Vaccination with a Single-Cycle Respiratory Syncytial Virus Is Immunogenic and Protective in Mice

This information is current as Megan E. Schmidt, Antonius G. P. Oomens and Steven M. of October 2, 2021. Varga J Immunol published online 19 April 2019 http://www.jimmunol.org/content/early/2019/04/18/jimmun ol.1900050 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2019/04/18/jimmunol.190005 Material 0.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average by guest on October 2, 2021

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 19, 2019, doi:10.4049/jimmunol.1900050 The Journal of Immunology

Vaccination with a Single-Cycle Respiratory Syncytial Virus Is Immunogenic and Protective in Mice

Megan E. Schmidt,* Antonius G. P. Oomens,† and Steven M. Varga*,‡,x

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory tract infection in infants and young children, but no vaccine is currently available. Live-attenuated vaccines represent an attractive immunization approach; however, balancing attenuation while retaining sufﬁcient immunogenicity and efﬁcacy has prevented the successful development of such a vaccine. Recently, a recombinant RSV strain lacking the gene that encodes the matrix (M) protein (RSV M-null) was developed. The M protein is required for virion assembly following infection of a host cell but is not necessary for either genome replication or gene expression. Therefore, infection with RSV M-null produces all viral proteins except M but does not generate infectious virus progeny, resulting in a single-cycle infection. We evaluated RSV M-null as a potential vaccine candidate by determining its pathogenicity, immunogenicity, and protective capacity in BALB/c mice compared with its recombinant wild-type control virus (RSV recWT). RSV

M-null–infected mice exhibited significantly reduced lung viral titers, weight loss, and pulmonary dysfunction compared with Downloaded from mice infected with RSV recWT. Despite its attenuation, RSV M-null infection induced robust immune responses of similar magnitude to that elicited by RSV recWT. Additionally, RSV M-null infection generated serum Ab and memory T cell responses that were similar to those induced by RSV recWT. Importantly, RSV M-null immunization provided protection against secondary viral challenge by reducing lung viral titers as efficiently as immunization with RSV recWT. Overall, our results indicate that RSV M-null combines attenuation with high immunogenicity and efficacy and represents a promising novel live-attenuated RSV vaccine candidate. The Journal of Immunology, 2019, 202: 000–000. http://www.jimmunol.org/

espiratory syncytial virus (RSV) is the most common children become infected with RSV at least once by 2 y of age (3). cause of acute lower respiratory tract infection in infants Additionally, approximately half of all children are infected with R and young children (1). An estimated 33 million episodes RSV multiple times by the age of 2 y, although the most severe of RSV-associated lower respiratory tract infection occur annually disease is observed in children under 1 y of age experiencing a in children under 5 y of age, with over 3 million episodes re- primary RSV infection (3). Despite the immense healthcare bur- quiring hospitalization (2). RSV infection is responsible for up to den attributed to RSV infection, there remains no licensed RSV 150,000 deaths in young children each year (2). Nearly all vaccine. Given the high risk of severe disease associated with a by guest on October 2, 2021 primary RSV infection, RSV seronegative infants are an important target population for RSV vaccine development. *Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, The challenges of developing an efficacious RSV vaccine are IA 52242; †Center for Veterinary Health Sciences, Oklahoma State University, Still- highlighted by the failure of a formalin-inactivated RSV (FI-RSV) water, OK 74078; ‡Department of Microbiology and Immunology, University of x vaccine in the 1960s. FI-RSV vaccination failed to induce steril- Iowa, Iowa City, IA 52242; and Department of Pathology, University of Iowa, Iowa City, IA 52242 izing immunity against a subsequent natural RSV infection (4–7). ORCIDs: 0000-0003-0434-6626 (M.E.S.); 0000-0001-7332-4290 (S.M.V.). Surprisingly, FI-RSV–vaccinated children exhibited severe respi- ∼ Received for publication January 15, 2019. Accepted for publication April 2, 2019. ratory disease following a natural RSV infection with 80% re- quiring hospitalization at one of the trial sites, compared with This work was supported by the Department of Microbiology and Immunology at the University of Iowa (to S.M.V.), the Oklahoma Center for the Advancement of 5% of a control vaccine group (4–7). Furthermore, two of the Science and Technology under Award HR08-1395 (to A.G.P.O.), and the National FI-RSV–vaccinated children died as a result of a subsequent Institute of Allergy and Infectious Diseases and the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under Awards natural RSV infection (7). Given the disastrous results of the R01AI124093 (to S.M.V.), T32AI007485 (to M.E.S.), and P20GM103648 (to FI-RSV vaccine trial, the development of a nonlive viral vaccine A.G.P.O.). Data presented in this study were obtained at the Genomics Division of for use in the RSV seronegative infant population has been ham- the Iowa Institute of Human Genetics, which is supported in part by the University of Iowa Carver College of Medicine, the Holden Comprehensive Cancer Center, and the pered because of lingering safety concerns. Live-attenuated RSV National Cancer Institute of the NIH under Award P30CA086862. The content is vaccines are a particularly attractive vaccine platform because of solely the responsibility of the authors and does not necessarily represent the official their opportunity to be administered through the intranasal route, views of the NIH. allowing for direct stimulation of the immune response within the Address correspondence and reprint requests to Dr. Steven M. Varga, University of Iowa, 51 Newton Road, 3-532 Bowen Science Building, Iowa City, IA 52242. E-mail respiratory tract (8, 9). Importantly, live-attenuated RSV vaccines address: [email protected] have been tested in seronegative infants for many years without The online version of this article contains supplemental material. any evidence of enhanced respiratory disease following a natural Abbreviations used in this article: EF50, midtidal expiratory flow; EGFP, enhanced RSV infection (10, 11). However, achieving an appropriate GFP; FI-RSV, formalin-inactivated RSV; GC, germinal center; IAV, influenza A balance between sufficient attenuation and retention of immu- virus; M, matrix; mLN, mediastinal lymph node; N, nucleoprotein; ORF, open reading frame; Penh, enhanced pause; p.i., postinfection; RSV, respiratory syncytial virus; nogenicity has remained a primary obstacle in the successful RSV-A2, A2 strain of RSV; RSV recWT, recombinant WT control virus; SH, small development of a live-attenuated RSV vaccine. hydrophobic; Tfh, T follicular helper; TRM, tissue-resident memory CD8 T cell; WT, Single-cycle viruses have been developed as potential live- wild-type. attenuated vaccine candidates for a variety of viruses, including Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 influenza A virus (IAV) (12–24). Single-cycle viruses are attenuated

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900050 2 SINGLE-CYCLE RSV VACCINATION IS IMMUNOGENIC AND PROTECTIVE through a genetic modification that prevents the formation of cells) (25). The second-generation RSV M-null and the H2-M helper cell mature virions capable of spreading to neighboring uninfected line will be described in detail in a future manuscript. The second- cells. This approach is advantageous because an antiviral immune generation RSV M-null behaves identically to the previously published first generation RSV M-null virus in vitro, as the only difference between response may be initiated in the absence of the corresponding them is the genetic method for M ablation (25). Both RSV recWT and RSV pathology that occurs following a wild-type (WT) virus infection. M-null were generated from the A2 strain of RSV (RSV-A2). Mice were A novel single-cycle recombinant RSV strain lacking the gene infected with 1.0 3 106 PFU of either RSV recWT or RSV M-null in- encoding the matrix (M) protein (RSV M-null) was recently cre- tranasally in 100 ml vol. RSV-A2 was a gift from Dr. B. Graham (National Institutes of Health, Bethesda, MD) and was purified as previously de- ated (25). Although not required for either genome replication or scribed (26). Recombinant IAV-M282 was provided by Dr. R. Langlois gene expression, the M protein is essential for assembly of the (University of Minnesota, Minneapolis, MN) and was generated as pre- mature RSV virion following infection of a host cell (25). viously described (26, 27). Naive mice or mice that were infected 6 or Therefore, RSV M-null is not able to form infectious viral parti- 12 wk prior with either RSV recWT or RSV M-null were challenged with 3 6 cles following the initial infection of a host cell and undergoes either 1.5 10 PFU of purified RSV-A2 or a 5 LD50 dose of IAV-M282 intranasally. only a single round of infection. Indeed, RSV M-null infection in vitro elicited robust expression of multiple RSV proteins in the Real-time PCR absence of virus spread beyond the initially infected cell, con- Lungs were harvested from either RSV recWT– or RSV M-null–infected firming that RSV M-null behaves as a single-cycle virus in vitro mice, and RNA was isolated as previously described (28). cDNA was (25). Because M is poorly tolerated, RSV M-null could not be prepared using SuperScript VILO Master Mix (Invitrogen, Carlsbad, CA) sufficiently amplified in M-expressing cells to enable in vivo and used as a template for real-time PCR. Real-time PCR to detect the RSV nucleoprotein (N) or M genes was performed with TaqMan Universal studies. Therefore, a second-generation RSV M-null virus strain

