EClinicalMedicine 27 (2020) 100517

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EClinicalMedicine
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Research Paper

Safety and immunogenicity evaluation of recombinant BCG vaccine

against respiratory syncytial virus in a randomized, double-blind,
placebo-controlled phase I clinical trial
Katia Abarcaa,b,*, Emma Rey-Juradoa,d, Natalia Mun ~ oz-Durangoa,c,d, Yaneisi Va
zqueza,d,
Jorge A. Sotoa,d, Nicola s M.S. Galveza,d, Javier Valde
s-Ferradaa,d, Carolina Iturriagaa,b,
Marcela Urzu  a , Arturo Borzutzky , Jaime Cerda , Luis Villarroele, Victoria Madrida,d,
a a,b e

Pablo A. Gonza leza,d, Jose  V. Gonza

lez-Aramundiza,f, Susan M. Buenoa,d,*,
a,c,d,
Alexis M. Kalergis *
a
Millennium Institute on Immunology and Immunotherapy, Pontificia Universidad Cato lica de Chile, Av. Libertador Bernardo O’Higgins No. 340, Santiago, Chile
b
Departamento de Enfermedades Infecciosas e Inmunología Pedia tricas, Divisio
n de Pediatría, Escuela de Medicina, Pontificia Universidad Cato lica de Chile,
Santiago, Chile
c
Departamento de Endocrinología, Escuela de Medicina, Pontificia Universidad Cato lica de Chile, Santiago, Chile
d
Departamento de Genetica Molecular y Microbiología, Facultad de Ciencias Biolo
gicas, Pontificia Universidad Cato lica de Chile, Santiago, Chile
e 
Departamento de Salud Publica, Escuela de Medicina, Pontificia Universidad Cato lica de Chile, Santiago, Chile
f
Departamento de Farmacia, Facultad de Química y de Farmacia, Pontificia Universidad Cato lica de Chile, Santiago, Chile

A R T I C L E I N F O A B S T R A C T

Article History: Background: Respiratory syncytial virus (RSV) is responsible for most respiratory tract infections and hospi-
Received 10 June 2020 talizations in infants and represents a significant economic burden for public health. The development of a
Revised 23 July 2020 safe, effective, and affordable vaccine is a priority for the WHO.
Accepted 5 August 2020
Methods: We conducted a double-blinded, escalating-dose phase 1 clinical trial in healthy males aged 18-
Available online 6 October 2020
50 years to evaluate safety, tolerability, and immunogenicity of a recombinant Mycobacterium bovis BCG vac-
cine expressing the nucleoprotein of RSV (rBCG-N-hRSV). Once inclusion criteria were met, volunteers were
Keywords:
enrolled in three cohorts in an open and successive design. Each cohort included six volunteers vaccinated
Human respiratory syncytial virus
BCG vaccine
with 5 £ 103, 5 £ 104, or 1 £ 105 CFU, as well as two volunteers vaccinated with the full dose of the standard
Phase I clinical trial BCG vaccine. This clinical trial (clinicaltrials.gov NCT03213405) was conducted in Santiago, Chile.
Safety Findings: The rBCG-N-RSV vaccine was safe, well-tolerated, and no serious adverse events related to the vac-
Transmissibility cine were recorded. Serum IgG-antibodies directed against Mycobacterium and the N-protein of RSV
Immunogenicity increased after vaccination, which were capable of neutralizing RSV in vitro. Additionally, all volunteers dis-
played increased cellular response consisting of IFN-g and IL-2 production against PPD and the N-protein,
starting at day 14 and 30 post-vaccination respectively.
Interpretation: The rBCG-N-hRSV vaccine had a good safety profile and induced specific cellular and humoral
responses.
Funding: This work was supported by Millennium Institute on Immunology and Immunotherapy from Chile
(P09/016), FONDECYT 1190830, and FONDEF D11E1098.
© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)

1. Introduction

Respiratory syncytial virus (RSV) is one of the leading causes of acute

respiratory tract infections (ARTIs) in children worldwide and is associ-
ated with significant health and economic burden, frequently leading to
* Corresponding authors at: Millennium Institute on Immunology and Immunother-
lica de Chile. Av. Libertador Bernardo O’Higgins No.
severe diseases such as bronchiolitis and pneumonia [1 3]. Extrapul-
apy. Pontificia Universidad Cato
340, Santiago 8331010, Santiago, Chile. monary symptoms have also been associated with RSV infection [4,5].
E-mail addresses: [email protected] (K. Abarca), [email protected] (S.M. Bueno), Currently, the only prophylactic approach against RSV consists of a
[email protected] (A.M. Kalergis).

