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Article Neonatal Genetic Delivery of Anti-Respiratory Syncytial (RSV) Antibody by Non- Primate-Based Adenoviral Vector to Provide Protection against RSV

Rika Gomi 1, Anurag Sharma 1, Wenzhu Wu 1 and Stefan Worgall 1,2,*

1 Department of Pediatrics, Weill Cornell Medicine, New York, NY 10065, USA; [email protected] (R.G.); [email protected] (A.S.); [email protected] (W.W.) 2 Department of Genetic Medicine, Weill Cornell Medicine, New York, NY 10065, USA * Correspondence: [email protected]; Tel.: +1-646-962-6236

 Received: 1 December 2018; Accepted: 22 December 2018; Published: 29 December 2018 

Abstract: Respiratory syncytial virus (RSV) is one of the leading causes of lower respiratory tract in infants. Immunoprophylaxis with the anti-RSV monoclonal antibody, palivizumab, reduces the risk for RSV-related hospitalizations, but its use is restricted to high-risk infants due to the high costs. In this study, we investigated if genetic delivery of anti-RSV antibody to neonatal mice by chimpanzee adenovirus type 7 expressing the murine form of palivizumab (AdC7αRSV) can provide protection against RSV. Intranasal and intramuscular administration of AdC7αRSV to adult mice resulted in similar levels of anti-RSV IgG in the serum. However, only intranasal administration resulted in detectable levels of anti-RSV IgG in the bronchoalveolar lavage fluid. Intranasal administration of AdC7αRSV provided protection against subsequent RSV challenge. Expression of the anti-RSV antibody was prolonged following intranasal administration of AdC7αRSV to neonatal mice. Protection against RSV was confirmed at 6 weeks of age. These data suggest that neonatal genetic delivery of anti-RSV antibody by AdC7αRSV can provide protection against RSV.

Keywords: respiratory syncytial virus (RSV); immunoprophylaxis; palivizumab; chimpanzee adenovirus type 7 (AdC7); neonate; passive immunization; genetic gene delivery

1. Introduction Respiratory syncytial virus (RSV) is one of the leading causes of lower respiratory tract in children, with a high morbidity and mortality, especially in developing countries [1–3]. Most children are infected during their first year of life, and infants under six months of age are at highest risk for severe disease, especially those with bronchopulmonary dysplasia or cyanotic heart disease [4,5]. Despite the extensive efforts over decades, there are no effective vaccines or treatments for RSV. Immunoprophylaxis with the neutralizing monoclonal antibody, palivizumab, is the only available drug to prevent infections with RSV [6–8]. Although palivizumab is safe and well-tolerated for prophylactic use, its use is limited to high-risk infants due to the high costs and the need for monthly intramuscular injections during the RSV season [9]. Since most deaths attributed to RSV occur in low-income countries where expensive prophylaxis is not affordable [3], more cost-effective strategies are needed to spare more infants at risk from severe RSV disease. Genetic delivery of neutralizing antibodies using gene transfer vectors is an alternative strategy to achieve sustained expression of neutralizing antibodies. Among all currently available viral vectors, adenovirus (Ad) is one of the most efficient gene delivery systems, but pre-existing immunity against common human serotypes, such as Ad5, is hampering its use in . Non-human primate-derived

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Ad, such as chimpanzee Ad serotype 7 (AdC7), are alternative vectors and are less likely to be affected by pre-existing immunity [10–12]. We have previously reported that anti-Ad5 neutralizing antibodies do not cross-neutralize AdC7, and that maternal immunization of mice with the AdC7 vector expressing RSV vaccine can protect their pups against RSV infection [13]. Therefore, we adopted AdC7 as a gene transfer vector to deliver anti-RSV neutralizing antibody to infants, expecting a successful application in the presence of maternal anti-human Ad antibodies. Anti-Ad immunity induced by the vector limits long-term expression of the transgene and is thus another limitation of Ad vectors. However, immune tolerance may allow longer persistence when the vector is administered to neonates. The neonatal immune system is much less likely to develop a vigorous immune response to transgenic proteins [14,15]. In this study, we administered AdC7 vector expressing anti-RSV antibody (AdC7αRSV) to neonatal mice and evaluated the efficacy of the delivered anti-RSV antibody to protect against RSV infection.

2. Materials and Methods

2.1. Mice BALB/c mice were purchased from The Jackson Laboratories (Bar Harbor, ME, USA) housed under specific pathogen-free conditions, and bred to obtain neonatal mice. Adult female BALB/c mice were used at 8 weeks of age, and neonatal mice were used between 24 and 48 h after the birth. All animal studies were conducted in accordance with the protocols reviewed and approved by the Weill Cornell Institutional Animal Care and Use Committee (protocol number 2015-0011). All efforts were made to minimize the suffering of the animals.