PCR Master Mix (Applied Biosystems, Foster City, CA) on an ABI Downloaded from and production cell line were generated in which RSV M-null 7900 Real-Time PCR System (Applied Biosystems) using universal contains a Tet transactivator gene in place of the M protein thermocycling parameters. RSV N and M gene primers and probe se- open reading frame (ORF), and the M gene in the M-expressing quences have been described previously (29, 30). Probes were synthe- cell line is preceded by a Tet-responsive promoter. In this manner, sized with a 59-6FAM reporter dye and a 39-TAMRA quencher dye (Integrated DNA Technologies, Coralville, IA). Results were analyzed RSV M-null viral titers can be readily generated at similar levels using Sequence Detection System analysis software (Applied Bio- to WT virus and used for in vivo studies. systems). Samples were compared with known standard dilutions of a In this study, we evaluate the second-generation RSV M-null as a plasmid containing either the RSV N or M gene. The number of RSV N http://www.jimmunol.org/ potential RSV vaccine candidate in vivo by assessing its patho- and M gene copies per lung was calculated based on the number of RSV gene copies in the sample and the total RNA isolated from the lung. genicity, immunogenicity, and protective capacity in BALB/c mice, which have been widely used to study RSV-induced pathogenesis Plaque assay for RSV and IAV and immunity. In comparison with its recombinant WT control Whole lungs were harvested from mice, weighed, and mechanically ho- virus (RSV recWT), RSV M-null–infected mice exhibited sig- mogenized, and supernatants were flash-frozen in liquid nitrogen prior to nificantly reduced lung viral titers, weight loss, and pulmonary storage at 280˚C. Plaque assays were performed using either H2-M helper dysfunction. Remarkably, RSV M-null infection elicited robust cells or VERO cells (American Type Culture Collection, Manassas, VA) acute and late memory immune responses that were similar as indicated in the figure legends to determine RSV titers as previously described (26). Plaque assays on MDCK cells (American Type Culture by guest on October 2, 2021 in magnitude to RSV recWT infection. Importantly, RSV M-null Collection) to determine IAV titers were performed as previously immunization provided protection against a secondary viral described (26). challenge as efficiently as immunization with RSV recWT. Therefore, to our knowledge, RSV M-null combines, for the first Assessment of weight loss and pulmonary function time, an effective and highly stable attenuation phenotype without Mice were evaluated daily following infection for weight loss and pul- a loss of immunogenicity resulting in fully protective immunity. monary function using an unrestrained, whole-body plethysmograph The stable attenuation because of the absence of the entire M ORF (Buxco Electronics, Wilmington, NC). Whole-body plethysmography parameters were measured as changes in respiration from baseline values prior may overcome the past difficulties experienced with attempting to infection and did not use methacholine administration. Pressure and to balance the level of attenuation by constraining genome repli- volume changes in the chamber caused by respiration were averaged over a cation while maintaining a high level of efficacy. Together, our 5-min period and used to calculate enhanced pause (Penh), midtidal ex- results indicate that RSV M-null is a promising prototype from piratory flow (EF50), and respiratory rate. which to develop a safe, live-attenuated RSV vaccine candidate. Flow cytometry analysis Lungs and mediastinal lymph nodes (mLN) were harvested from mice, and Materials and Methods single-cell suspensions were generated as previously described (31, 32). Mice Cells were stained extracellularly with CD8 tetramers (all tetramers made in-house) and mAbs specific to CD90.2 (clone 53-2.1; BioLegend, San Female BALB/cAnNCr and C57BL/6NCr mice between 6 and 8 wk old Diego, CA), CD4 (clone RM4-5), CD8 (clone 53-6.7), CD11a (clone M17/4), were purchased from the National Cancer Institute (Frederick, MD). First- CD49d (clone R1-2), CD45 (clone 30-F11; BioLegend), CD3 (clone 145- generation C57BL/6 x BALB/c (CB6F1) mice were bred at the University of 2C11), CD19 (clone 6D5), Fas (clone 15A7), GL-7 (clone GL7; Bio- Iowa Animal Facility and used between 6 and 8 wk old. All experimental Legend), CD69 (clone H1.2F3; BioLegend), and CD103 (clone 2E7) in procedures using mice were approved by the University of Iowa Animal FACS buffer (PBS, 2% FCS, and 0.02% sodium azide) for 30 min at 4˚C Care and Use Committee under Animal Protocols no. 4101196 and no. and fixed using 1-step Fix/Lyse Solution for 10 min at room temperature 7041999. The experiments were performed under strict accordance to the (eBioscience, San Diego, CA). For CXCR5 staining, cells were incubated Office of Laboratory Animal Welfare guidelines and the Public Health with the mAb specific to CXCR5 (biotin; BD Biosciences, San Jose, CA) Service Policy on Humane Care and Use of Laboratory Animals. at room temperature for 30 min, followed by streptavidin/PE in FACS Viruses and infection buffer. For transcription factor staining, cells were fixed with Foxp3 staining buffer set (eBioscience) and stained intracellularly for Bcl6 (clone RSV recWT in which the small hydrophobic (SH) ORF was replaced with BCL-DWN) according to manufacturer’s instructions. For intracellular that of enhanced GFP (EGFP) was generated as previously described (25). cytokine staining, cells were stimulated with 1 mM of either M282–90 A second-generation RSV M-null in which the SH ORF was replaced with or G183–195 peptides (Bio-Synthesis, Lewisville, TX) in the presence of that of EGFP and the M ORF was replaced with that of a Tet transactivator 10 mg/ml brefeldin A (Sigma-Aldrich, St. Louis, MO) for 5 h at 37˚C in sequence was propagated in HEp-2 cells containing a plasmid in which the 10% FCS-supplemented RPMI 1640. Stimulated cells were stained for surface M ORF was expressed behind a Tet-inducible promoter (H2-M helper markers as indicated above and subsequently stained intracellularly for IFN-g The Journal of Immunology 3