https://doi.org/10.1016/j.eclinm.2020.100517
2589-5370/© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
2 K. Abarca et al. / EClinicalMedicine 27 (2020) 100517

immunogenicity of three different doses of cGMP rBCG-N-hRSV in

Research in Context
healthy males aged 18 50 years.
The vaccine was well tolerated in healthy adults with no severe
Evidence before this study
adverse effects related to the immunization, while inducing both T
In 2008, two recombinant Mycobacterium bovis BCGs were able cell and antibody responses against RSV -either viral antigens or a
to induce a protective response against the infection caused by clinical isolate- with increasing neutralizing capacities. Our results
human respiratory syncytial virus (RSV), mediated by a T cell suggest that rBCG-N-hRSV is a safe vaccine that could induce protec-
protective response, in a murine model. Reports from 2010 tion against RSV-infection.
indicate that this protective response was best with the recom-
binant BCG expressing the N protein (rBCG-N-hRSV) and that it 2. Material and methods
was mediated by both CD4+ and CD8+ T cells, in mice. In 2017, a
single, low dose of the cGMP rBCG-N-hRSV vaccine in mice was 2.1. Vaccine
able to protect against the disease caused by RSV, as shown in
previous reports. Also, immunization was safe with no adverse The study vaccine is a live attenuated recombinant Mycobacterium
events in mice; and a PPD response could be detected up to 6 bovis BCG based on the Danish strain 1331 that expresses the nucleo-
months after immunization. Finally, in 2018 immunization of protein (N) of RSV (rBCG-N-hRSV) [14,19,21]. The vaccine was
mice with rBCG-N-hRSV was able to induce the secretion of designed at the Pontificia Universidad Cato lica de Chile and manufac-
antibodies against several viral proteins, with increased neu- tured under cGMP conditions at IDT Biologika (USA). The control vac-
tralizing capacities. Moreover, sera transfer assays were able to cine is a live attenuated Mycobacterium bovis BCG Serum Institute of
protect naïve mice from infection with RSV. India (SII) strain. Both vaccines were administered intradermally
over the deltoid area.
Added value of this study
2.2. Ethical statements and clinical trial information
This is the first time that the cGMP rBCG-N-hRSV is adminis-
tered to humans (healthy male adults), and safety and immuno-
This clinical trial (clinicaltrials.gov NCT03213405) was conducted
genicity responses are evaluated. We show that the vaccine is
in Santiago, Chile, at a single site in the Clinical Research Center of
safe and well-tolerated, and the immune response elicited by lica de Chile (CICUC). The final posting
the Pontificia Universidad Cato
the highest dose tested can last for at least six months.
date of the study in clinicaltrails.gov was defined in compliance of
regulatory requirements from local authorities.
Implications of all the available evidence
The study protocol was conducted according to the current guide-
This study suggests that this vaccine is an adequate candidate lines of the Tripartite Guidelines for Good Clinical Practices, the Dec-
to proceed with new clinical trials that may allow this vaccine laration of Helsinki [22], and local regulations. The clinical trial was
candidate to be eventually administered to newborns to pre- approved by both the Institutional Ethical Committee (number 15-
vent the disease caused by RSV. 216) and the Chilean Public Health Institute (ISP Chile, number
EC819077/16). Written informed consent was obtained from each
participant before study entry. Volunteers did not receive any pay-
humanized IgG monoclonal antibody (palivizumab) that prevents epi- ment for their participation.
thelial cell infection; although it is mainly aimed for high-risk infants
and its protection is short-lived [6]. Therefore, vaccines against RSV are 2.3. Study design
highly needed [7]. Ideally, these vaccines should avoid T helper (Th)-2
cell responses, that lead to enhanced pulmonary disease as previously This phase 1 study was double-blinded and with dose-escalation
seen [8 12]. According to PATH (www.path.com), currently, there are to evaluate the safety, tolerability, and immunogenicity of rBCG-N-
approximately fifteen vaccine prototypes against RSV being evaluated hRSV in healthy males, aged 18-50 years.
in clinical trials [13]. rBCG-N-hRSV is a recombinant Mycobacterium
bovis bacillus Calmette-Gue rin (BCG) that expresses the nucleoprotein 2.3.1. Inclusion/exclusion criteria and cohorts
(N) of RSV [14]. BCG, the vector of this vaccine, is currently routinely A sample of 24 subjects was recruited, considering a usual phase I
administered to newborns in most countries -such as Japan, Mexico, studies sample size (20-100 subjects). Once inclusion criteria were met,
China, Brazil, and Chile, among others [15]- and has been used for volunteers were enrolled in one of three cohorts, each containing eight
almost a century to prevent tuberculosis meningitis and miliary disease subjects (6 receiving the study vaccine and two the standard BCG as
in children [16]. A final volume of 0.05 mL of a reconstituted vial (which control vaccine) in an open and ascending dose cohort design (Table S1
usually contains about 2-8 £ 106 CFU of the bacteria per 0.1 mL) of this and Fig. A). In each cohort, they were vaccinated with 0.1 mL of the vac-
vaccine is administered intradermally [17], resulting in the administra- cine, as follows: 6 volunteers in Cohort A were vaccinated with the low-
tion of about 1 4 £ 105 CFU of BCG to a newborn. BCG induces a strong est dose (5 £ 103 CFU); 6 volunteers in Cohort B received the middle
Th-1 immune response and therefore is likely a suitable vector for a vac- dose (5 £ 104 CFU); 6 volunteers in Cohort C received the highest dose
cine against RSV, since a Th-1 response may be better for the clearance (1 £ 105 CFU) of the study vaccine. Each cohort included two volunteers
of viral pathogens [14,18]. In murine models, rBCG-N-hRSV vaccination vaccinated with 0.1 mL of the standard BCG vaccine (BCG-WT; a full
prevented RSV associated-lung damage, decreased pro-inflammatory dose of 1-33 £ 105 CFU) (Fig. 1A). As the adult dose of BCG vaccine is in
infiltration into this tissue, and induced early recruitment of CD4+ and the order of 105 CFU, we decided to use this dose as the highest, with
CD8+ T cells into the lungs [14,19,20]. rBCG-N-hRSV vaccination also 103 and 104 as the lower doses.
induced protective T cells and the secretion of antibodies against several
RSV proteins other than the N-protein upon infection -a response asso- 2.3.2. Randomization
ciated with a mechanism previously described as Linked Recognition Volunteers in each cohort were randomized to study vaccine
[21] with neutralizing capacity in vitro and an antibody isotype suit- (n=6) or to BCG control vaccine (n=2), using a statistical application
able for viral clearance [19,21]. (random.org). The study had one unblinded nurse who only vacci-
Here, we report the results of a double-blind dose-escalation nated the participants. The rest of the study team was kept blinded. A
phase 1 clinical trial to evaluate the safety, tolerability, and Data and Safety Monitoring Board (DSMB) evaluated the safety data
 K. Abarca et al. / EClinicalMedicine 27 (2020) 100517 3