2.2. RSV The RSV strains A2 (VR-1540: ATCC), Line19 and rA2-F19 (kindly provided by ML Moore, Emory University, Atlanta, GA, USA) were propagated in Hep-2 cells, purified and quantified as described previously [16].

2.3. Generation of an AdC7 Vector Expressing Murine Anti-RSV IgG (AdC7αRSV) The recombinant Ad vectors used in this study are replication defective E1-, E3-deleted Ad vectors based on the chimpanzee AdC7. The AdC7 plasmid pPan-GFP (kindly provided by JM Wilson, University of Pennsylvania, Philadelphia, PA, USA) was digested with I-Ceul and PI-Scel, and the expression cassette of anti-RSV antibody carrying (50 to 30) the / followed by cDNAs encoding the anti-RSV light chain, the internal ribosomal entry site (IRES), the anti-RSV heavy chain, and the SV40 signal [17] was inserted into the E1 region using the same restriction enzyme sites. AdC7GFP, an AdC7 vector with the green fluorescent protein cDNA under control of a prokaryotic promoter which does not lead to transgene expression in mammalian cells, were used as controls. The AdC7αRSV vectors were propagated in HEK-293 cells and purified by centrifugation twice through a CsCl gradient as previously described [18], and the particle units (pu) were determined spectrophotometrically [19].

2.4. Western Blot Analysis To confirm the expression of anti-RSV IgG in vitro, supernatants of A549 cells infected with AdC7αRSV were separated by SDS-PAGE under both non-reducing and reducing conditions. Following transfer to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA, USA) murine IgG was detected using a horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG antibody (Sigma, St. Louis, MO, USA) and Immobilon Western Chemiluminescent HRP substrate (EMD Millipore, Burlington, MA, USA). The supernatant of mock-infected cells was used as negative control. Mouse serum obtained 8 weeks following RSV infection was used as a positive control. Vaccines 2019, 7, 3 3 of 10

2.5. Dot Blot To evaluate binding of anti-RSV IgG to RSV, RSV Line19 (1.2 × 104 pfu/spot) was blotted to the PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA) and then developed with culture supernatant of HEK-293 cells infected with AdC7αRSV followed by the sheep anti-mouse IgG-peroxidase antibody as described above. Ad5 (2.0 × 106 pu/spot) was blotted as control.

2.6. Plaque Reduction Assay Serial dilutions of supernatants from HEK-293 cells that had been infected for 48 h with AdC7αRSV were incubated with RSV Line19 (5 × 103 pfu/mL) for 1 h at 37 ◦C, and then added to Vero cells. The number of plaques was quantified after 4 days as previously described [16].

2.7. Expression of anti-RSV IgG In Vivo AdC7αRSV or AdC7GFP (5 × 1010 pu each) diluted in 40 µl PBS were administered intranasally or intramuscularly to 8-week-old female BALB/c mice. Neonatal mice received either 3 × 1010 pu (5µL) or 6 × 1010 pu (10 µL) of the vectors intranasally. The levels and kinetics of anti-RSV IgG following administration of AdC7αRSV were quantified in serum and bronchoalveolar lavage (BAL) by ELISA. Serial dilutions of serum and BAL were added to flat-bottomed 96-well EIA/RIA plates (Corning, Corning, NY, USA) coated with 1µg/mL of human anti-palivizumab clone AbD23967 (HCA261, Bio-Rad-Antibodies, Hercules, CA, USA), followed by PBST + 5% blotting grade blocker (Bio-Rad Laboratories, Hercules, CA, USA. Detection was performed using an HRP-conjugated sheep anti-mouse IgG (Sigma, St. Louis, MO, USA) in PBS + 1% blotting grade blocker and substrate (hydrogen peroxide/tetramethylbenzidine) (R&D systems, Minneapolis, MN, USA) and the absorbance at 450 nm was measured. Titers were calculated with a log (OD)–log(dilution) interpolation model, with detection cut-off equal to 2-fold the background absorbance. Half-life (t1/2) was calculated by the formula: t1/2 = t ∗ ln(2)/ln(N0/Nt), (1) where t = time elapsed, N0 = titer at 1 week, and Nt = titer at 4 weeks after the administration of AdC7αRSV.

2.8. Protection Against RSV Infection Following AdC7αRSV Administration Mice were challenged 3 days or 6 weeks following Ad vector administration with RSV Line19 or RSV A2 (each 1 × 106 pfu) via the intranasal route. Four days after the challenge, RSV was quantified in lung homogenates by plaque assay, or viral RNA was quantified by real-time PCR as described previously [13]. For the detection of RSV Line19 viral RNA, Line19 NS1-specific primers (Forward: 50-CAGCGCTACAAAATGGAGGTTA-30, Reverse: 50-TTAGACCATTAGGTTGAGAGCAATGT-30) and FAM-labeled TaqMan MGB probes (50-ATATGGGAAATGATGGAATT-30) were used.