(clone XMG1.2) in FACS buffer containing 0.5% saponin (Sigma-Aldrich) express viral genes. Interestingly, RSV N gene copy numbers for 30 min at 4˚C. All Abs were purchased from eBioscience unless other- within the lung were similar following infection with RSV recWT wise indicated. Samples were run on a BD LSRFortessa and analyzed using and RSV M-null at all timepoints evaluated (Fig. 1D). Impor- FlowJo software (Tree Star, Ashland, OR). Cell types were phenotyped as follows: CD4 T cells (CD90.2+CD4+), CD8 T cells (CD90.2+CD8+), and tantly, RSV M gene copies were only detected in the lungs of RSV B cells (CD45+CD32CD19+). recWT–infected mice, and not in RSV M-null–infected mice, confirming that production of the M gene is absent following RSV RSV Ab ELISA M-null infection (Fig. 1E). Therefore, RSV M-null exhibits Mouse serum was collected on days 14, 28, and 84 following infection with robust viral gene expression of the N gene, despite its inability either RSV recWTor RSV M-null. RSV-specific serum Ab was measured by to generate infectious particles and spread beyond the initially ELISA as previously described (33). Briefly, serum was serially diluted 1:2 starting at 1:32 over 8 total dilutions and incubated overnight at 4˚C on infected cell. plates that were previously coated with 1 3 105 PFU/well of RSV-A2 and Because of its demonstrated attenuation of in vivo, we next blocked with 5% nonfat dry milk in PBS. RSV-specific Ab was de- evaluated whether RSV M-null induced lower magnitude clinical tected using biotinylated goat anti-mouse Ab specific for total Ig, IgG1, or signs of disease following infection compared with RSV recWT IgG2a (SouthernBiotech, Birmingham, AL). Plates were incubated with infection. RSV recWT infection induced weight loss up to 10% of streptavidin/horse radish peroxidase conjugate (Sigma-Aldrich), developed in 3,39,5,59-tetramethylbenzidine solution (Sigma-Aldrich), and the reac- the animal’s total body weight prior to recovery, as is typical of an tion was stopped with 2 M sulfuric acid. Absorbance values (450 nm) were acute RSV infection of BALB/c mice (Fig. 2A) (32, 36, 37). In measured and assessed using Gen5 software (BioTek, Winooski, VT). contrast, RSV M-null–infected mice exhibited significantly reduced Intravascular staining weight loss at all timepoints examined (Fig. 2A). Additionally, whole-body plethysmography was used to measure several param- Mice were injected intravascularly with 1 mg CD45-PE Ab (anti-CD45 eters of pulmonary function, including Penh as an indicator of Downloaded from labeled with PE) (clone 30-F11) 3 min prior to euthanasia. Cells from the lung were processed as previously described (34). airway obstruction, EF50 as a measure of bronchoconstriction, and respiratory rate (38–40). Infection with RSV recWT induced pul- Statistical analysis monary dysfunction, as defined by an elevation in Penh and EF50, All statistical analyses are described in each figure legend and were per- and a decline in respiratory rate from baseline (Fig. 2B–D). In formed using Prism software (GraphPad Software, San Diego, CA). Data contrast, RSV M-null–infected mice exhibited little change in Penh, were evaluated using either unpaired, two-tailed Student t tests between two EF50, and respiratory rate values from baseline, indicating a sig- http://www.jimmunol.org/ groups or one-way ANOVAwith Tukey–Kramer posttest for more than two nificant reduction in pulmonary dysfunction compared with RSV groups to determine a statistical significance of at least a = 0.05. recWT infection (Fig. 2B–D). Overall, these results indicate that RSV M-null infection significantly reduces clinical signs of disease, Results including weight loss and pulmonary dysfunction, compared with RSV M-null infection does not produce infectious progeny RSV recWT. in vivo and induces lower magnitude clinical signs of disease The single-cycle virus RSV M-null was generated such that the M RSV M-null infection induces robust immune responses of protein ORF was replaced with a Tet transactivator sequence to similar magnitude to RSV recWT by guest on October 2, 2021 facilitate its growth in vitro in the H2-M helper cell line, which Because of the strong attenuation of RSV M-null’s production of drives expression of the M gene by a Tet-responsive promoter infectious viral progeny and pathogenesis, we evaluated the im- (Fig. 1A). Additionally, EGFP was inserted in place of the SH munogenicity of RSV M-null vaccination. First, we determined ORF, which is dispensable for virus replication both in vitro and the ability of RSV M-null to induce a T cell response, as both CD4 in vivo, to allow for visualization of the virus via microscopy and CD8 T cells are critical for viral clearance following an acute (Fig. 1A) (35). In all experiments, RSV M-null was compared RSV infection (37, 41, 42). Remarkably, the total numbers of with RSV recWT, a recombinant WT RSV-A2 strain that encodes activated CD4 (Fig. 3A) and CD8 T cells (Fig. 3B) in the lung, as the RSV M gene but also contains EGFP in place of the SH ORF determined by surrogate activation marker staining, were similar (Fig. 1A). To confirm that RSV M-null behaves as a single-cycle between RSV recWT– and RSV M-null–infected BALB/c mice on virus in vivo, plaque assays using H2-M helper cells were per- days 6, 8, and 10 p.i. (43, 44). RSV M-null infection also induced + formed to quantify both EGFP focus–forming units (Fig. 1B) and CD8 T cells specific to the immunodominant M282 CD8 T cell infectious PFU (Fig. 1C) in the lungs of RSV recWT– and RSV epitope in the lung to a similar magnitude as observed following M-null–infected mice. RSV recWT was detected by both EGFP+ RSV recWT infection (Fig. 3C) (45–47). Similar results were foci and PFU on day 1 postinfection (p.i.), increased approxi- observed for CD8 T cell responses in the lung against the sub- mately two additional logs to peak viral titers on day 4 p.i., and dominant CD8 T cell epitopes M2127,F85, and F249 (Fig. 3D–F) was completely cleared by day 7 p.i. (Fig. 1B, 1C). In contrast, (48–50). The only significant difference observed between T cell RSV M-null EGFP+ foci were visualized on day 1 p.i. and were no responses induced by RSV recWT and RSV M-null infection was longer detected by day 2 p.i. (Fig. 1B). Although RSV M-null the number of F85-specific CD8 T cells in the lung on day 10 p.i. EGFP+ foci were seen on day 1 p.i., no infectious PFU were (Fig. 3E). Lung CD4 and CD8 T cells induced by RSV M-null present on day 1 p.i. (Fig. 1B, 1C). However, the number of infection were also capable of producing effector cytokines, as EGFP+ cells were not significantly different between RSV RSV recWT– and RSV M-null–infected mice exhibited similar recWT– and RSV M-null–infected mice on day 1 p.i., confirming numbers of IFN-g+ CD4 (Fig. 3G) and CD8 T cells (Fig. 3H) in that the number of initially infected cells within the lung were the lung following ex vivo stimulation with G183 and M282 pep- similar between infections (Fig. 1B). Importantly, infectious PFU tides, respectively. Overall, these results indicate that despite its were undetectable in the lungs of RSV M-null–infected mice at inability to produce infectious progeny following infection, RSV all timepoints evaluated (Fig. 1C). These results confirm that RSV M-null vaccination induces robust T cell responses of similar M-null does not produce infectious progeny following infection magnitude to that of RSV recWT. and behaves as a single-cycle virus in vivo. We next used C57BL/6 mice to evaluate the magnitude of T cell Given its lack of infectious progeny production in vivo, we responses directed against the RSV M protein following RSV performed real-time PCR to evaluate RSV M-null’s ability to M-null vaccination, as BALB/c mice do not contain any defined 4 SINGLE-CYCLE RSV VACCINATION IS IMMUNOGENIC AND PROTECTIVE

FIGURE 1. No infectious progeny is detected following RSV M-null infection in vivo. (A) Diagram depict- ing the genome content of RSV recWT and RSV M-null. (B–E) BALB/c mice were infected with either RSV recWT or RSV M-null, and lungs were harvested on days 1, 2, 4, and 7 p.i. (B) EGFP+ focus– forming units and (C) infectious PFU were quantiﬁed in the lung by plaque assay using H2-M helper cells. Open (recWT) and closed (M-null) circles represent values for individual mice, and lines indicate mean 6 SEM of two independent experiments (n = 8).

(D) RSV N gene and (E) RSV M gene Downloaded from copy numbers per lung were determined by real-time PCR. Data are presented as mean 6 SEM of representative results from one of two independent experiments (n =4).Dashed lines denote the limit of detection for each assay. Groups were compared http://www.jimmunol.org/ using one-way ANOVA. **p , 0.01, ***p , 0.001.

CD4 or CD8 T cell epitopes within the RSV M protein (50). Similar CD8 T cells (Supplemental Fig. 1B) in the lung following infec- by guest on October 2, 2021 to what was observed in BALB/c mice, C57BL/6 mice exhibited a tion with either RSV recWT or RSV M-null. In contrast, the similar number of total activated CD4 (Supplemental Fig. 1A) and evaluation of M protein–speciﬁc CD8 T cell responses by staining

FIGURE 2. RSV M-null infection reduces weight loss and pulmonary dysfunction. BALB/c mice were infected with either RSV recWT or RSV M-null and monitored daily for (A) weight loss and respiratory parameters including (B) Penh, (C)EF50,and(D) respiratory rate by whole-body plethysmography. Data are presented as mean 6 SEM of six independent experiments (n = 35 for recWT and n = 36 for M-null). Groups were compared using Student t test. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 5

BALB/c and C57BL/6 mice, CB6F1 mice infected with RSV M-null exhibited similar numbers of total activated CD4 (Supplemental Fig. 2A) and CD8 T cells (Supplemental Fig. 2B) as those infected with RSV recWT on day 8 p.i. M282-specific CD8 T cells in the lung were also similar in both frequency (Supplemental Fig. 2C) and total number (Supplemental Fig. 2D) following infection with either RSV recWT or RSV M-null. In contrast, M187-specific CD8 T cell responses were significantly reduced in RSV M-null– infected CB6F1 mice compared with mice infected with RSV recWT (Supplemental Fig. 2C, 2D). Therefore, M-specific CD8 T cell responses are significantly reduced following infection with RSV M-null, but the magnitude of non-M protein–specific CD8 T cell responses are largely unaf- fected by RSV M-null’s inability to produce infectious viral progeny. Given that overall CD4 T cell activation following RSV M-null infection was similar to that of RSV recWT infection, we next evaluated whether T follicular helper (Tfh) cells were generated by RSV M-null infection in BALB/c mice. Tfh cells Downloaded from are a subset of CD4 T cells that interact with germinal center (GC) B cells and are critical for the generation of long-lived plasma cells and memory B cells (57–59). We evaluated Tfh generation in the mLN by staining for the Tfh marker CXCR5 and the Tfh lineage–defining transcription factor Bcl6 (Fig. 4A) (60–64). Tfh cells were present in the mLN 14 d following RSV http://www.jimmunol.org/ M-null infection at a similar magnitude to that observed following RSV recWT infection (Fig. 4A, 4B). The Tfh populations in the mLN for both RSV recWT and RSV M-null declined in both frequency (Fig. 4A) and total number (Fig. 4B) on day 21 but remained comparable between the two infections. In addition, a small, but detectable, population of Tfh cells in the lung was also induced by RSV M-null infection, which was similar in magnitude to that of RSV recWT infection (Fig. 4C, by guest on October 2, 2021 4D). Overall, RSV M-null infection generates Tfh cells in the mLN and lung of BALB/c mice comparable to RSV recWT infection. FIGURE 3. RSV M-null infection induces T cell responses of similar In addition to T cell responses, we also determined the ability magnitude to recWT infection. BALB/c mice were infected with either of RSV M-null infection to generate B cells in the mLN and RSV recWT or RSV M-null, and lungs were harvested on days 6, 8, and 10 lung. The number of B cells in the mLN were similar in p.i. Total numbers of (A) CD11ahiCD49d+ CD4 T cells, (B) CD11ahiCD8lo magnitude between RSV recWT and RSV M-null infection on C D E F + CD8 T cells, and ( )M282-, ( )M2127-, ( )F85-, and ( )F249-tetramer day 14 p.i., but RSV M-null–infected mice exhibited fewer G H CD8 T cells were quantified. Total numbers of ( )CD4Tcellsand( )CD8 B cells on day 21 p.i. compared with RSV recWT–infected T cells producing IFN-g after restimulation with G and M2 peptides, 183 82 mice (Fig. 5A). In contrast, the total number of B cells in the respectively, were determined by intracellular cytokine staining. Data are presented as mean 6 SEM of two to three independent experiments (n =8 lung of RSV M-null–infected mice was decreased on day 14 for days 6 and 10 and n = 12 for day 8). Groups were compared using p.i. compared with RSV recWT infection, but similar in mag- Student t test. **p , 0.01. nitude on day 21 p.i. (Fig. 5B). We also evaluated whether GC B cells, which differentiate into Ab-secreting B cells, were generated following infection with RSV M-null (58, 59). Using with a tetramer specific to the immunodominant M187 CD8 the GC B cell markers GL-7 and Fas, we detected a sizeable GC T cell epitope in C57BL/6 mice revealed a dramatically reduced B cell population in the mLN of both RSV recWT– and RSV frequency and total number of M-specific CD8 T cells in the M-null–infected mice on day 14 p.i. (Fig. 5C, 5D). The GC lung following RSV M-null infection compared with RSV B cell population in the mLN declined by day 21 p.i., but recWT infection (Supplemental Fig. 1C, 1D) (51). Despite RSV remained similar in magnitude between RSV recWT and RSV M-null’s inability to produce the M protein, a small frequency M-null infections on both days 14 and 21 p.i. (Fig. 5C, 5D). of M187-specific CD8 T cells were detected in the lungs fol- Similar results were also observed for GC B cells in the lung of lowing RSV M-null infection, indicating that the initial amount RSV recWT– and RSV M-null–infected mice (Fig. 5E, 5F). of M protein within the virus inoculum is sufficient to prime the Therefore, RSV M-null infection generates B cell responses, activation of a fraction of the normal M187-specific CD8 T cell including GC B cells, at a similar magnitude to that of RSV response (Supplemental Fig. 1C). recWT infection. Overall, immunization with the single-cycle Additionally, we evaluated the ability of RSV M-null to in- virus RSV M-null induces robust immune responses, including duce a T cell response in CB6F1 mice, a cross between C57BL/6 CD4 T cells, CD8 T cells, Tfh cells, and GC B cells, that are of and BALB/c mice, which are frequently used as a murine model similar magnitude to infection with the replication-competent for RSV infection (52–56). Similar to what was observed in RSV recWT virus. 6 SINGLE-CYCLE RSV VACCINATION IS IMMUNOGENIC AND PROTECTIVE