Fig. 1. Clinical study flow diagram and timeline. This phase 1 clinical study was double-blinded and dose-escalated. (A) Each cohort included 6 volunteers vaccinated with escalat-
ing doses of rBCG-N-hRSV (Cohort A: 5 £ 103 CFU (blue); Cohort B: 5 £ 104 CFU (red); Cohort C: 1 £ 105 CFU (green)) and 2 volunteers vaccinated with the standard BCG at full dose
(BCG-WT 2 £ 105 CFU (purple)). A DSMB evaluated the safety data of each cohort after the first 30 days of follow-up and decided if escalation could continue. (B) A timeline indicat-
ing the periods of immunization and end of visits is shown. RSV peaks reported during 2017 and 2018 are also indicated.

of each cohort after the first 30 days of follow up and decided if the congenital disability in the offspring of a study subject. For this study,
next cohort could be vaccinated. Further information of the study can grade 4 laboratory abnormalities were also considered as SAEs.
be found in the Study protocol included as Appendix 1. AEs intensity: pain and sensitivity were graded as I: mild pain or
tactile discomfort, II: pain or discomfort upon movement, the
2.4. Study schedule and follow-up requirement for analgesics, III: significant pain or discomfort at rest,
use of narcotic analgesics, or prevention of regular activities and IV:
Inclusion criteria included healthy -as determined by medical his- AEs requiring emergency unit consultation. Erythema and induration
tory, physical examination, and laboratory tests- male adults ranging were graded as I: 25 50 mm, II: 51 100 mm, III: >100 mm. Pustule
between 18-50 years old, previously vaccinated with 1 or 2 doses of and scab were graded as I: 1 9 mm, II: 10-30 mm, III: >30 mm, IV:
BCG. A detailed list of the inclusion and exclusion criteria is presented necrosis. Lymphadenopathy was graded as I: regional, up to 1 cm, II:
in the supplementary material (Table S1). regional 1,1 to 5 cm, III: multiple, or unique > 5 cm or suppurated,
Participants received a single dose of the vaccine, administered IV: disseminated. Other AEs were graded as I: does not interfere with
intradermally in the deltoid area following the standard national pro- normal activities, II: some interference, III: prevents normal activities,
tocol for BCG vaccination used in Chile, and were observed during the and IV: AEs requires emergency unit consultation [23].
following 3 hours. Participants were then evaluated at 1 3, 7, 14, 30,
60, 120, and 180 days post-vaccination (dpv) (Table 1). Follow up 2.5.2. Laboratory tests
phone calls were performed at 4, 21, 45, 90, and 150 dpv. The study Hematological and biochemical parameters included blood counts
was performed between July 2017 and June 2018 (Fig. 1B). (hemoglobin, leukocytes, neutrophils, lymphocytes, and platelet
count), transaminases, bilirubin, cholesterol, creatinine, ureic nitro-
2.5. Primary outcome: safety evaluation gen, plasmatic electrolytes, coagulation test (aPTT and prothrombin
time), creatine phosphokinase (CPK), and urine analyses. Laboratory
tests were performed at 7, 14, 30, 60, 120, and 180 dpv. Grading of
2.5.1. Clinical evaluation
laboratory adverse events was assessed by the Toxicity Grading Scale
Adverse events (AEs): Local solicited AEs included pain, sensitivity,
for Healthy Adult and Adolescent Volunteers Enrolled in Preventive
erythema, induration, pustule, scab, and axillary lymphadenopathy;
Vaccine Clinical Trials of the U.S. Department of Health and Human
these AEs were registered until the end of the study. General (sys-
Services [23].
temic) solicited AEs included fever, tachycardia, hypo/hypertension,
headache, fatigue, myalgia, nausea/vomiting, and diarrhea. These AEs
were recorded for 30 dpv. 2.5.3. Transmissibility assays
General AEs included general solicited AEs occurring beyond rBCG-N-hRSV in blood, urine, and saliva of vaccinated volunteers
30 dpv, and any other general AEs up to 180 dpv, as well as concomi- was evaluated by PCR-evaluating the presence of both Mycobacterium
tant medications administered to volunteers. AEs were collected by genes and the n gene of RSV- and by conventional mycobacterial cul-
self-report on diary cards, in study visits, and study phone calls. ture. DNA was extracted from body fluids by QIAamp kits (Qiagen).
Serious adverse event (SAEs) were defined as any untoward medi- Further information on molecular analysis is included in the Supple-
cal occurrence that: resulted in death; was life-threatening; required mentary Material. For conventional cultures, samples of blood, urine,
hospitalization or prolongation of existing hospitalization; resulted and saliva were seeded on selective culture media for Mycobacteria
in disability/incapacity or resulted in a congenital anomaly/ and observed 21 days after to evaluate the presence or absence of
4 K. Abarca et al. / EClinicalMedicine 27 (2020) 100517

