Vaccine 33 (2015) 1338–1344

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The respiratory syncytial virus fusion protein formulated with a novel combination adjuvant induces balanced immune responses in lambs with maternal antibodies R. Garg a , L. Latimer a , V. Gerdts a,c , A. Potter a,c , S. van Drunen Littel-van den Hurk a,b,∗ a

VIDO-Intervac, University of Saskatchewan, Saskatoon, SK, Canada S7N 5E3 Microbiology & Immunology, University of Saskatchewan, Saskatoon, SK, Canada S7N 5E3 c Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK, Canada S7N 5E3 b

a r t i c l e

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Article history: Received 16 November 2014 Received in revised form 12 January 2015 Accepted 14 January 2015 Available online 28 January 2015 Keywords: RSV Neonatal immunization Maternal immunization Maternal antibodies Lambs Animal model

a b s t r a c t Respiratory syncytial virus (RSV) causes severe respiratory illness in infants. There are no licensed vaccines to prevent RSV infection. The neonate receives short-term protection from maternally derived antibodies, which, however, can also interfere with the active response to vaccination. A RSV vaccine consisting of a truncated version of the fusion protein formulated with polyI:C, innate defense regulator peptide and polyphosphazene (F/TriAdj), was evaluated in two to three week-old lambs. When delivered intrapulmonary, F/TriAdj elicited IgA production in the lung in addition to a robust systemic response similar to that induced by intramuscular immunization. To investigate potential interference by maternal antibodies, pregnant ewes were vaccinated with F/TriAdj. Lambs born to RSV F-immune or non-immune ewes were then given three vaccinations with F/TriAdj at 3 days, 4 weeks and 8 weeks post-birth. Lambs immunized intramuscularly with F/TriAdj vaccine developed high-affinity F-specific serum IgG and virus neutralizing antibodies, and displayed an increase in the frequency of IFN-␥-secreting cells by in vitro restimulated peripheral blood mononuclear cells. Maternal antibodies did not interfere with the development of an immune response to F/TriAdj in the newborn lambs. These results indicate that immunization of neonates with F/TriAdj is effective even in the face of maternal antibodies. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Respiratory syncytial virus (RSV) is a major cause of respiratory tract illness in infants and children leading to hospitalization and thousands of deaths each year worldwide [1]. The successful development of a RSV vaccine poses many challenges such as immature immune responses of infants, the potential interference of maternal antibodies with vaccination, and earlier concerns of vaccine-induced enhanced disease severity. In the 1960s, a formalin-inactivated RSV (FI-RSV) vaccine failed to induce RSV-specific neutralizing antibodies and unexpectedly caused vaccine-enhanced disease after natural RSV infection [2]. Many vaccine candidates, including live attenuated strains, as well

∗ Corresponding author at: University of Saskatchewan, Vaccine and Infectious Diseae Organization, 120 Veterinary Rd., Saskatoon, SK, Canada S7N 5E5. Tel.: +1 306 966 1559; fax: +1 306 966 7478. E-mail address: [email protected] (S. van Drunen Littel-van den Hurk). http://dx.doi.org/10.1016/j.vaccine.2015.01.041 0264-410X/© 2015 Elsevier Ltd. All rights reserved.

as vector-based and subunit protein vaccines, have been evaluated in rodent and primate models, but there are still no safe and effective RSV vaccines or specific treatments other than prophylaxis with passive antibody therapy (Palivizumab) [3]. The RSV fusion protein (F) is highly conserved and facilitates penetration of the virus into the host cell and subsequent formation of syncytia thus making it a suitable subunit vaccine candidate [4]. It has been shown that immunization with purified F protein alone is insufficient to induce protection due to poor immunogenicity [5,6]. Thus, the F protein needs to be formulated with an adjuvant to induce robust immune responses including high-affinity neutralizing antibodies. Previously, we have demonstrated the induction of protective immunity by vaccination with a novel RSV vaccine candidate consisting of a truncated fusion (F) protein formulated with a TLR3 agonist (polyI:C), an immune defense regulator (IDR) peptide and a synthetic polymer, polyphosphazene (F/TriAdj) in rodents [6,7]. Recently, we also revealed that the duration of protective immunity induced with this vaccine is at least one year [8]. Lamb models have been increasingly used to study RSV pathogenesis, immune responses, vaccination and therapeutic strategies