2.9. Statistics Statistical analyses were performed using one-way ANOVA, followed by two-tailed unpaired Student’s t-tests. Statistical significance was determined at p < 0.05.

3. Results

3.1. Expression of Murine Anti-RSV In Vitro AdC7αRSV (Figure1) was generated and propagated in HEK-293 cells. Vaccines 2018, 6, x 4 of 10

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Figure 1. Schema of the chimpanzee adenovirus type 7 vector expressing murine anti-respiratory syncytial virus antibody (AdC7αRSV) . The E1/E3 genes of AdC7 are deleted (ΔE1/ΔE3) and replaced Figure 1. Schema of the chimpanzee adenovirus type 7 vector expressing murine anti-respiratory by the expression cassette of the anti-RSV antibody cDNAs using the restriction enzyme sites I-CeuI syncytial virus antibody (AdC7αRSV). The E1/E3 genes of AdC7 are deleted (∆E1/∆E3) and replaced Figureand PI-SceI. 1. Schema The ofexpression the chimpanzee cassette adenovirusincludes th etype cytomegalovirus 7 vector expressing promoter murine (P CMV), anti-respiratory followed by by the expression cassette of the anti-RSV antibody cDNAs using the restrictionΔ enzymeΔ sites I-CeuI syncytialcDNAs virusencoding antibody the anti-RSV (AdC7α lightRSV) chain . The (LC),E1/E3 the genes poliovirus of AdC7 internal are deleted ribosome ( E1/ entryE3) andsite (IRES),replaced the andby the PI-SceI. expression The expression cassette of cassettethe anti-RSV includes antibody the cytomegalovirus cDNAs using th promotere restriction (P CMV),enzyme followed sites I-CeuI by anti-RSV heavy chain (HC), and SV40 polyadenylation signal (SV40 pA). cDNAsand PI-SceI. encoding The expression the anti-RSV cassette light chainincludes (LC), the the cytomegalovirus poliovirus internal promoter ribosome (P CMV), entry followed site (IRES), by the anti-RSV heavy chain (HC), and SV40 polyadenylation signal (SV40 pA). cDNAsTo confirm encoding expression the anti-RSV of murinelight chain anti-RSV (LC), the IgG poliovirus in vitro, internal A549 ribosome cells were entry infected site (IRES), with the purified anti-RSV heavy chain (HC), and SV40 polyadenylation signal (SV40 pA). AdC7Toα confirmRSV, and expression cell culture of murinesupernatants anti-RSV were IgG assessin vitroed by, A549 Western cells Blot were analysis infected (Figure with purified2). Under non-reducingα conditions, a complex of 150 kDa, corresponding to the size of the completely AdC7ToRSV, confirm and expression cell culture of supernatants murine anti-RSV were assessedIgG in vitro, by Western A549 cells Blot were analysis infected (Figure with2). purified Under assembled murine IgG, was detected (Figure 2A, lane 1). Under reducing conditions, individual non-reducingAdC7αRSV, and conditions, cell culture a complex supernatants of 150 kDa, were corresponding assessed by Western to the size Blot of analysis the completely (Figure assembled 2). Under heavy chains (HC, 50 kDa) and light chains (LC, 25 kDa) were detected (Figure 2B; lane 4). murinenon-reducing IgG, was conditions, detected (Figurea complex2A, laneof 150 1). UnderkDa, corresponding reducing conditions, to the individual size of the heavy completely chains (HC,assembled 50 kDa) murine and light IgG, chains was (LC,detected 25 kDa) (Figure were 2A, detected lane 1). (Figure Under2B; reducing lane 4). conditions, individual heavy chains (HC, 50 kDa) and light chains (LC, 25 kDa) were detected (Figure 2B; lane 4).

Figure 2. Expression of murine anti-respiratory syncytial virus (anti-RSV) IgG in vitro. Anti-RSV IgG

