INFECTIOUS DISEASE
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
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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
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.
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12 August 2015
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
3
4
Regimen*
No.
Prime study
day (week)
Boost 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.
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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,
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).
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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).
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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).
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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).
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
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
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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
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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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