Fig. 2. PPD- and N-RSV-specific humoral immune response in rBCG-N-hRSV-immunized volunteers. Anti-PPD and anti-N-RSV IgG levels were measured in the sera of all the volun-
teers at 0, 14, 30, 60, 120, and 180 dpv with rBCG-N-hRSV doses 5 £ 103 (blue), 5 £ 104 (red), and 1 £ 105 (green) CFU and the full dose of BCG-WT (Purple). IgG levels are expressed
as the geometric mean (and geometric standard deviation) of the concentrations of IgG (ng/mL) on a logarithmic scale. The responses against (A) PPD and (B) N-RSV antigens are
shown. Statistical differences were evaluated by a two-way ANOVA with a posterior Tukey test. If different letters are shown above a specific time point (with their respective color-
bar representing the corresponding group), then statistical differences (p<0.05) were found among those groups. If no letters are indicated above a group, then no statistical differ-
ences were found among them. n.s. = no statistical differences were found among any of the groups.

colony-forming units. Transmissibility of the vaccine was evaluated these populations in PBMC samples. ELISPOT assays were performed to
in all cohorts at three different time points (7, 30, and 180 dpv). detect cells secreting either IL-2 or IFN-g .

2.5.4. Criteria for vaccine safety and tolerability 2.7. Statistical analyses
We determined that immunization was safe and well-tolerated if
no subjects presented: (1) grade 4 local or general solicited AEs; (2) A sample of 24 subjects was recruited, considering a usual phase I
grade 4 general or laboratory AEs related to the vaccine; (3) SAEs con- studies sample size (20 100 subjects). A power calculation to esti-
sidered related to the vaccine, or (4) transmissibility of any of the vac- mate frequency of AE was not performed, as we used a conventional
cine components demonstrated in body fluids. sample size for a phase 1 study (20 100 subjects). Descriptive analy-
Assessment of causality included the following categories: non- ses were performed for local solicited, general, and laboratory
related (occurrence of the AEs is not reasonably related temporarily adverse events (AEs). All immunogenicity statistical analyses were
with the vaccine, or there is a cause that explains the event), possibly- performed using GraphPad Prism version 7.0 Software. Statistical sig-
related (administration of the study vaccine and the AEs are reasonably nificances were assessed using one-way or two-way ANOVA test
related temporarily, but an alternative cause is more likely to be respon- with a posteriori Tukey test when corresponded. Additional informa-
sible for AEs), and probably-related (administration of the study vaccine tion is indicated in each figure legend.
and the AEs are reasonably related temporarily, and the vaccine is more
likely to be responsible for AEs than other causes). 2.8. Role of the funding sources

2.6. Secondary outcomes: immunogenicity assays The funding sources had no role during the study design; in the
collection, analysis, or interpretation of data; in the writing of the
2.6.1. Humoral immunogenicity assays report; or in the decision to submit the paper for publication.
All humoral immunogenicity assays were performed with serum
obtained from peripheral blood of volunteers at 0, 14, 30, 60, 120, 3. Results
and 180 dpv. Samples were used for indirect ELISA assays and neu-
tralization assays, as indicated in the Supplementary Material section. 3.1. Subjects
Quantification of specific anti-N and anti-PPD antibody concentra-
tions were performed by ELISA, and RSV neutralization capacities of 42 males were screened, and 24 healthy males with ages ranging
these antibodies were determined by plaque reduction assays with- from 19 to 44 were enrolled and vaccinated, 8 in each cohort (Fig. 1A).
out complement, as described in [24]. Further information can be All participants were Hispanic and Chilean. Screening failures were due
found in the Supplementary Material. to detection of Gilbert Syndrome [n=5], hepatitis B and latent Mycobac-
terium tuberculosis infection [n=1], HIV infection [n=1], BCG scars [n=1],
2.6.2. Cellular immunogenicity assays and screening tests out of the normal range [n=10]. Weight ranged from
All cellular immunogenicity assays were performed using PBMCs 57.3 to 91 kg, height from 167 to 183 cm, and body mass index (BMI)
obtained from volunteers. Briefly, 20 mL of venous blood were collected from 19.2 to 29.9 kg/m2 (Table S2). Follow up of vaccinated participants
in heparinized tubes (BD Vacutainer), and PBMCs were purified by a was performed as described in Table 1. The successive cohort vaccina-
gradient of centrifugation with Leukocyte Separation Medium (LSM, tions were conducted without interruption, as approved by the DSMB.
Corning, Virginia, USA). Cells were collected, washed, counted in a Neu-
bauer chamber, and finally frozen. All samples were stored in a 150 °C 3.2. Safety and reactogenicity
Ultrafreezer until ELISPOT and flow cytometry assays were performed.
Flow cytometry assays and ELISPOT assays were performed as indicated All participants were followed for six months after vaccination,
in the Supplementary Material section. Once used, all samples were with no withdrawals or lost-to-follow ups. All vaccines were well tol-
evaluated in a blinded manner. Flow cytometry assays were performed erated; there were two SAEs, none considered related to the vaccine;
to characterize cellular populations and the production of cytokines by no participants were discontinued due to an AEs.
 K. Abarca et al. / EClinicalMedicine 27 (2020) 100517 5