in supernatants of A549 cells infected with chimpanzee adenovirus type 7 vector expressing murine anti-RSVFigure 2. IgG Expression (AdC7αRSV) of murine was detected anti-respiratory by Western syncytial Blot analysis. virus (anti-RSV) (A) Expression IgG in vitro. of the Anti-RSV full-length IgG in supernatants of A549 cells infected with chimpanzee adenovirus type 7 vector expressing murine murine IgG under non-reducing conditions. (B) Expression of the heavy chain (HC) and light chain (LC)anti-RSV of murine IgG IgG(AdC7 underαRSV) reducing was detected conditions. by Western The supernatant Blot analysis. of mock-infected (A) Expression cells of the was full-length used as Figure 2. Expression of murine anti-respiratory syncytial virus (anti-RSV) IgG in vitro. Anti-RSV IgG a negativemurine IgG control under (lanes non-reducing 2, 5). Mouse conditions. serum 8(B weeks) Expression post infection of the heavy with chain RSV (HC) was usedand light as a positivechain (LC) in supernatants of A549 cells infected with chimpanzee adenovirus type 7 vector expressing murine controlof murine (lanes IgG 3, 6). under Detection reducing was withconditions. a horseradish The supernatant peroxidase of (HRP)-conjugated mock-infected cells sheep was anti-mouse used as a anti-RSV IgG (AdC7αRSV) was detected by Western Blot analysis. (A) Expression of the full-length IgGnegative and HRP control chemiluminescence (lanes 2, 5). Mouse substrate. serum 8 weeks post infection with RSV was used as a positive murinecontrol IgG (lanes under 3, 6).non-reducing Detection was conditions. with a horseradish (B) Expression peroxidase of the heavy (HRP)-conjugated chain (HC) and sheep light chain anti-mouse (LC) TheofIgG murine binding and HRPIgG ability underchemiluminescence of reducing the expressed conditions. substrate. anti-RSV The supernatant IgG to RSV of was mock-infected confirmed bycells dot was blot used ELISA as a using RSV Line19negative binding control to(lanes cell culture2, 5). Mouse supernatants serum 8 weeks of HEK-293 post infection cells infected with RSV with was AdC7 usedα asRSV a positive (Figure 3A, control (lanes 3, 6). Detection was with a horseradish peroxidase (HRP)-conjugated sheep anti-mouse lanes 1,The 2). binding Plaque-reduction ability of the assay expressed showed anti-RSV dose-dependent IgG to RSV neutralization was confirmed of RSVby dot by blot the ELISA antibodies using IgG and HRP chemiluminescence substrate. (FigureRSV Line193B). binding to cell culture supernatants of HEK-293 cells infected with AdC7αRSV (Figure The binding ability of the expressed anti-RSV IgG to RSV was confirmed by dot blot ELISA using

RSV Line19 binding to cell culture supernatants of HEK-293 cells infected with AdC7αRSV (Figure

Vaccines 2018, 6, x 5 of 10 Vaccines 2018, 6, x 5 of 10 3A, lanes 1, 2). Plaque-reduction assay showed dose-dependent neutralization of RSV by the 3A, lanes 1, 2). Plaque-reduction assay showed dose-dependent neutralization of RSV by the Vaccinesantibodies2019, 7, 3(Figure 3B). 5 of 10 antibodies (Figure 3B).

Figure 3. Assessment of anti-respiratory syncytial virus (anti-RSV) IgG expression in vitro. FigureFigure 3.3. AssessmentAssessment of of anti-respiratory anti-respiratory syncytial syncytial vi virusrus (anti-RSV) (anti-RSV) IgG IgG expression expression inin vitrovitro.. Supernatants of HEK-293 cells infected with AdC7αRSV were assessed for the presence of functional SupernatantsSupernatants of of HEK-293 HEK-293 cells cells infected infected with with AdC7 AdC7ααRSVRSV were were assessed assessed for for the the presence presence of of functional functional anti-RSV IgG. (A) Binding to RSV. Supernatants were incubated with RSV Line19 or Ad5 (control) anti-RSVanti-RSV IgG.IgG. (A(A)) Binding Binding to to RSV. RSV. Supernatants Supernatants were were incubated incubated withwith RSVRSV Line19Line19 oror Ad5Ad5 (control)(control) immobilized on a polyvinylidene difluoride (PVDF) membrane followed by an HRP-conjugated immobilizedimmobilized onon aa polyvinylidene polyvinylidene difluoridedifluoride (PVDF)(PVDF) membranemembrane followedfollowed byby an an HRP-conjugated HRP-conjugated sheep anti-mouse IgG. Mouse serum 8 weeks post infection with RSV was used as a positive control sheepsheep anti-mouse anti-mouse IgG. IgG. Mouse Mouse serum serum 8 8 weeks weeks post post infection infection with with RSV RSV was was used used as as a a positive positive control control (lane 4). (B) Plaque-reduction assay. Serial dilutions of supernatants were incubated with RSV Line19 (lane(lane 4). 4). ( B(B)) Plaque-reduction Plaque-reduction assay. assay. Serial Serial dilutions dilutions of of supernatants supernatants were were incubated incubated with with RSV RSV Line19 Line19 (5x1033 pfu/ml) for 1 hour, followed by infection of Vero cells. Data are shown as % reduction of (5(5x10× 103 pfu/ml)pfu/mL for) for 1 1hour, h, followed followed by infectionby infection of Vero of cells.Vero Datacells. are Data shown are asshown % reduction as % reduction of plaques of plaques after 4 days, with mean ± SEM of 4 replicates. afterplaques 4 days, after with 4 days, mean with± SEM mean of ± 4 SEM replicates. of 4 replicates. 3.2.3.2. Assessment Assessment of of Anti-RSV Anti-RSV IgG IgG Delivered Delivered by by AdC7 AdC7αRSVαRSV In In Vivo Vivo 3.2. Assessment of Anti-RSV IgG Delivered by AdC7αRSV In Vivo TheThe kinetics kinetics of of anti-RSV anti-RSV IgG IgG in in the the serum serum of of adult adult mice mice following following intranasal intranasal or or intramuscular intramuscular The kinetics of anti-RSV IgG in the serum of adult mice following intranasal or intramuscular administrationadministration of of AdC7 AdC7αRSVαRSV showed showed that that the the anti-RSV anti-RSV IgG IgG titer titer peaked peaked at at one one week week following following administration of AdC7αRSV showed that the anti-RSV IgG titer peaked at one week following administration,administration, andand then then gradually gradually decreased decreased to non-specific to non-specific response response levels by levels eight byweeks eight (Figure weeks 4A). administration, and then gradually decreased to non-specific response levels by eight weeks (Figure 4A). (FigureThe estimated4A). The estimatedhalf-life was half-life 19 days. was Although 19 days. th Althoughe titers were the titershigher were following higher intramuscular following The estimated half-life was 19 days. Although the titers were higher following intramuscular intramuscularadministration, administration, the kinetics of the anti-RSV kinetics levels of anti-RSV over time levels were over similar time werebetween similar the betweentwo routes the of administration, the kinetics of anti-RSV levels over time were similar between the two routes of twoadministration. routes of administration. In contrast, Inanti-RSV contrast, IgG anti-RSV in the IgGBAL in at the one BAL week at one was week only was detectable only detectable following administration. In contrast, anti-RSV IgG in the BAL at one week was only detectable following followingintranasal intranasal administration administration (Figure 4B). (Figure 4B). intranasal administration (Figure 4B).