FIGURE 4. Tfh cells are generated by RSV M-null infection. BALB/c mice were infected with either RSV recWT or RSV M-null, and mLN and lungs were harvested on days 14 and 21 p.i. Cells were gated on CD4 T cells, and gates were placed using ﬂuorescence minus one controls. Representative staining panels of Bcl6+CXCR5+ Tfh cells in the (A) mLN and (C) lung. Total numbers of Bcl6+CXCR5+ Tfh cells in the (B) mLN and (D) lung. Data are Downloaded from presented as mean 6 SEM of two independent experiments (n = 8). Groups were compared using Student t test. http://www.jimmunol.org/

RSV M-null infection generates long-lasting Abs and memory RSV recWT–infected mice at the late memory timepoint of 100 d by guest on October 2, 2021 T cell responses p.i. These results indicate that although the T cell contraction The induction of RSV-specific Abs in the serum has been correlated kinetics may be accelerated in RSV M-null–infected mice, with a reduced risk of secondary RSV infection and amelioration of memory T cells contract to form a stable memory T cell pop- RSV-associated disease in humans (65–68). Therefore, we deter- ulation of similar magnitude to RSV recWT infection. Similar mined the degree to which RSV M-null infection induces RSV- results were observed for M282-specific memory CD8 T cells in the lung parenchyma (Fig. 7C). Additionally, we evaluated the specific Abs in the serum compared with RSV recWT infection of ability of RSV M-null immunization to generate RSV-specific BALB/c mice. Early p.i. on days 14 and 28, RSV M-null–infected tissue-resident memory CD8 T cells (T ), which have been mice exhibited a robust induction of RSV-specific serum Abs, RM shown to be elicited following RSV infection (69, 70). As ex- including total Ig-, IgG1-, and IgG2a-specific Abs (Fig. 6). The pected, RSV recWT infection induced a large proportion of serum of RSV M-null–infected mice exhibited an elevated level of M2 -specific memory CD8 T cells within the lung parenchyma both total Ig- and IgG1-specific Abs at the lower Ab dilutions com- 82 expressing both CD69 and CD103, the canonical markers of T , pared with RSV recWT–infected mice (Fig. 6A, 6B). Importantly, the RM on day 30 p.i. (Fig. 7D) (71, 72). The T population generated RSV-specific serum Abs generated by RSV M-null infection were RM by RSV recWT infection contracted to a lower number by 100 d long-lasting, with Ab levels similar to that of RSV recWT–infected p.i. (Fig. 7D). RSV M-null infection also induced a population of mice at the late memory timepoint of day 84 p.i. (Fig. 6). Overall, M2 -specific T on day 30 p.i. (Fig. 7D). Similar to both total RSV M-null infection induces serum Ab responses that are long- 82 RM activated and M282-specific memory CD8 T cells, the number of lasting and similar in magnitude to RSV recWT infection. M282-specific TRM was decreased in magnitude as compared with In addition to evaluating serum Abs, we also determined the RSV recWT infection (Fig. 7E). Importantly, both the frequency ability of RSV M-null to generate memory T cell responses, which (Fig. 7D) and total number (Fig. 7E) of RSV-specific TRM generated are important for providing protection against secondary infection by RSV M-null infection were similar to RSV recWT infection on (26, 69). We used an intravascular staining technique to ensure day 100 p.i., indicating that RSV M-null vaccination generates long- that the memory T cells were located within the lung parenchyma lived, RSV-specific TRM. Overall, RSV M-null infection induces rather than the pulmonary vasculature (34, 70). The number of memory CD4 and CD8 T cell responses that are similar in magni- total activated CD4 T cells (Fig. 7A) and CD8 T cells (Fig. 7B) tude to RSV recWT infection at a late memory timepoint. within the lung parenchyma were decreased in RSV M-null– infected mice at the early memory timepoint of 30 d p.i. com- RSV M-null immunization provides protection against pared with RSV recWT–infected mice. However, the number of secondary viral challenge activated CD4 (Fig. 7A) and CD8 T cells (Fig. 7B) in RSV We next evaluated the ability of RSV M-null vaccination to provide M-null–infected mice contracted down to a similar magnitude as protection against a secondary RSV infection. BALB/c mice were The Journal of Immunology 7

FIGURE 5. RSV M-null infection induces similar numbers of GC B cells as recWT infection. BALB/c mice were infected with either

RSV recWT or RSV M-null, and mLN and lungs Downloaded from were harvested on days 14 and 21 p.i. To- tal numbers of CD19+ B cells in the (A) mLN and (B) lung. Representative staining panels of Fas+GL-7+ GC B cells in the (C) mLN and (E) lung. Total numbers of Fas+GL-7+ GC B cells in the (D) mLN and (F) lung. Data are presented as mean 6 SEM of two independent experiments http://www.jimmunol.org/ (n = 8). Groups were compared using Student t test. *p , 0.05. by guest on October 2, 2021

immunized with either RSV recWT or RSV M-null and challenged with either RSV recWT or RSV M-null 6 wk prior were chal- with RSV 6 wk later (Fig. 8A). Although naive mice receiving a lenged with IAV-M282, a recombinant IAV strain expressing the primary RSV infection exhibited high levels of virus in their lungs RSV M282 CD8 T cell epitope. Using this approach, we can on day 4 postchallenge, immunization with either RSV recWT or evaluate the isolated ability of M282-specific CD8 T cells to re- RSV M-null resulted in complete viral clearance in the lungs duce viral titers, as the only RSV-specific cells that will specif- (Fig. 8A). Importantly, the protection afforded by RSV M-null ically respond to IAV-M282 challenge are the M282-specific CD8 vaccination is long lasting, as mice challenged with RSV at T cells generated by vaccination. M282-specific memory CD8 12 wk postvaccination also completely cleared the virus from T cells generated by immunization with either RSV recWT or the lungs (Fig. 8B). Therefore, RSV M-null vaccination pro- RSV M-null significantly reduced lung viral titers on day 6 tects against a secondary RSV infection by reducing lung viral postchallenge compared with naive mice receiving a primary titers after RSV challenge. IAV-M282 infection (Fig. 8C). However, there was no difference in RSV-specific memory CD8 T cells have previously been shown IAV-M282 lung viral titers between mice vaccinated with either to significantly reduce lung viral titers following a secondary RSV RSV recWT or RSV M-null, indicating that memory CD8 T cells challenge independently of both RSV-specific Abs and memory generated by RSV M-null vaccination provided protection as ef- CD4 T cells (26). Given the protective potential of RSV-specific ficiently as those induced by RSV recWT infection (Fig. 8C). memory CD8 T cells, we wanted to determine the specific ability Importantly, the protection provided by RSV-specific memory of RSV-specific memory CD8 T cells induced by RSV M-null CD8 T cells was long term, as mice challenged with IAV-M282 vaccination to provide protection against a secondary viral chal- at 12 wk postimmunization also exhibited a significant reduc- lenge. Therefore, BALB/c mice that were previously immunized tion in lung viral titers compared with naive controls (Fig. 8D). 8 SINGLE-CYCLE RSV VACCINATION IS IMMUNOGENIC AND PROTECTIVE