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as they have similar lung development and mucosal immune responses to human infants [9]. Moreover, lambs develop similar lesions to those of human infants after RSV infection. Another advantage is that the neonatal period of lambs is longer than that in rodents providing a better opportunity to evaluate vaccine efficacy in the context of a newborn immune system. In the present study, we evaluated the immunogenicity of the F/TriAdj vaccine formulation via two different routes of delivery in lambs. Additionally, we examined whether the F/TriAdj vaccine is susceptible to interference by maternal antibodies (MatAbs), as well as the role of a booster dose, as a means of trying to overcome potential interference. The efficacy of the F/TriAdj was equivalent when administered parenterally and mucosally, as well as in newborn lambs with and without MatAbs, demonstrating the potential of this vaccine in neonates. 2. Materials and methods

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by streptavidin-AP (Jackson Immuno Research Laboratories Inc., Westgrove, PA). The assay was developed with p-nitrophenyl phosphate (Sigma-Aldrich Inc., St Louis, MO) substrate. RSV F-specific IgG avidity was determined by incubating F protein-coated ELISA plates with serum, followed by washing with PBS or different concentrations of urea (7 M, 8 M and 9 M). The subsequent steps were performed in accordance with the standard ELISA protocol described above. The percentage of bound antibodies was calculated by comparing titers of bound antibodies after urea washes with titers after PBS washes. A competitive ELISA was performed with biotinylated Palivizumab monoclonal antibody. Briefly, two-fold serially diluted sera (starting with 1:10 dilution) together with 100 ng/ml of biotinylated Palivizumab were applied to F-coated plates and incubated for 2 h at room temperature. Bound Palivizumab was detected using streptavidin-AP and developed with p-nitrophenyl phosphate substrate. Optical densities (OD) of the wells were determined at 405–490 nm.

2.1. Vaccine formulation 2.4. Virus neutralization (VN) assay The F protein was produced and purified as described previously [6]. The F protein was co-formulated with PCEP (250 ␮g), IDR1002 (500 ␮g) and polyI:C (250 ␮g) in PBS. PolyI:C (Invivogen, San Diego, CA) and IDR 1002 (Genscript, Piscataway, NJ) were formulated in a 1:2 ratio at 37 ◦ C; poly[di(sodium carboxylatoethylphenoxy)]-phosphazene (PCEP) (Idaho National Laboratory, Idaho Falls, ID) along with F protein was added after 30 min to make a final 1:2:1 ratio of polyI:C, IDR and PCEP (F/TriAdj).

RSV-specific neutralization titers were determined by plaque reduction assays. Heat-inactivated sera were serially diluted in 96well plates (Corning Incorporated, Corning, NY), and then mixed with 500 PFU/well of RSV strain A2 for 1 h at 37 ◦ C. The serumvirus mixtures were transferred to duplicate HEp-2 cell monolayers and incubated at 37 ◦ C, and after four days the cells were fixed and stained with 0.5% crystal violet. 2.5. Enzyme-linked immunospot (ELISPOT) assay