Figure 4. Assessment of anti-respiratory syncytial virus (anti-RSV) IgG expression in adult mice. (A) FigureFigure 4. 4. Assessment of of anti-respiratory anti-respiratory syncytial syncytial virus virus (anti-RSV) (anti-RSV) IgG IgG expression expression in inadult adult mice. mice. (A) (AKinetics) Kinetics of anti-RSV of anti-RSV IgG in IgG the inserum the following serum following intranasal intranasal (i.n.) or intramuscular (i.n.) or intramuscular (i.m.) administration (i.m.) Kinetics of anti-RSV IgG in the serum following intranasal (i.n.) or intramuscular (i.m.) administration administrationof AdC7αRSV of (5 AdC7x 1010α pu).RSV Titers (5 × were1010 measuredpu). Titers by were ELISA. measured Data are by shown ELISA. as mean Data are± SEM shown of 4 mice as of AdC7αRSV (5 x 1010 pu). Titers were measured by ELISA. Data are shown as mean ± SEM of 4 mice meanper group.± SEM (B of) Anti-RSV 4 mice per IgG group. in the ( Bbronchoalveolar) Anti-RSV IgG lavage in the (BAL) bronchoalveolar 1 week following lavage administration (BAL) 1 week of per group. (B) Anti-RSV IgG in the bronchoalveolar lavage (BAL) 1 week following administration of followingAdC7αRSV administration (5 x 1010 pu). of Titers AdC7 αwereRSV measured (5 × 1010 pu). by EL TitersISA.were Datameasured are shown by with ELISA. mean Data ± SEM. are shown AdC7αRSV (5 x 1010 pu). Titers were measured by ELISA. Data are shown with mean ± SEM. with mean ± SEM.

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3.3. Protection against RSV Infection following AdC7αRSV Administration in Adult Mice 3.3. Protection Against RSV Infection Following AdC7αRSV Administration in Adult Mice To evaluate if AdC7αRSV can deliver sufficient antibody to protect against RSV infection, α AdC7Toα evaluateRSV, AdC7GFP if AdC7 or RSVPBS (No can AdC7 deliver control) sufficient were antibody intranasally to protectadministered against to RSVeight-week-old infection, α AdC7femaleRSV, BALB/c AdC7GFP mice, followed or PBS (No by AdC7RSV Line19 control) infection were intranasally three days later. administered RSV viral to loads eight-week-old in the lungs femaleof mice BALB/c that had mice, received followed AdC7 byα RSV Line19were lower infection (below three detection days later. level) RSV compared viral loads to mice in the that lungs had α ofreceived mice that AdC7GFP had received (Figure AdC7 5A; RSVp < 0.01). were Likewise, lower (below RSV detection viral level) comparedalso decreased to mice in thatmice had that receivedhad received AdC7GFP AdC7 (FigureαRSV compared5A; p < 0.01). to AdC7GFP Likewise, (Figure RSV viral 5B; genomesp < 0.05). alsoThis decreasedsuggests that in miceintranasal that α hadadministration received AdC7 of AdC7RSVα comparedRSV provides to AdC7GFP protection (Figure against5B; RSVp < infection 0.05). This in suggestsadult mice. that intranasal administration of AdC7αRSV provides protection against RSV infection in adult mice.