FIGURE 6. RSV M-null infection generates long-lasting RSV-speciﬁc serum Ab responses. BALB/c mice were infected with either RSV recWT or RSV M-null, and serum was collected on days 14, 28, and 84 p.i. Serum was assessed Downloaded from for levels of RSV-speciﬁc (A) total Ig, (B) IgG1, and (C) IgG2a by ELISA. Data are presented as mean 6 SEM of representative results from one of two independent experiments (n = 5). Groups were compared using Student t test. *p , 0.05, **p , 0.01, ***p , 0.001. http://www.jimmunol.org/ by guest on October 2, 2021

Overall, RSV M-null vaccination provides long-lasting protection day 4 p.i. It is not clear why RSV N gene copy levels are similar against both secondary RSV and IAV-M282 challenges by signif- between RSV recWT– and RSV M-null–infected mice. In vitro, icantly reducing lung viral titers. the ﬁrst-generation RSV M-null replicated and transcribed geno- mic RNA at higher levels than RSV recWT because of the absence Discussion of replication inhibition by the RSV M protein (25, 73). Therefore, The RSV M protein is critical for the production of infectious viral the lack of replication inhibition by RSV M may also contribute to progeny and the formation of viral ﬁlaments in vitro (25). Thus, the unexpectedly high copy levels of the RSV N gene following infection with the single-cycle virus RSV M-null, which does in vivo infection with the second generation RSV M-null, which not contain the RSV M gene, cannot generate infectious viral may be masking potential differences. Alternatively, the large viral particles in WT cell lines (25). However, in the H2-M helper cell inoculum required for RSV infection in mice, which are semi- line, which provides expression of the M protein via a trans- permissive hosts, may be the primary driver of viral gene ex- complementing plasmid, RSV M-null is capable of spreading to pression (74, 75). The amount of input virus for RSV recWT and neighboring uninfected cells (25). These results correspond to RSV M-null (1 3 106 PFU) was substantially larger than the what we observed following in vivo infection with RSV M-null in amount of virus detected in the lungs of RSV recWT–infected BALB/c mice, which also did not produce infectious viral progeny mice (1 3 105 PFU/gram of lung, ∼2.5 3 104 PFU/whole lung). in the lung at any of the timepoints evaluated following infection. Thus, it is likely that viral gene expression is primarily a result of Although RSV M-null infection in vivo did not result in mature genome replication by the initial viral inoculum, and the relatively virion formation, viral gene expression of RSV N was robust and small amount of infectious progeny produced following infection similar in magnitude to RSV recWT infection. Given that the peak with a replication-competent virus has minimal impact on gene of infectious virus replication is day 4 p.i., it is particularly in- expression levels. If this is the case, the lack of difference in RSV teresting that the magnitude of RSV N gene expression remains N gene expression observed following RSV recWT and RSV similar between RSV recWT– and RSV M-null–infected mice on M-null infection would be expected given that the initial infection The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 7. RSV M-null infection induces RSV-specific TRM. BALB/c mice were infected with either RSV recWT or RSV M-null and administered anti- CD45 Ab intravascularly 3 min prior to euthanasia on days 30 and 100 p.i. Cells negative for staining with the CD45 intravascular Ab (IV2) were gated, and hi + hi lo + total numbers of (A) CD11a CD49d CD4 T cells, (B) CD11a CD8 CD8 T cells, and (C)M282-tetramer CD8 T cells within the lung parenchyma are + + 2 + shown. (D) Representative staining panels and (E) total numbers of CD69 CD103 IV M282-tetramer CD8 T cells. Data are presented as mean 6 SEM of two independent experiments (n = 10 for day 30 and n = 9 for day 100). Groups were compared using Student t test. *p , 0.05, **p , 0.01, ***p , 0.001. dose was identical. We are currently examining this further using the Although attenuation of pathogenesis following vaccination is by guest on October 2, 2021 RSV M-null virus as a tool to evaluate the mechanisms involved. critical, it often corresponds with a marked reduction in immu- Regardless of the underlying mechanism, the data clearly demon- nogenicity. Indeed, the immune responses generated following strate that a single-cycle live RSV is sufficient to induce a compre- immunization with single-cycle virus vaccine candidates against hensive, protective, and long-lasting immune response. IAVand CMV were substantially reduced compared with their WT The attenuation of live viral vaccine candidates to reduce counterparts (16, 80). In contrast, single-cycle adenovirus, rabies pathogenesis following immunization is a critical component of virus, and vesicular stomatitis virus vectors induced robust im- vaccine development. Importantly, RSV M-null–infected mice mune responses that were comparable to WT controls (15, 81, 82). exhibited significantly reduced weight loss and pulmonary dys- Therefore, the immunogenicity of single-cycle virus vaccine function, as measured by the respiratory parameters Penh, EF50, candidates varies widely. Remarkably, RSV M-null immunization and respiratory rate, compared with RSV recWT infection. These induced robust acute immune responses, including CD4 T cells, results are similar to what was previously observed following vac- CD8 T cells, B cells, and serum Abs, that were similar in mag- cination of mice with single-cycle virus vaccines against IAV, HSV, nitude to those generated by RSV recWT infection. These results and vesicular stomatitis virus (16, 19, 20, 22). Clinical signs of correlated with the robust expression of the RSV N gene in the disease including early weight loss and pulmonary dysfunction were lung following RSV M-null infection, despite its lack of infectious almost completely abrogated following RSV M-null infection with progeny production. These results suggest that sustained viral little change from preimmunization baseline levels. Although RSV gene expression, rather than the production and spread of infec- M-null infection induced a small amount of weight loss and pul- tious viral progeny, is responsible for the majority of the immune monary dysfunction at the later phase of infection between days 5 activation that occurs following RSV infection in mice. Interest- and 7 p.i., it was dramatically reduced compared with infection with ingly, although significantly reduced compared with RSV recWT RSV recWT. The clinical signs of disease that occur following RSV infection, a small population of M187-specific CD8 T cells were infection of BALB/c mice between day 5 and day 7 p.i. is largely detectable in the lungs of RSV M-null–infected C57BL/6 and attributed to the induction of the RSV-specific T cell response (37, CB6F1 mice. This observation suggests that a portion of the CD8 41, 42). However, the acute T cell response induced by RSV M-null T cell response is primed solely by the RSV M protein that is vaccination was similar in magnitude to that of an RSV recWT in- contained within the initial viral inoculum. It is possible that fection. Thus, it is likely that inflammatory cytokines, such as IFN-g APCs in the lung and the airway that were directly infected with and TNF, induced by continued RSV production of infectious the initial RSV M-null inoculum primed the small M187-specific progeny contribute to the T cell–mediated pathology observed CD8 T cell response observed following infection of C57BL/6 following RSV recWT infection (76–79). Overall, RSV M-null is and CB6F1 mice. RSV M-null provides an interesting tool to sufficiently attenuated to prevent pathogenesis, as the induction of further examine the degree to which immune responses are clinical signs of disease following vaccination is minimal. primed by the original inoculum prior to the production of viral 10 SINGLE-CYCLE RSV VACCINATION IS IMMUNOGENIC AND PROTECTIVE

FIGURE 8. RSV M-null infection provides protection against secondary viral challenge. (A and B) Naive BALB/c mice were infected with either RSV recWT or RSV M-null and challenged with RSV either (A) 6 or (B) 12 wk p.i. Lung RSV titers on day 4 p.i. were determined by plaque assay using VERO cells. (C and D) Naive BALB/c mice were infected with either RSV recWT or RSV M-null and challenged with

IAV-M282 either (C) 6 or (D) 12 wk p.i. Lung IAV titers on day 6 p.i. were determined by plaque assay using MDCK cells. Data are presented as mean 6 SEM of two independent experiments (n = 8). Groups were compared using , one-way ANOVA. ***p 0.001. Downloaded from http://www.jimmunol.org/ progeny. Overall, these results indicate that despite its signifi- in mice. RSV M-null behaved as a single-cycle virus in vivo, as it cant attenuation, RSV M-null infection is strongly immunoge- did not produce infectious viral particles following infection. RSV nic in mice. M-null is sufficiently attenuated, as RSV M-null–infected mice In addition to robust acute immune responses, RSV M-null exhibited significantly reduced weight loss and pulmonary vaccination also induced serum Ab and memory T cell re- dysfunction compared with infection with RSV recWT. Sur- sponses that were similar in magnitude to RSV recWT infection at a prisingly, despite its inability to produce infectious progeny and late memory timepoint. A portion of the memory CD8 T cells spread to neighboring cells, RSV M-null vaccination induced induced by RSV M-null vaccination were RSV-specific T . robust immune responses, including long-lasting serum Abs and

