Antiviral Research 125 (2016) 46e50

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Short communication

Agonistic 4-1BB antibody fails to reduce disease burden during acute respiratory syncytial virus (RSV) infection M.J. Norris a, b, W. Duan b, Y. Cen b, T.J. Moraes a, b, * a b

Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada Physiology and Experimental Medicine, SickKids, Toronto, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 April 2015 Received in revised form 16 September 2015 Accepted 5 October 2015 Available online 17 November 2015

Respiratory Syncytial Virus (RSV) remains a leading cause of infant morbidity and mortality worldwide. Despite this, there are limited therapeutic options. CD8 T cells have an integral role in controlling viral infections; strategies to enhance these responses may be clinically relevant. The T cell costimulatory receptor, 4-1BB, is known to play a role in expansion of antiviral CD8 T cells. In this study, we examined the effect of agonistic 4-1BB antibody at the time of RSV infection in mice. We show that this antibody did not improve outcomes in the setting of RSV infection but rather, led to increased weight loss and a reduction in RSV specific CD8 T cells in the lung. This work suggests caution in the use of agonistic 4-1BB antibody in the setting of viral infections. © 2015 Elsevier B.V. All rights reserved.

Keywords: Respiratory syncytial virus (RSV) Agonistic 4-1BB antibody CD8 T cells Costimulation

Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family of viruses, is a leading cause of bronchiolitis and hospitalization in infants (Samson, 2009). Despite disease burden, no commercial vaccine is available and treatment remains nonspecific. CD8 T cells are critical for clearance of viruses (Wong and Pamer, 2003). With respect to RSV, human and animal studies demonstrate that CD8 T cell deficiency results in poor outcomes (Hall et al., 1986; Wong et al., 1985; Zhou et al., 2008). In animal models, immunization with CD8 epitopes generates protective CD8 T cell based immunity (Connors et al., 1992; Bueno et al., 2008; Simmons et al., 2001). Strategies that boost CD8 T cell responses may therefore provide a therapeutic strategy for RSV. The receptor 4-1BB is a member of the TNFR family expressed on activated T cells (Watts, 2005). 4-1BB is a costimulatory receptor; T cell stimulation via the T cell receptor combined with additional message provided by 4-1BB signaling improves T cell activation and proliferation. Notably, 4-1BB signaling plays an important role in CD8 T cell responses to several viruses (Wortzman et al., 2013). Agonist 4-1BB antibody (Ab) has been used to stimulate 4-1BB signaling in vivo (Sun et al., 2004). We tested the hypothesis that

* Corresponding author. The Hospital for Sick Children, Peter Gilgan Centre for Research and Learning, 686 Bay St., Room 09.9714, Toronto, Ontario, M5G 0A4, Canada. E-mail address: [email protected] (T.J. Moraes). http://dx.doi.org/10.1016/j.antiviral.2015.10.007 0166-3542/© 2015 Elsevier B.V. All rights reserved.

agonist 4-1BB Ab could improve outcomes in RSV infection by enhancing cytotoxic CD8 T cell responses. We examined the in vivo effect of 4-1BB costimulation on the primary CD8 T cell response to RSV. Pathogen free 6e8 week old female BALB/c mice (Charles River Laboratories) were sedated with isoflurane and infected intranasally with 5
106 plaque forming units (PFU) of RSV-A2 on day 0. This methodology has been described previously (Tayyari et al., 2011). Within 20 min of inoculation with RSV, mice received PBS or 100 mg of monoclonal (clone 3H3) agonistic 4-1BB Ab (gift from Dr. Tania Watts and Dr. Robert Mittler) intraperitoneally (i.p.). Mice were euthanized at day 4 post RSV in order to measure RSV titers in lung homogenates. The 4-1BB agonist Ab was not associated with a reduction in lung viral titers 4 days post infection (Fig. 1A) or a change in histological appearance of the lung (Supplementary Fig. 1). However, mice treated with agonist 4-1BB Ab lost more weight than control mice post RSV (Fig. 1B). This finding suggested more morbidity with agonist 4-1BB Ab treatment. Inflammation post RSV infection is associated with changes in lung mechanics peaking on day 6 (Schwarze et al., 1997). Both 4-1BB agonist Ab treated and non-treated RSV infected mice exhibited increased resistance (Fig. 1C) and decreased dynamic compliance (Fig. 1D) as compared to control uninfected mice. Although there was no statistically significant difference in lung compliance between the non-treated group and agonist 4-1BB Ab treated group, a trend was seen with the agonist 4-1BB Ab group having higher resistance and