Figure 5. Protection against RSV infection following AdC7αRSV administration to adult mice. AdC7FigureαRSV, 5. Protection AdC7GFP against (5 × 10 10RSVpu) infection or PBS (No following AdC7 control)AdC7αRSV were administration intranasally administered to adult mice. to 8-week-oldAdC7αRSV, BALB/c AdC7GFP mice, (5 followed x 1010 pu) by or RSV PBS Line19 (No AdC7 (106 pfu) control) challenge were 3 intranasally days later. (A administered) RSV viral loads to 8- inweek-old the lungs BALB/c 4 days aftermice, the followed RSV challenge by RSV Line19 by plaque (106 assay. pfu) challenge (B) RSV genome3 days later. expression (A) RSV in theviral lungs loads 4in days the after lungs the 4 challengedays after bythe RT-qPCR. RSV challenge Data are by shownplaque with assay. mean (B) RSV± SEM. * and expression ** denote p in< 0.05the lungs and p 4< days 0.01, respectively.after the challenge by RT-qPCR. Data are shown with mean ± SEM. * and ** denote p < 0.05 and p < 0.01, respectively. 3.4. Protection Against RSV Following AdC7αRSV Administration to Neonatal Mice 3.4. ToProtection evaluate against if AdC7 RSVα RSVfollowing administered AdC7αRSV to Administration neonatal mice to can Neonatal lead toMice protection against RSV, α AdC7ToRSV evaluate or AdC7GFP if AdC7 wereαRSV intranasally administered administered to neonatal tomice one-day-old can lead to mice. protection Anti-RSV against IgG wasRSV, α detectedAdC7αRSV in 4 outor AdC7GFP of 5 mice thatwere had intranasally received AdC7administRSVered at to four one-day-old and six weeks mice. of Anti-RSV age (Figure IgG6 A).was Thedetected kinetics in of 4 anti-RSVout of 5 mice IgG inthat the had serum received of neonatal AdC7 miceαRSV after at four four weeksand six of weeks age following of age (Figure intranasal 6A). administrationThe kinetics of AdC7anti-RSVαRSV IgG showed in the that serum the titers of neonatal were the mice highest after at four four weeks weeks and of then age graduallyfollowing decreased,intranasal butadministration stayed at detectable of AdC7 levelsαRSV untilshowed nine that weeks the (Figuretiters were S1A). the Serum highest anti-RSV at four IgGweeks titers and werethen also gradually at detectable decreased, levels but 14 stayed weeks at following detectable intranasal levels until administration nine weeks of(Figure AdC7 S1A).αRSV. Serum The mice anti- thatRSV had IgG received titers awere higher also dose at ofdetectable AdC7αRSV levels tended 14 toweeks have following higher anti-RSV intranasal IgG titers,administration particularly of inAdC7 lungα homogenateRSV. The mice supernatants that had received (Figure a S1B). higher Importantly, dose of AdC7 challengeαRSV tended with RSV to have at six higher weeks anti-RSV of age showedIgG titers, a reduction particularly of viral in lung loads homogenate only in the supern mice thatatants had (Figure received S1B). AdC7 Importantly,αRSV, with challenge undetectable with RSVRSV level at six in threeweeks out of ofage five showed mice, compared a reduction with of the viral mice loads that hadonly receivedin the mice AdC7GFP that had or thatreceived had notAdC7 receivedαRSV, any with Ad undetectable vector (Figure RSV6B). level Protection in three against out of RSV five was mice, also compared evaluated with at a the later mice time that point; had Anti-RSVreceived IgGAdC7GFP expression or that was had confirmed not received in the any mice Ad that vector had (Figure received 6B). AdC7 ProtectionαRSV at against four and RSV eight was weeksalso evaluated of age (Figure at a later S2A). time Challenge point; withAnti-RSV RSV atIgG 10 expression weeks of age was also confirmed showed in a reductionthe mice ofthat viral had loadsreceived in three AdC7 outα ofRSV four at micefour thatand hadeight received weeks of AdC7 age α(FigureRSV, compared S2A). Challenge with the with mice RSV that at had 10 receivedweeks of AdC7GFPage also showed or that had a reduction not received of viral any Adloads vector in three (Figure out S2B). of four These mice data that suggest had received that administration AdC7αRSV, ofcompared AdC7αRSV with to the neonatal mice that mice had can received lead to AdC7GFP protection or against that had RSV, not and received prolonged any Ad protective vector (Figure immunity S2B). comparedThese data to suggest adult mice that can administration be provided. of AdC7αRSV to neonatal mice can lead to protection against RSV, and prolonged protective immunity compared to adult mice can be provided.