RM by guest on October 2, 2021 TRM persist in the lung and rapidly respond to pathogen exposure memory T cell responses, that were similar in magnitude to that by producing inflammatory cytokines, such as IFN-g (71, 83, 84). of an RSV recWT infection. Importantly, RSV M-null vacci- Importantly, TRM in the lung have been demonstrated to provide nation provided protection against secondary viral challenges protection against secondary infection with IAV (83–85). Therefore, by reducing lung viral titers as effectively as immunization vaccination to induce RSV-specific TRM may provide a critical with RSV recWT. Therefore, a single immunization with RSV component of the protective immune response. Interestingly, the M-null is strongly immunogenic and protective in mice. Hu- magnitude of memory T cell responses induced by RSV M-null mans are more susceptible to RSV infection than mice. Fur- vaccination was reduced on day 30 p.i., but was similar by day thermore, intranasal vaccination of humans results in virus 100 p.i., compared with RSV recWT vaccination. These results deposition primarily in the upper respiratory tract, whereas in- indicate that the T cell contraction kinetics may be accelerated fection of mice results in direct virus deposition in the lungs. following infection with RSV M-null compared with RSV recWT. Thus, whether RSV M-null would be similarly efficacious fol- Importantly, the serum Abs and memory T cells induced by RSV lowing intranasal vaccination in humans will need to be deter- M-null vaccination provided protection against secondary viral mined in future studies. Our data indicate that RSV M-null may challenge with both RSV-A2 and IAV-M282. These results indicate provide a safety advantage over other line-attenuated vaccine that both Ab and T cell responses contribute to the protection candidates for use in humans. As the entire M gene sequence is afforded by RSV M-null vaccination. The ability of RSV M-null to absent from the viral genome, the RSV M-null vaccine cannot provide protection is particularly striking given that, despite its in- regain virulence by restoring M function. This is in contrast to ability to produce infectious progeny, a single immunization was live vaccines attenuated by point mutations, which have the sufficient to provide protection as efficiently as was observed with potential to revert and regain virulence. Overall, our results immunization with the replication-competent RSV recWT virus. suggest that the single-cycle virus RSV M-null has potential to The current RSV M-null vaccine does not improve on cross- overcome the tremendous hurdle of balancing vaccine efficacy protection against RSV B strains compared with other RSV-A2– with safety and therefore represents a promising novel live- based, live-attenuated vaccines. Improvement of cross-protection attenuated RSV vaccinate candidate. remains an important consideration for the future development of any live-attenuated vaccines against RSV. Overall, RSV M- Acknowledgments null vaccination induced serum Ab and memory T cell re- We thank Stacey Hartwig for excellent technical assistance and Garry sponses that were able to provide protection against secondary Hauser for assistance with real-time PCR. viral challenges. In summary, we have evaluated the novel single-cycle virus Disclosures strain RSV M-null as a potential live-attenuated vaccine candidate The authors have no financial conflicts of interest. The Journal of Immunology 11