2.2. Animals and immunizations Suffolk lambs were purchased from the Department of Animal and Poultry Science, University of Saskatchewan, SK, Canada. The trials were carried out according to the guidelines provided by the Canadian Council for Animal Care. In the first trial, 42 lambs (two to three weeks old) were randomly allocated into six groups of seven animals each. Lambs were immunized twice intramuscularly (IM) at a three-week interval, with vaccine containing different amounts of F (12.5, 25, 50 or 100 ␮g) formulated with TriAdj (250 ␮g polyI:C, 500 ␮g IDR and 250 ␮g PCEP). Two additional groups of lambs received either F (100 ␮g) (Th2 control) or PBS (control). In the second trial, 13 lambs (two to three weeks old) were randomly allocated to three groups of five animals, and one group of three animals (control). Lambs were immunized twice IM or intrapulmonary (IP) at a three-week interval with vaccine containing F (50 ␮g) formulated with TriAdj. The control group of lambs received PBS. In the third trial, 24 pregnant ewes were randomly allocated to two groups of 12 animals each, half of which were vaccinated twice IM with a three-week interval with F/TriAdj, while the other half received TriAdj only. The lambs born to RSV F-immune or non-immune ewes were then given three IM vaccinations with either TriAdj or F/TriAdj at three days, four weeks and eight weeks post-birth. Serum, colostrum and whole blood were collected at regular intervals, and bronchoalveolar lavages (BAL) at the end of the trial, to assess the humoral and cell-mediated immune responses. 2.3. Enzyme-linked immunosorbent assays (ELISAs) RSV F-specific IgG and IgA were evaluated in serum, colostrum and BAL by standard ELISA. Briefly, four-fold serially diluted samples were applied to F-coated plates (50 ng/ml) and bound F-specific antibodies were detected using alkaline phosphatase (AP)-conjugated goat anti-sheep IgG (Kirkegaard & Perry Laboratories, Gaitherburg, MD), and biotin-labeled IgA followed

Isolation of peripheral blood mononuclear cells (PBMCs) and lymph node (LN) cells, and ELISPOT assays were performed by standard technique. Briefly, ELISPOT plates (Millipore, Billerica, MA) were coated overnight with a bovine gamma interferon (IFN-␥)-specific monoclonal antibody. PBMCs and LN cells (106 cells/well) were stimulated with either F protein (2 ␮g/ml) or medium. The plates were incubated at 37 ◦ C, and after 24 h bovine IFN-␥-specific rabbit serum [10] was added. Spots were developed using AP-conjugated goat anti-rabbit IgG (Kirkegaard & Perry Laboratories) and BCIP/NBT (Sigma-Aldrich) as the substrate. 2.6. Statistical analysis All data were analyzed using GraphPad PRISM version 6 for Windows (GraphPad Software). Differences among all groups were examined using one-way ANOVA, followed by a Newman-Keuls post-test. Differences were considered significant if P < 0.05. 3. Results 3.1. Effect of the dose of RSV F protein on the magnitude of the immune response The goal of the first trial was to determine the optimal dose of F protein in lambs. The lambs were immunized IM with 12.5, 25, 50 or 100 ␮g of F protein formulated with TriAdj. Control lambs received 100 ␮g of F protein in PBS or PBS alone. To determine the humoral immune responses, the RSV F-specific serum IgG titers were determined. The lambs immunized with F(50 ␮g)/TriAdj produced significantly higher F-specific serum IgG titers compared to the other vaccinated groups, after both the first and the second immunization (Fig. 1A and B). The F-induced secretion of IFN-␥ by in vitro restimulated PBMCs was measured two weeks after the second vaccination. The F(50 ␮g)/TriAdj formulation generated a significantly higher frequency of IFN-␥ secreting cells