Fig. 3. Evaluation of the neutralizing capacity of antibodies obtained from vaccinated subjects. The neutralizing capacities of the sera obtained from the immunized subjects were
tested and are shown as fold change (Normalized to Day 0 for each subject). The sera samples were tested at 0, 14, 30, 60, 120, and 180 dpv with rBCG-N-hRSV doses 5 £ 103 (A -
Blue), 5 £ 104 (B - Red), and 1 £ 105 (C - Green) CFU and the full dose of BCG-WT (D - Purple). Neutralization was tested with amounts of IgG ranging from 10 mg to 0.1 mg, which
are equivalent to dilutions 1/160 and 1/20.480, respectively. Highest amounts resulted in 100% neutralization, while lowest amounts resulted in 0% neutralization. Therefore, the
level of neutralization for 0.75 mg of total antibodies -dilution 1/2.560- is shown. Statistical differences were evaluated by a one-way ANOVA with a posterior Tukey test. n.s.= No sta-
tistical differences; *=P<0.05.

3.2.1. Clinical adverse events (AEs) or grade 2; 8 (6.5%) as severe or grade 3; and 1 (0.8%) as grade 4,
Local solicited AEs: most frequent were sensitivity, pustule, and scab, which was classified as SAEs (see below). Grade 3 and 4 AEs are pre-
occurring in all participants (N=24). Pain was present in 23 individuals, sented more in detail in Table S5.
erythema in 19, induration in 13, and lymphadenopathy in 2 (in both
cases, lymphadenopathy was less than 2 cm, non-suppurative, and 3.2.3. Severe adverse events (SAEs)
resolved without treatment). Erythema was detected more frequently Two SAEs were reported, neither considered related to study vac-
with increasing doses of the study vaccine (Table 2, sub-table 1). Days cine: (i) a grade 4 increase in CPK 15 days after vaccination with the
(median) post-vaccination for the onset of pustule and scab decreased control BCG, related to intense physical exercise two days before the
with increasing doses of the study vaccine (Table 2, sub-table 2). The exam. CPK values returned to normal levels in an analysis performed
median duration of pustule and pain increased with increasing doses of 16 days later; (ii) a one-day hospitalization due to thoracic pain second-
the study vaccine (Table 2, sub-table 3). The intensity of local solicited ary to supraventricular tachycardia (previous condition of the subject)
AEs was mostly mild (grade 1: 70.9%) and moderate (grade 2: 27.6%). detected 51 days after vaccination with the control BCG (not included).
Only one participant, who received the highest dose of the study vac-
cine, reported severe intensity (grade 3) for sensitivity and pain; how- 3.2.4. Transmissibility
ever, his symptoms lasted for just one day as grade 3. When analyzing None of the subjects evaluated were positive at any of the time
the subset of local solicited AEs with moderate (grade 2) intensity, sub- points for the n gene of RSV in the samples evaluated. Non-patho-
jects with pustule and scab increased in number with increasing doses genic Mycobacteria were identified by culture in saliva samples in 3
of the study vaccine (Table 2, sub-table 4). subjects; in one of them, PCR and sequencing were also performed.
General solicited AEs: eighty general solicited AEs were reported, In addition, urine samples at 30 and 180 dpv of one subject of the
72 (90.0%) were classified as mild intensity, and 8 (10.0%) as moder- intermediate cohort were positive for the 16S RNA of M. bovis. In this
ate intensity. The most frequent were headache (37.5%), fatigue subject, two extra control genes were tested to evaluate a possible
(17.5%), diarrhea (15.0%), and myalgia (12.5%). Considering all general relationship to the immunization: the is6110 and the esat6 genes,
solicited AEs, we found no association with increasing doses of the which have been previously reported to be used for the characteriza-
study vaccine (Table 2, sub-table 5). tion of Mycobacterium, specifically indicating whether the identified
General AEs: there was a total of 77 general AEs (Table S3). There bacterium belongs to the Tuberculosis complex -such as M. bovis- or
was no tendency in the frequency of general AEs with the dose of the not. In this sample, these genes were undetected, suggesting that the
vaccine received. Sixty-eight were considered not related to the vac- Mycobacteria was not M. bovis, and therefore should not be associ-
cine, and nine were considered possibly related. General AEs consid- ated with the study vaccine [25,26]. Table S6 shows the non-patho-
ered possibly related to the vaccine were diarrhea: (2), nausea (1), genic Mycobacteria species identified, and additional information can
bilateral otalgia (1), retro-orbital pain (1), headache (1), leg pain (1), be found in the Supplementary Material.
scalp pain (1), malaise (1). Urticaria occurred in one subject of the
highest dose vaccine group 12 days after vaccination, which was con-
3.3. Immunogenicity
sidered not related to the vaccine.