M.J. Norris et al. / Antiviral Research 125 (2016) 46e50

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Fig. 1. RSV associated disease outcomes. BALB/c mice were infected i.n. with 5
106 PFU of RSV on day 0. Mock infected mice were included as a control and received PBS on day 0. Within 20 min of inoculation, mice received PBS or 100 mg of monoclonal (clone 3H3) agonistic 4-1BB Ab i.p. or i.n. (A) On day 4, the peak of viral replication, lung homogenates were analyzed by standard plaque assay for viral load. (B) Weight loss of the infected mice was monitored daily. Results are representative of 4 experiments with N ¼ 3e4 mice per group. (C, D) Airway hyperresponsiveness (AHR) was assessed in response to increasing doses of nebulized methacholine (SigmaeAldrich, Oakville, ON, Canada) on day 6 following RSV infection. Airway resistance (C) and dynamic compliance (D) were measured. Results are shown as mean ± SEM and p values were derived from a 1-way ANOVA analysis with a Tukey's post hoc test for group comparisons.

lower compliance in response to inhaled methacholine. This suggests that agonist 4-1BB Ab does not attenuate RSV associated disease outcomes but rather is associated with more morbidity. Agonist 4-1BB Ab should enhance antiviral CD8 T cell generation, which in turn should improve viral clearance. 4-1BB expression on T cells is inducible and transient (Watts, 2005), thus, it was possible that lack of benefit was related to a lack of effect on T cell numbers. Alternatively, agonist 4-1BB Ab may have led to an overly aggressive T cell response associated with increased morbidity. To test this, spleens and lungs were removed on day 6 after RSV challenge and CD8 T cell numbers enumerated by FACS analysis. This data confirmed that i.p. agonist 4-1BB Ab increased the percentage and numbers of CD8 T cells in the spleen (Fig. 2A and B) when compared to RSV infection alone. We also examined antigen specific CD8 T cells (RSV M2 e H-2K(d)/SYIGSINNI; NIH Tetramer Facility). In the spleen, there was no difference in the percentage of CD8 T cells that were RSV specific between the mice that received i.p. agonist 4-1BB Ab and those that did not (Fig. 2C). However, the numbers of RSV specific CD8 T cells were higher in the spleen (Fig. 2D). This supports the hypothesis that agonist 4-1BB Ab enhances CD8 T cell responses and can do so in an antigen specific manner. The CD4 percentages decreased correspondingly in the spleen though the absolute numbers did not change significantly (data not shown). In the lung, the percent and number of CD8 T cells were not altered by i.p. agonist 4-1BB Ab (Fig. 2E and F). Most surprising, we found the RSV specific CD8 T cells were reduced in the lung (Fig. 2G and H) in mice treated with i.p. 4-1BB Ab as compared to RSV infection alone. We questioned if these findings were related to the route of costimulation and so repeated experiments delivering agonist 4-

1BB Ab intranasally following RSV infection. The route of agonist 41BB Ab administration did not significantly affect the viral titers (Fig. 1A) or weight loss (Fig. 1B). Intranasal agonist 4-1BB Ab resulted in similar enhancement of CD8 T cells in the spleen suggesting that the route of costimulation did not alter this systemic effect (Fig. 2A and B). However, RSV specific CD8 T cells in the spleen were slightly reduced with intranasal versus i.p. agonist 4-1BB Ab. Further, in the lung, intranasal agonist 4-1BB Ab was superior to i.p agonist 4-1BB Ab at boosting CD8 T cells numbers (Fig. 2F). Despite this, the percentage of CD8 T cells that were RSV specific was reduced in the lung (Fig. 2G) in mice treated with intranasal agonist 4-1BB Ab as compared to RSV infection alone. The number of RSV specific CD8 T cells trended lower but given the overall increase in CD8 T cell numbers there was not a statistical difference seen (Fig. 2H). These data suggest that route of delivery of agonist 4-1BB Ab does lead to different effects; i.p. delivery provides slightly improved antigen specific splenic CD8 T cells whereas intranasal delivery results in slightly improved lung CD8 populations. However, the important finding remained that regardless of route of delivery, agonist 4-1BB Ab did not improve outcomes and resulted in reduced RSV specific CD8 T cells in the lung. Differences associated with agonist 4-1BB Ab administration were not seen with administration of isotype IgG control Ab. Thus the effects reported are related to the agonist 41BB Ab and not a non-specific effect of IgG. We questioned whether the reduction in antigen specific T cells in the lung was related to upregulation of regulatory T cell populations. In the lung, there was a trend towards higher percentage CD4/CD25 þ T cells with agonist 4-1BB Ab (Fig. 3A); this pattern was also seen with absolute numbers and a statistically significant difference was seen between mice that received intranasal agonist

