
The data were normally distributed as determined by the DAgostino-Pearson
omnibus normality test. We considered P < 0.05 as statistically significant. All statistical analyses were performed using the GraphPad Prism
software.
SUPPLEMENTARY MATERIALS
www.sciencetranslationalmedicine.org/cgi/content/full/7/300/300ra127/DC1
Fig. S1. BRSV replication in the nasopharynx.
Fig. S2. Analysis of cells in BAL 6 days after BRSV challenge.
Fig. S3. Kinetics of BRSV IgG and IgA antibodies in serum and nasal secretions.
Fig. S4. Vaccination induces BRSV-specific antibody-secreting B cells.
Fig. S5. Comparison of serum nAb titers against HRSV and BRSV.
Fig. S6. Breadth of T cell response induced by vaccination.
Fig. S7. Principal components analysis.
Table S1. Vaccine efficacy in cotton rats.
Table S2. Vaccine efficacy in calves.
Table S3. Effect of vaccination on clinical scores.
Table S4. Effect of vaccination on histopathology scores.
Table S5. Vaccine-induced immune responses in calves.
Table S6. Clinical score.
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Acknowledgments: We thank the animal service staff at Animal and Plant Health Agency
(Addlestone, Surrey, UK) for their care of the experimental calves; Sigmovir Biosystems Inc.
(Rockville, MD) for expert assistance in the cotton rat studies, including analysis of the cotton
rat histopathology; and M. Peeples (The Research Institute at Nationwide Childrens Hospital,
Columbus, OH) for providing the recombinant HRSV-GFP virus. Funding: This work was
supported by Medical Research Council grant MR/J014648/1 and the Biotechnology and
Biological Sciences Research Council Institute Strategic Programme grant on Livestock Viral Diseases at The Pirbright Institute. Author contributions: G.T. was the chief investigator. G.T., M.T.,
R.H., and E.G. performed the calf studies, and G.T. analyzed the calf histopathology. G.T., M.T., A.V.,
R.C., S. Colloca, A.N., A.F., and E.S. contributed to the design of the experiments. G.T., M.T., R.H., E.G., F.N.,
and A.P. optimized and performed the assays to evaluate the immunogenicity and protective
efficacy of the vaccine regimens in calves. G.T., M.T., E.G., S. Capone, and A.V. performed the data
analysis. A.G. performed the PCA, and S. Gubbins (The Pirbright Institute) provided other statistical advice. G.T., A.V., and A.N. wrote the manuscript. All authors had input into the manuscript
and have approved the manuscript for publication. Competing interests: A.V., R.C., and A.N. are
named inventors on patent applications named Expression systems covering RSV antigen expression system (WO 2012/089833). The other authors declare that they have no competing
interests.
Submitted 14 May 2015
Accepted 2 July 2015
Published 12 August 2015
10.1126/scitranslmed.aac5757
Citation: G. Taylor, M. Thom, S. Capone, A. Pierantoni, E. Guzman, R. Herbert, E. Scarselli,
F. Napolitano, A. Giuliani, A. Folgori, S. Colloca, R. Cortese, A. Nicosia, A. Vitelli, Efficacy of a
virus-vectored vaccine against human and bovine respiratory syncytial virus infections. Sci.
Transl. Med. 7, 300ra127 (2015).
12 August 2015
13
RESEARCH ARTICLE
Efficacy of a virus-vectored vaccine against human and bovine

respiratory syncytial virus infections
Geraldine Taylor et al.
Sci Transl Med 7, 300ra127 (2015);
DOI: 10.1126/scitranslmed.aac5757
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RSV vaccine cows infection

Respiratory syncytial virus (RSV) causes a severe lower respiratory tract disease that affects both children and
the elderly. Vaccines have shown promise in rodents and nonhuman primates, but it remains unclear if these models
reflect human RSV infection. Now, two papers by Taylor et al. and Green et al. translate one vaccine strategy first into
calves, which are natural hosts of bovine RSV (BRSV), and then into humans in a phase 1 clinical trial. A prime-boost
strategy protected against upper and lower respiratory tract infection and pulmonary disease induced by heterologous
BRSV challenge in calves, and demonstrated safety and immunogenicity in humans. These data support further trials
to test vaccine efficacy in human patients.

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RESEARCH ARTICLE

Efficacy of a virus-vectored vaccine against human and

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receiving FI-HRSV, developed neutralizing antibody (nAb) titers well

Prime/boost vaccine regimens protect cotton rats against

Prime/boost vaccine regimens protect against BRSV

Fig. 1. Vaccine immunogenicity, efficacy, and safety in cotton rats.

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misconducted vaccination course, such as missing prime or boost. In

which the interval between the two administrations was reduced to

Table 1. Design of calf studies. n.a., not available.

BRSV challenge study

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vaccinated calves were completely protected against BRSV replication

Fig. 2. Vaccine efficacy and safety in calves challenged with BRSV.

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Prime/boost vaccine regimens induced BRSV-specific

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(IgG) and mucosal IgA responses, induction of nAbs, circulating BRSV-specific

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Fig. 4. Vaccination protects against clinical signs of BRSV respiratory

As seen for BRSV-specific serum IgG, there was little or no increase

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Despite the importance of HRSV as a respiratory pathogen in man,

Fig. 5. Vaccine-induced BRSV-specific antibody and cell-mediated

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two administrations of PanAd3-RSV or a heterologous prime/boost

may be predominantly in the prefusion conformation, which is known

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prime/MVA boost regimens. Notably, heterologous PanAd3-RSV

ogy, reminiscent of a recent report on protection against influenza infection (36).

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MATERIALS AND METHODS

PanAd3-RSV and MVA-RSV vectors

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heterologous prime/boost vaccine regimens can completely prevent

BRSV and cells

of heat-inactivated calf sera were incubated with HRSV-GFP (0.1 MOI)

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were challenged by IN and IT administration of 104 pfu of BRSV, Snook

REFERENCES AND NOTES

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23. P. A. Piedra, A. M. Jewell, S. G. Cron, R. L. Atmar, W. P. Glezen, Correlates of immunity to

38. G. Taylor, L. H. Thomas, J. M. Furze, R. S. Cook, S. G. Wyld, R. Lerch, R. Hardy, G. W. Wertz,

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Efficacy of a virus-vectored vaccine against human and bovine

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RSV vaccine cows infection

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