3.3.1. Humoral immune response

3.2.2. Laboratory examinations We evaluated antibody production against BCG (anti-PPD) and
Overall, 123 laboratory AEs were reported, as shown in detail in RSV (anti-N-RSV) in response to rBCG-N-hRSV immunization (Fig. 2).
Table S4. None was considered probably related to the study vaccine. For PPD, we detected antibodies in subjects vaccinated with all the
92 (74.8%) were classified as mild or grade 1; 22 (17.9%) as moderate doses tested for rBCG-N-hRSV, as well as in the subjects vaccinated
6 K. Abarca et al. / EClinicalMedicine 27 (2020) 100517

a b b
b,c b,c b,c b,c

A 1500
b,c B a
a,c a,c a,c
1000 b b
a,c
b b b
SFC/million cells

SFC/million cells
b,c b,c b,c
800
1000
(IFN-γ)

(IL-2)
600

400
500 Response
200 to PPD
0 0

rBCG-N-hRSV 5x10 3
D 14
30

60
ay 0

D 14
30

60
ay 0

0
C 12 D

18

12

18
D ay

D ay
ay

ay

rBCG-N-hRSV 5x10 4

ay

ay
ay

ay
D

ay

ay
D

D
D

D
b rBCG-N-hRSV 1x10 5
800 150 a b a
BCG-WT 2x105
SFC/million cells

SFC/milion cells
600
100
(IFN-γ)

(IL-2)
400 Response
50 to N-RSV
200

0 0

D 14
30

60
ay 0

0
D 14
30

60
ay 0

12

18
12

18

D ay
D ay

ay

ay
ay

ay

IFN- IL-2

ay

ay
D
D

ay

ay

D
D

D
D

Fig. 4. PPD- and N-RSV-specific cellular immune response in the rBCG-N-hRSV-immunized subjects evaluated by ELISPOT. Cellular responses to PPD (A and B) and N-RSV (C and D)
antigens in all PBMCs samples were measured at 0, 14, 30, 60, 120, and 180 dpv with rBCG-N-hRSV doses 5 £ 103 (blue), 5 £ 104 (red), and 1 £ 105 (green) CFU and the full dose of
BCG-WT (Purple). The IFN-g - (A and C) and IL-2- (B and D) secreting cells were detected by ELISPOT and plotted as spot-forming cells (SFC) per million cells. Statistical differences
were evaluated by a two-way ANOVA with a posterior Tukey test. If different letters are shown above a specific time point (with their respective color-bar representing the corre-
sponding group), then statistical differences (p<0.05) were found among those groups. If no letters are indicated above a group, then no statistical differences were found among
them.