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Fig. 2. Administration of 4-1BB agonist Ab with primary RSV infection increases total CD8 T cells in the spleen but reduces RSV M2 specific CD8 T cells in the lung. Samples were analyzed using a Gallios Flow Cytometer (Beckman Colter) and FlowJo (TreeStar Inc., Ashland, OR, USA) software. Where indicated, cells were labeled with antibodies to CD8a and CD3 (eBioscience). RSV M2 specific CD8 T cells (M2 H-2K(d)/SYIGSINNI) were enumerated using MHC tetramers (obtained from the NIH tetramer facility). Bar graphs show percent (A, C, E, G) and number (B, D, F, H) of lymphocytes positive for indicated marker in spleen or lung. Cells from uninfected mice in open bars, RSV infected mice shown in black bars, RSV infection with i.p. 4-1BB agonist Ab in dark gray bars and RSV infection with i.n. 4-1BB agonist Ab in light gray bars. All samples are from mice at day 6 post RSV infection. N ¼ 3e15 for each panel. p values were derived from a 1-way ANOVA analysis with a Tukey's post hoc test for group comparisons.

4-1BB Ab versus no treatment (Fig. 3B). These findings were not seen in the spleen (data not shown). We also examined dendritic cell (DC) populations and saw a reduction in inflammatory (Fig. 3C and D; CD3-, CD19-, CD14-, CD11cþ,CD11bþ) and plasmacytoid DCs (Fig. 3E and F; CD3-, CD19, CD14-, CD11c dim, CD11b-,CD45þ) with agonist 4-1BB Ab administration in both i.n. and i.p. groups. No significant change was seen in conventional DCs (data not shown; CD3-, CD19-, CD14-, CD11cþ,CD11b-,CD103þ). These findings suggest that administration of 4-1BB agonist Ab at the time of primary RSV infection does not influence morbidity from RSV in a favorable manner. This manuscript was not aimed at documenting the importance of 4-1BB signaling during RSV infection. The role of the 4-1BB pathway in boosting T cell responses, and especially secondary memory T cell responses, has

been documented (Watts, 2005; Bertram et al., 2004). Rather we sought to explore if exploiting this pathway could acutely improve outcomes in the setting of RSV. We chose the current model in order to keep the antigen (RSV) and the costimulatory signal (4-1BB) in close proximity to each other. Previously, 4-1BBL has been coupled to an influenza epitope in the form of viral vectors (Moraes et al., 2011). In this setting, antiviral CD8 T cell production was enhanced to influenza. Physically uncoupling antigen from the 4-1BB costimulatory signal (despite temporal proximity) may have led to a less specific effect of 4-1BB signaling (i.e. expansion of non RSV or non M2 specific CD8 T cells), however, there was no benefit seen in outcomes to RSV to support an enhanced non M2 RSV response. Lee et al. did study the use of agonist 4-1BB Ab in aged mice in order to boost CD8 responses in an RSV vaccination model. In this setting multiple

M.J. Norris et al. / Antiviral Research 125 (2016) 46e50

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Fig. 3. Administration of 4-1BB agonist Ab with primary RSV infection alters CD25 and dendritic cell populations in the lung. Samples were analyzed using a Gallios Flow Cytometer (Beckman Colter) and FlowJo (TreeStar Inc., Ashland, OR, USA) software. For these experiments inflammatory dendritic cells (DC) were defined as CD3-, CD19-, CD14-, CD11cþ,CD11bþ; plasmacytoid DCs were defined as CD3-, CD19-, CD14-, CD11c dim, CD11b-,CD45þ. All antibodies were from eBioscience. Bar graphs show percent (A, C, E) and number (B, D, F) of indicated populations. All cells from the lungs of mice day 6 post RSV infection. RSV infected mice without treatment shown in black bars, RSV infection with i.p. 4-1BB agonist Ab in dark gray bars and RSV infection with i.n. 4-1BB agonist Ab in light gray bars. N ¼ 6e8 for each panel. p values were derived from a 1-way ANOVA analysis with a Tukey's post hoc test for group comparisons.