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Figure 6. ProtectionProtection against against RSV RSV infection infection following following AdC7 AdC7ααRSVRSV administration administration to to neonatal neonatal mice. mice. 10 AdC7αRSVRSV or AdC7GFP (6 ×x 1010 pu)pu) were were intranasally intranasally administer administereded to 1-day-old BALB/c mice, mice, 6 followedfollowed by by RSV A2 (10 6 pfu)pfu) challenge challenge at at 6 6 weeks weeks of of age. age. ( (AA)) Anti-RSV Anti-RSV IgG IgG titer titer in in serum serum before before the the RSV challenge. Serum Serum was was collected collected at at 4 4 and and 6 6 weeks, weeks, and and titers titers were were measured by by ELISA. Data are ± shownshown with with mean ± SEM.SEM. ( (BB)) RSV RSV viral viral loads loads in in the the lungs lungs 4 4 days days after after the the RSV RSV challenge challenge by by plaque ± assay. Data are shown with mean ± SEM.SEM. * * and and *** *** denote denote pp << 0.05 0.05 and and pp << 0.005, 0.005, respectively. respectively. 4. Discussion 4. Discussion In this study, we showed that intranasal administration of AdC7αRSV could provide protection againstIn this RSV study, infection. we showed The administration that intranasal route administration of Ad vector of may AdC7 greatlyαRSV contribute could provide to the protection efficiency againstof local RSV delivery infection. to the The airway. administration Intravenous route administration of Ad vector of may palivizumab greatly contribute was shown to tothe be efficiency effective ofin local rodents delivery and humans, to the airway. effectively Intravenous reducing administration lung RSV load of in palivizumab the lung of was cotton shown rats [to20 be] and effective being indetectable rodents inand human humans, nasal effectively washes [21 reducing]. It is known lung thatRSV intramuscularlyload in the lung injected of cotton palivizumab rats [20] and is slowlybeing detectableabsorbed andin human maximum nasal serum washes concentrations [21]. It is known are reached that intramuscularly at three to five days injected [22], butpalivizumab the kinetics is slowlyof lung absorbed and airway and levels maximum are less serum well-known. concentrations Our data are showed reached that at thethree anti-RSV to five days IgG was[22], detected but the kineticsin the BAL of lung after and intranasal airway administrationlevels are less well-known. but not intramuscular Our data showed administration that the atanti-RSV the time IgG of peakwas detectedserum IgG in levelsthe BAL (at after one week).intranasal The administration intranasal administration but not intramuscular route may administration be advantageous at the through time ofthe peak production serum IgG of the levels neutralizing (at one week). antibodies The intranasal directly by administration the respiratory route mucosal may cells,be advantageous the primary throughtarget of the RSV. production Future studies of the should neutralizing examine antibodies the cellular directly source ofby thethe antibody respiratory within mucosal the respiratory cells, the primarymucosa, target and include of RSV. more Future analyses studies of should the nose-associated examine the lymphoidcellular source tissue of (NALT) the antibody immune within cells. the respiratoryImmune mucosa, responses and ininclude the respiratory more analyses tract of can the be nose-associated altered by prior lymphoid viral infections tissue (NALT) [23,24]. immuneThe recent cells. study [25] revealed that nasal priming by can influence lung immunity and can induceImmune protective responses immunity in the against respiratory heterologous tract can viral be infection.altered by Our prior data viral in theinfections adult mice [23,24]. showed The recentsome protection study [25] against revealed RSV that also nasal in the priming mice that by viruses had received can influence the control lung AdC7GFP immunity vector and (Figurecan induce5A). protectiveAlthough theimmunity protection against against heterologous subsequent viral RSV infection. infection Our was data much in the greater adult in mice the miceshowed that some had protectionreceived AdC7 againstαRSV, RSV the also AdC7 in the vector mice itself that seemed had received to have the a primingcontrol AdC7GFP effect to the vector lung immunity.(Figure 5A). AlthoughNeonatal the protection adaptive against immune subsequent responses RSV show infection a great wasdeal much of greater variability, in the rangingmice that fromhad receivednon-responsiveness AdC7αRSV, to the fully AdC7 mature vector function. itself seemed It has beento have shown a priming that newborn effect to micethe lung can immunity. be tolerized to AdNeonatal vectors, adaptive and that repeatimmune administration responses show of Ad a great vector deal could of bevariability, possible [26ranging]. Thus, from immune non- responsivenesstolerance can be to expected fully mature when function. vectors areIt has administered been shown to that neonates. newborn Administration mice can be tolerized of AdC7 toαRSV Ad vectors,to one-day-old and that mice repeat resulted administration in prolonged of Ad antibody vector could expression be possible compared [26]. Thus, to adult immune mice. tolerance Another canpotential be expected advantage when of vectors neonatal are gene administ deliveryered is to the neonates. higher vector Administration particles-to-cell of AdC7 ratio,αRSV requiring to one- a day-oldlower relative mice resulted dose [15 ].in In prolonged addition, antibody Ad vector expr deliveryession seems compared to be more to adult efficient mice. in Another the neonatal potential lung advantagecompared toof adultneonatal lungs gene [27 ].delivery Our data is showedthe higher that vector the serum particles-to-cell anti-RSV IgG ratio, titers requiring are higher a lower after relativeneonatal dose administration [15]. In addition, compared Ad tovector adult delivery administration, seems to albeit be more with efficient the same in dose. the neonatal lung comparedTo reduce to adult the burdenlungs [27]. of disease Our data caused showed by RSV, thatthere the serum is a strong anti-RSV consensus IgG titers that are focus higher should after be neonatalplaced on administration children in their compared first six monthsto adult ofadministration, life when the riskalbeit of with severe the RSV-associated same dose. respiratory diseaseTo reduce is highest the burden [8]. Immunization of disease caused to provide by RSV, the there protective is a strong immunity consensus lasting that focus throughout shouldthe be placed on children in their first six months of life when the risk of severe RSV-associated respiratory