References 25. Mitra, R., P. Baviskar, R. R. Duncan-Decocq, D. Patel, and A. G. Oomens. 2012. The human respiratory syncytial virus matrix protein is required for maturation 1.Nair,H.,D.J.Nokes,B.D.Gessner,M.Dherani,S.A.Madhi, of viral filaments. J. Virol. 86: 4432–4443. R. J. Singleton, K. L. O’Brien, A. Roca, P. F. Wright, N. Bruce, et al. 2010. 26. Schmidt, M. E., C. J. Knudson, S. M. Hartwig, L. L. Pewe, D. K. Meyerholz, Global burden of acute lower respiratory infections due to respiratory syn- R. A. Langlois, J. T. Harty, and S. M. Varga. 2018. Memory CD8 T cells mediate cytial virus in young children: a systematic review and meta-analysis. Lancet severe immunopathology following respiratory syncytial virus infection. PLoS 375: 1545–1555. Pathog. 14: e1006810. 2. Shi, T., D. A. McAllister, K. L. O’Brien, E. A. F. Simoes, S. A. Madhi, 27. Hoffmann, E., G. Neumann, Y. Kawaoka, G. Hobom, and R. G. Webster. 2000. A B. D. Gessner, F. P. Polack, E. Balsells, S. Acacio, C. Aguayo, et al; RSV Global DNA transfection system for generation of influenza A virus from eight plas- Epidemiology Network. 2017. Global, regional, and national disease burden mids. Proc. Natl. Acad. Sci. USA 97: 6108–6113. estimates of acute lower respiratory infections due to respiratory syncytial virus 28. Castilow, E. M., D. K. Meyerholz, and S. M. Varga. 2008. IL-13 is required for in young children in 2015: a systematic review and modelling study. Lancet 390: eosinophil entry into the lung during respiratory syncytial virus vaccine- 946–958. enhanced disease. J. Immunol. 180: 2376–2384. 3. Glezen, W. P., L. H. Taber, A. L. Frank, and J. A. Kasel. 1986. Risk of primary 29. Hu, A., M. Colella, J. S. Tam, R. Rappaport, and S. M. Cheng. 2003. Simulta- infection and reinfection with respiratory syncytial virus. Am. J. Dis. Child. 140: neous detection, subgrouping, and quantitation of respiratory syncytial virus A 543–546. and B by real-time PCR. J. Clin. Microbiol. 41: 149–154. 4.Chin,J.,R.L.Magoffin,L.A.Shearer,J.H.Schieble,andE.H.Lennette. 30. Kuypers, J., N. Wright, and R. Morrow. 2004. Evaluation of quantitative and 1969. Field evaluation of a respiratory syncytial virus vaccine and a trivalent type-specific real-time RT-PCR assays for detection of respiratory syncytial virus parainfluenza virus vaccine in a pediatric population. Am. J. Epidemiol. 89: in respiratory specimens from children. J. Clin. Virol. 31: 123–129. 449–463. 5. Fulginiti, V. A., J. J. Eller, O. F. Sieber, J. W. Joyner, M. Minamitani, and 31. Fulton, R. B., M. R. Olson, and S. M. Varga. 2008. Regulation of cytokine G. Meiklejohn. 1969. Respiratory virus immunization. I. A field trial of two production by virus-specific CD8 T cells in the lungs. J. Virol. 82: 7799–7811. inactivated respiratory virus vaccines; an aqueous trivalent parainfluenza virus 32. Weiss, K. A., A. F. Christiaansen, R. B. Fulton, D. K. Meyerholz, and vaccine and an alum-precipitated respiratory syncytial virus vaccine. Am. J. S. M. Varga. 2011. Multiple CD4+ T cell subsets produce immunomodulatory Epidemiol. 89: 435–448. IL-10 during respiratory syncytial virus infection. J. Immunol. 187: 3145–3154. 6. Kapikian, A. Z., R. H. Mitchell, R. M. Chanock, R. A. Shvedoff, and 33. Knudson, C. J., S. M. Hartwig, D. K. Meyerholz, and S. M. Varga. 2015. RSV vaccine-enhanced disease is orchestrated by the combined actions of distinct C. E. Stewart. 1969. An epidemiologic study of altered clinical reactivity to Downloaded from respiratory syncytial (RS) virus infection in children previously vaccinated with CD4 T cell subsets. PLoS Pathog. 11: e1004757. an inactivated RS virus vaccine. Am. J. Epidemiol. 89: 405–421. 34. Anderson, K. G., H. Sung, C. N. Skon, L. Lefrancois, A. Deisinger, V. Vezys, 7. Kim, H. W., J. G. Canchola, C. D. Brandt, G. Pyles, R. M. Chanock, K. Jensen, and D. Masopust. 2012. Cutting edge: intravascular staining redefines lung CD8 and R. H. Parrott. 1969. Respiratory syncytial virus disease in infants despite T cell responses. J. Immunol. 189: 2702–2706. prior administration of antigenic inactivated vaccine. Am. J. Epidemiol. 89: 422– 35. Bukreyev, A., S. S. Whitehead, B. R. Murphy, and P. L. Collins. 1997. 434. Recombinant respiratory syncytial virus from which the entire SH gene has been 8. Ambrose, C. S., X. Wu, and R. B. Belshe. 2010. The efficacy of live attenuated deleted grows efficiently in cell culture and exhibits site-specific attenuation in and inactivated influenza vaccines in children as a function of time postvacci- the respiratory tract of the mouse. J. Virol. 71: 8973–8982. nation. Pediatr. Infect. Dis. J. 29: 806–811. 36. Fulton, R. B., D. K. Meyerholz, and S. M. Varga. 2010. Foxp3+ CD4 regulatory http://www.jimmunol.org/ 9. Hoft, D. F., E. Babusis, S. Worku, C. T. Spencer, K. Lottenbach, S. M. Truscott, T cells limit pulmonary immunopathology by modulating the CD8 T cell re- G. Abate, I. G. Sakala, K. M. Edwards, C. B. Creech, et al. 2011. Live and sponse during respiratory syncytial virus infection. J. Immunol. 185: 2382–2392. inactivated influenza vaccines induce similar humoral responses, but only live 37. Graham, B. S., L. A. Bunton, P. F. Wright, and D. T. Karzon. 1991. Role of vaccines induce diverse T-cell responses in young children. J. Infect. Dis. 204: T lymphocyte subsets in the pathogenesis of primary infection and rechallenge 845–853. with respiratory syncytial virus in mice. J. Clin. Invest. 88: 1026–1033. 10. Karron, R. A., U. J. Buchholz, and P. L. Collins. 2013. Live-attenuated respi- 38. Glaab, T., M. Ziegert, R. Baelder, R. Korolewitz, A. Braun, J. M. Hohlfeld, ratory syncytial virus vaccines. Curr. Top. Microbiol. Immunol. 372: 259–284. W. Mitzner, N. Krug, and H. G. Hoymann. 2005. Invasive versus noninvasive 11. Wright, P. F., R. A. Karron, R. B. Belshe, J. R. Shi, V. B. Randolph, P. L. Collins, measurement of allergic and cholinergic airway responsiveness in mice. Respir. A. F. O’Shea, W. C. Gruber, and B. R. Murphy. 2007. The absence of enhanced Res. 6: 139. disease with wild type respiratory syncytial virus infection occurring after re- 39. Huck, B., D. Neumann-Haefelin, A. Schmitt-Graeff, M. Weckmann, J. Mattes, ceipt of live, attenuated, respiratory syncytial virus vaccines. Vaccine 25: 7372– S. Ehl, and V. Falcone. 2007. Human metapneumovirus induces more severe by guest on October 2, 2021 7378. disease and stronger innate immune response in BALB/c mice as compared with 12. Choi, K. Y., M. Root, and A. McGregor. 2016. A novel non-replication- respiratory syncytial virus. Respir. Res. 8: 6. competent cytomegalovirus capsid mutant vaccine strategy is effective in re- 40. Van Schaik, S. M., G. Enhorning, I. Vargas, and R. C. Welliver. 1998. Respi- ducing congenital infection. J. Virol. 90: 7902–7919. ratory syncytial virus affects pulmonary function in BALB/c mice. J. Infect. Dis. 13. Crosby, C. M., and M. A. Barry. 2014. IIIa deleted adenovirus as a single-cycle 177: 269–276. genome replicating vector. Virology 462–463: 158–165. 41. Alwan, W. H., F. M. Record, and P. J. Openshaw. 1992. CD4+ T cells clear virus 14. Evans, D. T., J. E. Bricker, and R. C. Desrosiers. 2004. A novel approach for but augment disease in mice infected with respiratory syncytial virus. Com- producing lentiviruses that are limited to a single cycle of infection. J. Virol. 78: parison with the effects of CD8+ T cells. Clin. Exp. Immunol. 88: 527–536. 11715–11725. 42. Alwan, W. H., W. J. Kozlowska, and P. J. Openshaw. 1994. Distinct types of 15. Gomme, E. A., E. J. Faul, P. Flomenberg, J. P. McGettigan, and M. J. Schnell. lung disease caused by functional subsets of antiviral T cells. J. Exp. Med. 179: 2010. Characterization of a single-cycle rabies virus-based vaccine vector. J. 81–89. Virol. 84: 2820–2831. 43. McDermott, D. S., and S. M. Varga. 2011. Quantifying antigen-specific CD4 16. Guo, H., S. F. Baker, L. Martıńez-Sobrido, and D. J. Topham. 2014. Induction of T cells during a viral infection: CD4 T cell responses are larger than we think. CD8 T cell heterologous protection by a single dose of single-cycle infectious J. Immunol. 187: 5568–5576. influenza virus. J. Virol. 88: 12006–12016. 44. Rai, D., N. L. Pham, J. T. Harty, and V. P. Badovinac. 2009. Tracking the total 17. Ishikawa, T., D. G. Widman, N. Bourne, E. Konishi, and P. W. Mason. 2008. CD8 T cell response to infection reveals substantial discordance in magnitude Construction and evaluation of a chimeric pseudoinfectious virus vaccine to and kinetics between inbred and outbred hosts. J. Immunol. 183: 7672–7681. prevent Japanese encephalitis. Vaccine 26: 2772–2781. 45. Kulkarni, A. B., H. C. Morse, III, J. R. Bennink, J. W. Yewdell, and 18. Li, R., Y. Qin, Y. He, W. Tao, N. Zhang, C. Tsai, P. Zhou, and J. Zhong. 2011. B. R. Murphy. 1993. Immunization of mice with vaccinia virus-M2 recombinant Production of hepatitis C virus lacking the envelope-encoding genes for single- induces epitope-specific and cross-reactive Kd-restricted CD8+ cytotoxic T cells. cycle infection by providing homologous envelope proteins or vesicular sto- J. Virol. 67: 4086–4092. matitis virus glycoproteins in trans. J. Virol. 85: 2138–2147. 46. Kulkarni, A. B., P. L. Collins, I. Bacik, J. W. Yewdell, J. R. Bennink, J. E. Crowe, 19. Petro, C., P. A. Gonza´lez, N. Cheshenko, T. Jandl, N. Khajoueinejad, Jr., and B. R. Murphy. 1995. Cytotoxic T cells specific for a single peptide on the A. Beńard, M. Sengupta, B. C. Herold, and W. R. Jacobs. 2015. Herpes M2 protein of respiratory syncytial virus are the sole mediators of resistance simplex type 2 virus deleted in glycoprotein D protects against vaginal, skin induced by immunization with M2 encoded by a recombinant vaccinia virus. and neural disease. Elife 4. J. Virol. 69: 1261–1264. 20. Roberts, A., L. Buonocore, R. Price, J. Forman, and J. K. Rose. 1999. Attenuated 47. Openshaw, P. J., K. Anderson, G. W. Wertz, and B. A. Askonas. 1990. The vesicular stomatitis viruses as vaccine vectors. J. Virol. 73: 3723–3732. 22,000-kilodalton protein of respiratory syncytial virus is a major target for Kd- 21. Shan, C., X. Xie, J. Zou, R. Zu¨st, B. Zhang, R. Ambrose, J. Mackenzie, K. Fink, restricted cytotoxic T lymphocytes from mice primed by infection. J. Virol. 64: and P. Y. Shi. 2018. Using a virion assembly-defective dengue virus as a vaccine 1683–1689. approach. J. Virol. 92: e01002. 48. Chang, J., A. Srikiatkhachorn, and T. J. Braciale. 2001. Visualization and 22. Si, L., H. Xu, X. Zhou, Z. Zhang, Z. Tian, Y. Wang, Y. Wu, B. Zhang, Z. Niu, characterization of respiratory syncytial virus F-specific CD8(+) T cells during C. Zhang, et al. 2016. Generation of influenza A viruses as live but replication- experimental virus infection. J. Immunol. 167: 4254–4260. incompetent virus vaccines. Science 354: 1170–1173. 49. Lee, S., S. A. Miller, D. W. Wright, M. T. Rock, and J. E. Crowe, Jr. 2007. 23. Snyder, C. M., J. E. Allan, E. L. Bonnett, C. M. Doom, and A. B. Hill. 2010. Tissue-specific regulation of CD8+ T-lymphocyte immunodominance in respi- Cross-presentation of a spread-defective MCMV is sufficient to prime the ma- ratory syncytial virus infection. J. Virol. 81: 2349–2358. jority of virus-specific CD8+ T cells. PLoS One 5: e9681. 50. McDermott, D. S., C. J. Knudson, and S. M. Varga. 2014. Determining the 24. Widman, D. G., T. Ishikawa, R. Fayzulin, N. Bourne, and P. W. Mason. 2008. breadth of the respiratory syncytial virus-specific T cell response. J. Virol. 88: Construction and characterization of a second-generation pseudoinfectious West 3135–3143. Nile virus vaccine propagated using a new cultivation system. Vaccine 26: 2762– 51. Rutigliano, J. A., M. T. Rock, A. K. Johnson, J. E. Crowe, Jr., and B. S. Graham. 2771. 2005. Identification of an H-2D(b)-restricted CD8+ cytotoxic T lymphocyte 12 SINGLE-CYCLE RSV VACCINATION IS IMMUNOGENIC AND PROTECTIVE