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compared to all other vaccine formulations (Fig. 1C). A very low immune response was observed in lambs immunized with F in PBS. These results indicate that F (50 ␮g) formulated with TriAdj promotes a strong humoral and cell-mediated immune response compared to other F protein doses (12.5, 25, and 100 ␮g). This observation led to further experimentation where the F protein dose was 50 ␮g. 3.2. Influence of route of vaccination with F/TriAdj on the magnitude and quality of the immune response To determine whether the F/TriAdj can induce a mucosal immune response, lambs were vaccinated IM or IP with F/TriAdj. Systemic humoral immune responses induced by F/TriAdj were measured by evaluating the F-specific IgG titres in the serum. After the last immunization, significantly higher IgG production was observed in the serum of animals immunized with F/TriAdj delivered IM or IP (Fig. 2A). To evaluate the mucosal immune responses induced by the F/TriAdj adjuvant formulation, the secretion of IgG (Fig. 2B) and IgA (Fig. 2C) in BAL was measured. Lambs immunized IP with F/TriAdj developed significantly higher IgA levels compared to IM immunized animals, whereas the IgG responses were comparable for both routes of immunization. We further evaluated the function and quality of the antibody responses induced by the F/TriAdj vaccine via both routes of immunization. It is well established that neutralizing antibody plays a protective role against viral infections. To evaluate the biological activity of the F-specific serum antibodies, VN titers were determined. The F/TriAdj formulation, whether administered by the IP or IM route, elicited significantly higher neutralizing antibody titers when compared to PBS (Fig. 3A). Avidity plays a major role in antibody-mediated protection against many viruses, and a lack of antibody affinity maturation may have been responsible for the enhanced respiratory disease in the children vaccinated with the FI-RSV vaccine [11]. Therefore, we determined the avidity of the F-specific IgG induced by F/TriAdj. Interestingly, after the final vaccination, high-affinity F-binding antibodies were observed in lambs immunized with F/TriAdj irrespective of the delivery route (Fig. 3B). Furthermore, a Palivizumab competitive ELISA was performed to determine whether antibodies directed against antigenic site II were induced by vaccination with F/TriAdj. Animals immunized either IP or IM with F/TriAdj developed significantly higher levels of antibodies that inhibit the binding of Palivizumab to F protein. No inhibition of Palivizumab binding was observed in lambs immunized with PBS alone (Fig. 3C). The F-induced secretion of IFN-␥ by in vitro restimulated PBMCs and LN cells was measured two weeks after primary and secondary immunization. In PBMCs, IM or IP delivery of the F/TriAdj vaccine formulation resulted in increased numbers of IFN-␥ secreting cells both after the first and the second immunization (Fig. 4A) when compared to PBS administration. Furthermore, both group of lambs immunized with F/TriAdj developed significantly higher numbers of IFN-␥-secreting cells in the draining LNs, regardless of the delivery route (Fig. 4B). The potential to induce mucosal immune responses by IP immunization with F/TriAdj vaccine was confirmed in a second trial. 3.3. Effect of maternal antibodies on the immune response of newborn lambs induced by F/TriAdj Fig. 1. Systemic humoral and cell-mediated immune responses induced by different concentrations of RSV F protein formulated with TriAdj. Serum IgG titers after (A) 1st immunization, and (B) 2nd immunization, and (C) Numbers of IFN-␥ secreting PBMCs determined in response to in vitro restimulation with F protein two weeks after the 2nd immunization. Lambs were immunized twice IM with different doses of F formulated with either PBS or TriAdj. A control group received PBS. ELISA titers are expressed as the reciprocal of the highest dilution resulting in a value of two standard deviations above the negative control serum. ELISPOT results are expressed

Since ultimately, a RSV vaccine should be efficacious in neonates, newborn lambs three days of age were targeted in the

as the difference between the number of cytokine-secreting cells in F-stimulated wells and medium-control wells per 106 cells. Bars indicate median values with interquartile ranges. * P < 0.05; ** P < 0.01; *** P < 0.001.

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Fig. 2. Systemic and mucosal immune responses induced by RSV F protein with TriAdj via IM or IP route. (A) Serum IgG, (B) Lung IgG, and (C) Lung IgA titers. Lambs were immunized twice IM or IP with F/TriAdj or PBS. ELISA titers are expressed as the reciprocal of the highest dilution resulting in a value of two standard deviations above the negative control serum. Bars indicate median values with interquartile ranges. * P < 0.05; ** P < 0.01.

next trial. To evaluate whether the F/TriAdj vaccine can efficiently transfer from mother to off-spring, pregnant ewes were vaccinated with F/TriAdj prior to lambing, which was expected to result in transfer of MatAbs to the newborn lambs through the colostrum. F-specific antibodies were detected in serum (Fig. 5A) and colostrum (Fig. 5B) of vaccinated ewes. The existence of RSV

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Fig. 3. Function and quality of the antibody responses induced by the F/TriAdj vaccine via IM or IP route. (A) Serum VN antibody titers, (B) IgG antibody affinity, and (C) Palivizumab competitive ELISA. Lambs were immunized as described in the legend for Fig. 2. Virus neutralization titers are expressed as the highest dilution of serum that resulted in