Vaccines 2019, 7, 3 8 of 10 vulnerable period would be an ideal strategy to protect children from RSV infection. We focused on passive immunization in this study, but the combination of AdC7-based passive immunization and AdC7-based active immunization can be an attractive strategy to provide complete protective immunity throughout the vulnerable period. Since mature responses to vaccines can also be expected in the neonatal immune system [14], simultaneous administration of AdC7 which carries anti-RSV neutralizing antibody and RSV vaccine antigen may provide both passive and active immunization. An appropriate vaccine antigen that is not overlapped with the antigenic site for palivizumab would be required to make this strategy successful. We did not include wild-type RSV or other RSV vaccine controls in this study. Since our primary interest was to determine if genetic delivery of anti-RSV antibody by AdC7 vector can be effective in neonatal mice, we used AdC7 control vector without transgene expression (AdC7GFP). Future studies should include wild-type RSV or vaccine controls to further evaluate the effectiveness of this vector strategy, even if wild-type RSV is not a realistic vaccine alternative for neonates. The half-life of the delivered antibody in our study was 19 days, which is compatible with a known half-life of palivizumab. Prolonged expression was seen when the vectors were administered to neonatal mice, but we could not estimate the half-life of antibodies since a serial collection of neonatal serum was not feasible. Several explanations can be possible for the prolonged expression. The antibody may have had a longer half-life than in adult mice because of the unique immunological properties of neonatal mice. The efficiency of gene delivery may have been greater due to the higher susceptibility to Ad of neonatal lungs. Recently, the development of new anti-RSV monoclonal antibodies went toward prolongation of the serum half-life. The amino acid modification made it possible to extend the serum half-life of the antibody [28,29]. Modifying the expression cassette on the AdC7 to deliver more efficient anti-RSV monoclonal antibody with extended half-life itself may enable further long-term protection against RSV.

5. Conclusions In summary, our data showed that intranasal administration of AdC7αRSV to neonatal mice provided prolonged expression of anti-RSV antibody to protect against RSV infection. Neonatal immune responses have been mainly studied in mice, but there are some indications that a similar situation is true in humans in early life [14]. We demonstrated the potential of neonatal genetic delivery by non-human primate-based Ad vector that could efficiently deliver anti-RSV neutralizing antibody to neonatal lungs and could provide protection against RSV.

Supplementary Materials: The following are available online at http://www.mdpi.com/2076-393X/7/1/3/s1, Figure S1: Assessment of anti-RSV IgG expression in neonatal mice, Figure S2: Protection against RSV 10 weeks following AdC7αRSV administration to neonatal mice. Author Contributions: S.W. and A.S. conceptualized the study; R.G. and W.W. performed experimental procedures. A.S. supervised the experiments. R.G. analyzed data and wrote the manuscript. S.W. reviewed and edited the manuscript. Funding: This research was funded by National Institutes of Health grant number R21 AI113801 to S.W. Acknowledgments: We thank Martin L. Moore for providing us the RSV strain Line19 and rA2-F19, James M. Wilson for the Ad vectors. Conflicts of Interest: The authors declare no conflicts of interest.

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