with BCG-WT (Fig. 2A). The highest anti-PPD antibody production when the RSV outbreak was over for at least 2 months, fold-increase
was observed at 60 and 120 dpv, with the intermediate dose of the of anti-N antibodies started immediately at 14 dpv, and it was even
rBCG-N-hRSV (Cohort B: 5 £ 104 CFU) exhibiting the highest concen- more pronounced than the one seen for cohort A (Fig. S1C). There-
tration at 60 dpv, as compared to the other doses used and the BCG fore, we can suggest that rBCG-N-hRSV induce a sustained produc-
control (BCG-WT: 2 £ 105 CFU) (Fig. 2A). This point -60 dpv for tion of anti-N antibodies, at least for Cohorts A and C.
Cohort B- was statistically different as compared to all the other
groups at 0 dpv and also to the lowest dose of the study vaccine 3.3.2. Antibody neutralization assays
(Cohort A: 5 £ 103 CFU) and the BCG-WT control group, at 14 and Since the production of total anti-N antibodies in the immunized
30 dpv. For anti-N-RSV antibodies, we observed an increase in the subjects could be due to seasonal exposure to RSV (Fig. 1B), neutrali-
concentrations of all the groups at 60-, 120- and 180- dpv, although zation assays were performed as described previously [21], in order
this was not statistically significant. We also observed an early to determine whether immunization with rBCG-N-hRSV resulted in
increase in the levels of anti-N antibodies at 30 dpv in the subjects improved neutralizing capacities of the produced antibodies. Fig. 3
who received the highest dose of rBCG-N-hRSV (Cohort C: 1 £ 105), shows the neutralizing capacities -depicted as Fold Change normal-
as compared to the subjects in Cohort A or BCG-WT (Fig. 2B). Accord- ized to day 0- of the antibodies obtained from the escalating doses of
ingly, from 14 to 60 dpv, Cohort C exhibited the highest concentra- the rBCG-N-hRSV (Fig. 3A-C) and the BCG-WT (Fig. 3D) immunized
tion of anti-N antibodies, supporting our dose-escalated study. At subjects. These assays were performed evaluating the neutralizing
120 dpv, all cohorts exhibited increased levels of anti-N-RSV antibod- capacity detected in a fixed amount of total IgG for all the volunteers
ies, suggesting possible natural exposure to RSV -due to seasonal out- in the three cohorts (0.75 mg, equivalent to a dilution 1/2,560), using
break-, which would promote antibody production in all the cohorts a neutralization assay protocol described previously, with modifica-
for both vaccines (Fig. 2B). However, in BCG-WT immunized volun- tions [24]. We observed increased neutralization capacities of viral
teers antibodies concentrations starts to wane at 120 dpv, while particles, starting from 14 dpv, in the sera obtained from subjects
rBCG-N-hRSV immunized volunteers keep their antibody levels high, immunized with rBCG-N-hRSV, as compared to the neutralization
up until 180 dpv. To better understand the effect of natural exposure capacity detected at day 0. In agreement with the results shown in
to RSV in the anti-N antibody response, we determined the anti-N Fig. S1, higher increases of neutralization capacities were observed
antibody fold-increase -normalized to day 0- for each volunteer (Fig. for volunteers immunized with rBCG-N-hRSV in cohort A, as com-
S1). In cohort A, which was immunized in the middle of the RSV sea- pared to cohort B and C. These could be related to recent seasonal
sonal outbreak (Fig. 2B), we observed a more rapid and pronounced RSV exposure of some volunteers included in cohorts B and C, which
fold-increase of anti-N antibodies at 14 dpv in 4 out of 6 volunteers showed >50% neutralizing capacities at day 0 (data not shown). Nev-
immunized with the rBCG-N-hRSV vaccine, as compared to volun- ertheless, in each cohort immunized with rBCG-N-hRSV the neutrali-
teers of the same cohort immunized with BCG WT (Fig. S1A and D). zation capacity of the sera increased over time (Fig. 2A C). No
In Cohort B, which was immunized at the end of the RSV outbreak, significant differences in the neutralization ability of the sera were
fold-increase of anti-N antibodies started to rise at 60 dpv, suggesting detected for BCG-WT volunteers, although a subtle increase in the
that -probably due to recent RSV exposure- levels of anti-N antibod- percentage of neutralization was observed at 120 dpv. Fig. S2 shows
ies were already high (Fig. S1B). In cohort C, which was immunized the fold change of the neutralization capacities for each volunteer,
 K. Abarca et al. / EClinicalMedicine 27 (2020) 100517 7

Table 1
Follow up schedule.

Screening Va Follow up
Day -10 -3 0 1 2 3 4 7 14 21 30 45 60 90 120 150 180

Clinical evaluation x x x x x x x x x x x x
Informed Consent x
Transmissibility samples x x x
Immunogenicity samples x x x x x x
Adverse Events report x x x x x x x x x x x x x x x
*Va=Vaccination.