administrations (4 occasions) of agonist 4-1BB Ab did improve RSV specific CD8 T cell responses 20 days later (Lee et al., 2014). It is likely that the effects of 4-1BB stimulation will vary depending on the timing of administration and the timing of the assessment. These factors will influence which cells express 4-1BB or 4-1BBL, and how much time the cells have to respond to the signal. To approximate the antigen and costimulatory signal, we delivered agonist 4-1BB Ab intranasally. To our knowledge, intranasal agonist 4-1BB Ab has not been used previously. Both intransal and intraperitoneal agonist 4-1BB Ab enhanced CD8 T cell generation in a similar fashion. This work suggests some minor differences between routes of administration of the costimulatory signal, however, weight loss, viral titers and percentage of lung RSV specific CD8 T cells were not appreciable altered by route of delivery of

agonist 4-1BB Ab. We did explore the impact of agonist 4-1BB Ab on CD4/ CD25 þve T cells and DCs. Both cell types can express 4-1BB and can play a role in RSV infection (Zheng et al., 2004; Vinay et al., 2006; Wang et al., 2006; Lukens et al., 2009; Kim et al., 2011). The effects were minimal but did suggest some changes that will need future work with a kinetic study including examination of draining lymph nodes in order to better understand the role of these cells. Our findings presented here suggest caution in using agonist 41BB Ab in vivo. Although, there is literature to support agonist 41BB Ab in the setting of asthma and cancer (Polte et al., 2006; Sallin et al., 2014), our results suggest that agonist 4-1BB Ab may lead to increased morbidity with acute infections. In vivo use of agonist 41BB Ab should be considered carefully with close attention paid to

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monitoring for adverse events with infection. Conflicts of interest The authors declare no conflict of interest. Acknowledgments We acknowledge the NIH Tetramer Core Facility for provision of tetramers. TJM was funded through the SickKids Foundation/Institute for Human Development, Child and Youth Health e CIHR. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.antiviral.2015.10.007. References Bertram, E.M., Dawicki, W., Sedgmen, B., Bramson, J.L., Lynch, D.H., Watts, T.H., 2004. A switch in costimulation from CD28 to 4-1BB during primary versus secondary CD8 T cell response to influenza in vivo. J. Immunol. 172, 981e988 published online EpubJan. Bueno, S.M., Gonzalez, P.A., Cautivo, K.M., Mora, J.E., Leiva, E.D., Tobar, H.E., Fennelly, G.J., Eugenin, E.A., Jacobs Jr., W.R., Riedel, C.A., Kalergis, A.M., 2008. Protective T cell immunity against respiratory syncytial virus is efficiently induced by recombinant BCG. Proc. Natl. Acad. Sci. U. S. A. 105, 20822e20827 published online EpubDec 30. Connors, M., Kulkarni, A.B., Collins, P.L., Firestone, C.Y., Holmes, K.L., Morse 3rd, H.C., Murphy, B.R., 1992. Resistance to respiratory syncytial virus (RSV) challenge induced by infection with a vaccinia virus recombinant expressing the RSV M2 protein (Vac-M2) is mediated by CD8þ T cells, while that induced by Vac-F or Vac-G recombinants is mediated by antibodies. J. Virol. 66, 1277e1281 published online EpubFeb. Hall, C.B., Powell, K.R., MacDonald, N.E., Gala, C.L., Menegus, M.E., Suffin, S.C., Cohen, H.J., 1986. Respiratory syncytial viral infection in children with compromised immune function. N. Engl. J. Med. 315, 77e81 published online EpubJul 10. Kim, Y.H., Choi, B.K., Shin, S.M., Kim, C.H., Oh, H.S., Park, S.H., Lee, D.G., Lee, M.J., Kim, K.H., Vinay, D.S., Kwon, B.S., 2011. 4-1BB triggering ameliorates experimental autoimmune encephalomyelitis by modulating the balance between Th17 and regulatory T cells. J. Immunol. 187, 1120e1128. http://dx.doi.org/ 10.4049/jimmunol.1002681 published online EpubAug 1. Lee, S., Mittler, R.S., Moore, M.L., 2014. Targeting CD137 enhances vaccine-elicited anti-respiratory syncytial virus CD8þ T cell responses in aged mice. J. Immunol. 192, 293e299. http://dx.doi.org/10.4049/jimmunol.1300453 published online EpubJan 1. Lukens, M.V., Kruijsen, D., Coenjaerts, F.E., Kimpen, J.L., van Bleek, G.M., 2009. Respiratory syncytial virus-induced activation and migration of respiratory dendritic cells and subsequent antigen presentation in the lung-draining lymph

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