epitope in the matrix protein of respiratory syncytial virus. Virology 337: 335– 69. Kinnear, E., L. Lambert, J. U. McDonald, H. M. Cheeseman, L. J. Caproni, and 343. J. S. Tregoning. 2018. Airway T cells protect against RSV infection in the ab- 52. Malloy, A. M., T. J. Ruckwardt, K. M. Morabito, A. W. Lau-Kilby, and sence of antibody. Mucosal Immunol. 11: 249–256. B. S. Graham. 2017. Pulmonary dendritic cell subsets shape the respiratory 70. Knudson, C. J., K. A. Weiss, S. M. Hartwig, and S. M. Varga. 2014. The pul- syncytial virus-specific CD8+ T cell immunodominance hierarchy in neonates. monary localization of virus-specific T lymphocytes is governed by the tissue J. Immunol. 198: 394–403. tropism of infection. J. Virol. 88: 9010–9016. 53. Ruckwardt, T. J., K. L. Bonaparte, M. C. Nason, and B. S. Graham. 2009. 71. Gebhardt, T., L. M. Wakim, L. Eidsmo, P. C. Reading, W. R. Heath, and Regulatory T cells promote early influx of CD8+ T cells in the lungs of respi- F. R. Carbone. 2009. Memory T cells in nonlymphoid tissue that provide ratory syncytial virus-infected mice and diminish immunodominance disparities. enhanced local immunity during infection with herpes simplex virus. Nat. J. Virol. 83: 3019–3028. Immunol. 10: 524–530. 54. Ruckwardt, T. J., C. Luongo, A. M. Malloy, J. Liu, M. Chen, P. L. Collins, and 72. Turner, D. L., K. L. Bickham, J. J. Thome, C. Y. Kim, F. D’Ovidio, E. J. Wherry, B. S. Graham. 2010. Responses against a subdominant CD8+ T cell epitope and D. L. Farber. 2014. Lung niches for the generation and maintenance of protect against immunopathology caused by a dominant epitope. J. Immunol. tissue-resident memory T cells. Mucosal Immunol. 7: 501–510. 185: 4673–4680. 73. Ghildyal, R., J. Mills, M. Murray, N. Vardaxis, and J. Meanger. 2002. Respi- 55. Ruckwardt, T. J., A. M. Malloy, E. Gostick, D. A. Price, P. Dash, J. L. McClaren, ratory syncytial virus matrix protein associates with nucleocapsids in infected P. G. Thomas, and B. S. Graham. 2011. Neonatal CD8 T-cell hierarchy is distinct cells. J. Gen. Virol. 83: 753–757. from adults and is influenced by intrinsic T cell properties in respiratory syn- 74.Prince,G.A.,R.L.Horswood,J.Berndt,S.C.Suffin,andR.M.Chanock. cytial virus infected mice. PLoS Pathog. 7: e1002377. 1979. Respiratory syncytial virus infection in inbred mice. Infect. Immun. 26: 56. Ruckwardt, T. J., A. M. Malloy, K. M. Morabito, and B. S. Graham. 2014. 764–766. Quantitative and qualitative deficits in neonatal lung-migratory dendritic cells 75. Stark, J. M., S. A. McDowell, V. Koenigsknecht, D. R. Prows, J. E. Leikauf, impact the generation of the CD8+ T cell response. PLoS Pathog. 10e1003934. A. M. Le Vine, and G. D. Leikauf. 2002. Genetic susceptibility to respiratory 57. Crotty, S. 2014. T follicular helper cell differentiation, function, and roles in syncytial virus infection in inbred mice. J. Med. Virol. 67: 92–100. disease. Immunity 41: 529–542. 76. Ostler, T., W. Davidson, and S. Ehl. 2002. Virus clearance and immunopathology 58. Shlomchik, M. J., and F. Weisel. 2012. Germinal center selection and the de- by CD8(+) T cells during infection with respiratory syncytial virus are mediated velopment of memory B and plasma cells. Immunol. Rev. 247: 52–63. by IFN-gamma. Eur. J. Immunol. 32: 2117–2123. 59. Victora, G. D., and M. C. Nussenzweig. 2012. Germinal centers. Annu. Rev. 77. Rutigliano, J. A., and B. S. Graham. 2004. Prolonged production of TNF-alpha Immunol. 30: 429–457. exacerbates illness during respiratory syncytial virus infection. J. Immunol. 173: Downloaded from 60. Ansel, K. M., L. J. McHeyzer-Williams, V. N. Ngo, M. G. McHeyzer-Williams, 3408–3417. and J. G. Cyster. 1999. In vivo-activated CD4 T cells upregulate CXC chemokine 78. Van Schaik, S. M., N. Obot, G. Enhorning, K. Hintz, K. Gross, G. E. Hancock, receptor 5 and reprogram their response to lymphoid chemokines. J. Exp. Med. A. M. Stack, and R. C. Welliver. 2000. Role of interferon gamma in the path- 190: 1123–1134. ogenesis of primary respiratory syncytial virus infection in BALB/c mice. 61. Haynes, N. M., C. D. Allen, R. Lesley, K. M. Ansel, N. Killeen, and J. G. Cyster. J. Med. Virol. 62: 257–266. 2007. Role of CXCR5 and CCR7 in follicular Th cell positioning and appear- 79. Tregoning, J. S., P. K. Pribul, A. M. Pennycook, T. Hussell, B. Wang, N. Lukacs, ance of a programmed cell death gene-1high germinal center-associated sub- J. Schwarze, F. J. Culley, and P. J. Openshaw. 2010. The chemokine MIP1alpha/

population. J. Immunol. 179: 5099–5108. CCL3 determines pathology in primary RSV infection by regulating the balance http://www.jimmunol.org/ 62. Johnston, R. J., A. C. Poholek, D. DiToro, I. Yusuf, D. Eto, B. Barnett, A. L. Dent, of T cell populations in the murine lung. PLoS One 5: e9381. J. Craft, and S. Crotty. 2009. Bcl6 and Blimp-1 are reciprocal and antagonistic 80. Snyder, C. M., K. S. Cho, E. L. Bonnett, J. E. Allan, and A. B. Hill. 2011. regulators of T follicular helper cell differentiation. Science 325: 1006–1010. Sustained CD8+ T cell memory inﬂation after infection with a single-cycle 63. Nurieva, R. I., Y. Chung, G. J. Martinez, X. O. Yang, S. Tanaka, cytomegalovirus. PLoS Pathog. 7: e1002295. T. D. Matskevitch, Y. H. Wang, and C. Dong. 2009. Bcl6 mediates the devel- 81. Crosby, C. M., P. Nehete, K. J. Sastry, and M. A. Barry. 2015. Ampliﬁed and opment of T follicular helper cells. Science 325: 1001–1005. persistent immune responses generated by single-cycle replicating adenovirus 64. Yu, D., S. Rao, L. M. Tsai, S. K. Lee, Y. He, E. L. Sutcliffe, M. Srivastava, vaccines. J. Virol. 89: 669–675. M. Linterman, L. Zheng, N. Simpson, et al. 2009. The transcriptional repressor 82. Publicover, J., E. Ramsburg, and J. K. Rose. 2005. A single-cycle vaccine Bcl-6 directs T follicular helper cell lineage commitment. Immunity 31: 457–468. vector based on vesicular stomatitis virus can induce immune responses 65. Capella, C., S. Chaiwatpongsakorn, E. Gorrell, Z. A. Risch, F. Ye, S. E. Mertz, comparable to those generated by a replication-competent vector. J. Virol. 79: S. M. Johnson, M. Moore-Clingenpeel, O. Ramilo, A. Mejias, and M. E. Peeples. 13231–13238.

2017. Prefusion F, postfusion F, G antibodies, and disease severity in infants and 83. McMaster, S. R., J. J. Wilson, H. Wang, and J. E. Kohlmeier. 2015. Airway- by guest on October 2, 2021 young children with acute respiratory syncytial virus infection. J. Infect. Dis. resident memory CD8 T cells provide antigen-speciﬁc protection against 216: 1398–1406. respiratory virus challenge through rapid IFN-g production. J. Immunol. 195: 66. Falsey, A. R., and E. E. Walsh. 1998. Relationship of serum antibody to risk of 203–209. respiratory syncytial virus infection in elderly adults. J. Infect. Dis. 177: 463–466. 84. Wu, T., Y. Hu, Y. T. Lee, K. R. Bouchard, A. Benechet, K. Khanna, and 67. Lee, F. E., E. E. Walsh, A. R. Falsey, R. F. Betts, and J. J. Treanor. 2004. Ex- L. S. Cauley. 2014. Lung-resident memory CD8 T cells (TRM) are indispensable perimental infection of humans with A2 respiratory syncytial virus. Antiviral for optimal cross-protection against pulmonary virus infection. J. Leukoc. Biol. Res. 63: 191–196. 95: 215–224. 68. Piedra, P. A., A. M. Jewell, S. G. Cron, R. L. Atmar, and W. P. Glezen. 2003. 85. Slu¨tter, B., N. Van Braeckel-Budimir, G. Abboud, S. M. Varga, S. Salek- Correlates of immunity to respiratory syncytial virus (RSV) associated- Ardakani, and J. T. Harty. 2017. Dynamics of inﬂuenza-induced lung-resident hospitalization: establishment of minimum protective threshold levels of serum memory T cells underlie waning heterosubtypic immunity. Sci. Immunol. 2: neutralizing antibodies. Vaccine 21: 3479–3482. eaag2031.