while figure S3A shows pictures of the changes in the neutralization found. Mycobacterium bovis and the RSV component in the vaccine
capacities of one volunteer for each cohort, for all the time points were not observed in the different fluids evaluated, suggesting that
evaluated. Two positive controls -a polyclonal pool of anti-RSV anti- there is no vaccine excretion. These data suggest that this BCG based
bodies from BEI resources (NR-21973) and palivizumab- were vaccine has an adequate safety profile, similar to the routinely used
included in the assays to confirm reproducibility of the results, and BCG [27], and therefore it could be used in the future to replace the
their neutralizing effects are shown in Fig. S3B. regular vaccine administered to newborns.
The results obtained from these neutralization assays suggest that We measured immunogenicity against PPD and the N-RSV protein
immunization with rBCG-N-hRSV most-likely promotes the produc- by evaluating the production of specific IgGs and the T cell response
tion of antibodies with increasing neutralizing capacities -as com- against these antigens. Specific IgGs increased upon immunization in
pared to the BCG-WT group- as early as 14 dpv. all the subjects evaluated for antibodies against both PPD and N-RSV.
However, this response was dependent on the dose used and may be
3.3.3. Cellular immune response influenced by the time of the year when cohorts were immunized, as
PPD and N-RSV-specific immune responses in vaccinated subjects some of the subjects were probably exposed to natural RSV infection
were evaluated by ELISPOT (Fig. 4) and flow cytometry assays (Fig. during autumn and winter seasons. In this line, recruitment and
S4). The number of IFN-g +- and IL-2+-producing cells were evaluated immunization of volunteers per cohort are shown in Fig. 1B. Cohort A
by ELISPOT and are shown as the number of spot-forming-cells (SFC) was mostly recruited during July, the month when the outbreak of
per million cells (Fig. 4). The data obtained show a dose-dependent RSV was reported. This overlap could result in low levels of both cells
increase in the number of IFN-g + and IL-2+ secreting cells in response and antibodies specific for RSV at day 0 in Cohort A, as compared to
to PPD at 14 dpv, which was at least 4-fold times higher -and statisti- the levels found in Cohort B and C at the same time (Fig S3), and this
cally different- in Cohort C, as compared to the BCG-WT group could have a masking effect over the interpretation of the results
(Fig. 4A and B). A similar response was observed at 30 dpv, in which obtained by these particular cohorts. Despite this, subjects immu-
the response against PPD was dose-dependent. At later time points nized with the highest dose of rBCG-N-hRSV showed the earliest
-180 dpv-, the response detected in the volunteers from Cohort A increase of anti-N antibodies -14 dpv-, which lasted longer -up to
and B and BCG-WT was not higher than the one observed in Cohort C 180 dpv. Importantly, this effect in cohort C was unlikely affected by
volunteers. An increased response of IFN-g +-secreting cells upon seasonal exposure to RSV during the study (Figure 1B). Subjects
stimulation with N-RSV was detected in Cohorts B and C, while an immunized with BCG-WT were distributed among the three cohorts,
increased response in IL-2+-secreting cells was detected in volunteers which resulted in subjects exposed to the same conditions as each
included in Cohort C, particularly at 14 dpv (Fig. 4C and D). This cohort. Nevertheless, we observed increasing neutralization abilities
increased response was only detected in subjects vaccinated with in the subjects immunized with rBCG-N-hRSV, as compared to the
rBCG-N-hRSV, since vaccination with BCG-WT mostly exhibited an subjects immunized with BCG-WT, which was more evident for
invariable response. Also, levels of IFN-g + and IL-2+-secreting cells cohort A. This is depicted by the fold-increases at 60 and 120 dpv in
remained stable during the 180 days of monitoring (Fig. 4C and D). the rBCG-N-hRSV immunized subjects, as compared to the BCG-WT
Additionally, specific IFN-g +-IL-2+ (g 2) and TNF-a+-IL-2+ (a 2) immunized subjects. These results support the notion that immuni-
secreting CD4+ and CD8+ T cells were evaluated by flow cytometry zation with rBCG-N-hRSV could promote the production of anti-RSV
(Fig. S4). The highest numbers of g 2 and a 2 cells were detected antibodies more effectively. However, future clinical trials need to be
starting at 30 dpv against PPD, mainly in Cohort C (Fig. S3A D). This performed when the circulation of RSV is low, including more BCG-
response was similarly found in both CD4+ and CD8+ T cells. A lower WT immunized as well as non-immunized volunteers, in order to
but similar response was observed against N-RSV in Cohort C, exhib- evaluate immunogenicity specifically induced by the study vaccine,
iting higher numbers of g 2 and a 2 -both CD4+ and CD8+- secret- ruling out the effect of seasonal RSV exposure.
ing T cells (Fig. S3E-H). The response to both PPD and N-RSV antigens We found that cells from all volunteers responded to an in vitro
showed a constant increase in time when g 2 T cells were evaluated, stimulation with the RSV N protein, as determined by the secretion
as compared to a 2 T cells, which was not constant. of IFN-g , IL-2, and TNF-a (Figs. 3 and S6). Consistent with these data,
all participants exhibited increased numbers of SFC and higher fre-
4. Discussion quencies of IFN-g -secreting CD4+ and CD8+ T cells after in vitro stimu-
lation with the RSV N protein, as determined by ELISPOT. However,
The rBCG-N-hRSV vaccine candidate was safe, well-tolerated, and this general response may be due to a recall immune response after
immunogenic in healthy male adult volunteers. No SAEs related to seasonal RSV infection, which is known to occur throughout life
the vaccine were reported up to 180 dpv. Local response to the BCG [3,28,29]. This recall immunity could also have an impact on further
component of the vaccine (development of pustule and then a scab) studies that we could perform, and it will be particularly significant if
occurred as expected. Although none of the local reactions were we evaluate this vaccine in newborns, where no pre-existing RSV
severe, higher reactogenicity was observed in some parameters as immune response should be found. In that particular population, this
the study vaccine dose increased. All local reactions were well toler- could have further implications and lead to different outcomes like
ated and resolved spontaneously. Additionally, no general AEs nor the ones seen for the adult volunteers. As seen for PPD measure-
laboratory abnormalities probably-related to the study vaccine were ments, N-RSV immune responses increased with higher doses of the
8 K. Abarca et al. / EClinicalMedicine 27 (2020) 100517

vaccine, indicating that it is possible to detect a specific immune Data Sharing

response against the N-RSV protein. Importantly, polyfunctional T
cell immune responses, as well as IFN-g - and IL-2-positive cells were All the data reported in this manuscript is available upon request
more pronounced at 30 dpv, as seen in the cellular immunogenicity to the corresponding author.
assays (Fig. S6). Prior to vaccination, all participants displayed a mod-
est frequency of IFN-g and IL-2 secreting cells in response to PPD
stimulation, likely due to a prior vaccination in their childhood with Supplementary materials
BCG (Table S1). Therefore, we expected to find a specific recall
immune response after in vitro cell stimulation with PPD [30]. Upon Supplementary material associated with this article can be found
immunization, all the volunteers exhibited a sustained increase in in the online version at doi:10.1016/j.eclinm.2020.100517.
IFN-g and IL-2 secretion, starting from 14 dpv (Figs. 3 and S6).
Both immunogenicity and reactogenicity responses were lower in References
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