A New Master Donor Virus for the Development of Live-Attenuated Influenza B Virus Vaccines

Influenza B viruses (IBV) circulate annually, with young children, the elderly and immunocompromised individuals being at high risk. Yearly vaccinations are recommended to protect against seasonally influenza viruses, including IBV. Live attenuated influenza vaccines (LAIV) provide the unique opportunity for direct exposure to the antigenically variable surface glycoproteins as well as the more conserved internal components. Ideally, LAIV Master Donor Viruses (MDV) should accurately reflect seasonal influenza strains. Unfortunately, the continuous evolution of IBV have led to significant changes in conserved epitopes compared to the IBV MDV based on B/Ann Arbor/1/1966 strain. Here, we propose a recent influenza B/Brisbane/60/2008 as an efficacious MDV alternative, as its internal viral proteins more accurately reflect those of circulating IBV strains. We introduced the mutations responsible for the temperature sensitive (ts), cold adapted (ca) and attenuated (att) phenotype of B/Ann Arbor/1/1966 MDV LAIV into B/Brisbane/60/2008 to generate a new MDV LAIV. In vitro and in vivo analysis demonstrated that the mutations responsible of the ts, ca, and att phenotype of B/Ann Arbor/1/1966 MDV LAIV were able to infer the same phenotype to B/Brisbane/60/2008, demonstrating its potential as a new MDV for the development of LAIV to protect against contemporary IBV strains.


Introduction
Over the last century, influenza viruses have been responsible of both yearly seasonal and occasional pandemics infections of great consequences to humans. While Influenza A viruses (IAV) circulate within several zoonotic hosts, providing the opportunity for pandemic, Influenza B viruses (IBV) primarily circulate in humans, and are not considered a pandemic threat. However, IBV are still major human pathogens responsible for seasonal epidemics of respiratory illness [1][2][3]. IBV infect humans of all ages but is responsible of more severe disease in young children, elderly, and immunocompromised populations [2][3][4][5][6][7][8][9]. Notably, epidemiological evidences indicates that the number of IBV cases has increased during the last years [10][11][12].
IAV and IBV infections are most effectively prevented through vaccination [41,42]. However, and despite comprehensive and effective vaccination programs, the World Health Organization (WHO) estimates that the global disease burden from influenza results in 1 billion infections, 3-5 million cases of severe disease and between 300,000-500,000 deaths annually. United States (US) Food and Drug Administration (FDA)-approved vaccines for the prevention of seasonal IAV or IBV in humans include recombinant influenza vaccines (rIV), inactivated influenza vaccines (IIV), and live-attenuated influenza vaccines (LAIV). rIV and IIV are administered intramuscularly to induce protective humoral immunity by inducing the production of neutralizing antibodies that target the viral HA glycoprotein, and to a lesser extent NA, but are poor inducers of cellular immunity [43,44]. LAIV are given intranasally using a nasal spray and more closely mimic the natural route of influenza viral infection. Notably, LAIV have been shown to elicit broader cross-reactive humoral and cellular immune responses, providing better immunogenicity and protection efficacy than rIV and IIV, including protection against heterotypic viral infections [43][44][45][46][47]. For these reasons, LAIV are ideal to prevent and control influenza infections in humans as well as in other animals. Historically, influenza seasonal vaccines were made of trivalent formulations made of two IAV subtypes (H1N1 and H3N2) and one IBV lineage (Yamagata or Victoria) following WHO strain recommendations for each season [48]. However, since 2012, quadrivalent formulations of influenza vaccines containing representative strains of both IBV Victoria and Yamagata lineages, in addition to the two IAV H1N1 and H3N2 strains, have been approved and available to prevent seasonal influenza infections.
LAIV are generated using Master Donor Viruses (MDV) containing mutations that allow the virus to replicate at permissive low temperatures (33 • C), such as those in the upper respiratory tract (URT), but not at non-permissive high temperatures (37 • C) present in the lower respiratory tract (LRT). In the US, the MDV for IBV LAIV is B/Ann Arbor/1/1966. This MDV LAIV was generated by passaging wild-type (WT) B/Ann Arbor/1/1966 at progressive lower temperatures, resulting in a temperature sensitive (ts) variant that still replicate efficiently at low temperatures (cold-adapted, ca), but with an attenuated (att) phenotype in vivo [49][50][51]. The mutations responsible for the ts, ca and att phenotype of the IBV MDV used to generate LAIV have been mapped into the PB2 (S630R), PA (V341M), NP (V114A, P410H, and A509T), and M1 (H159Q and M183V) proteins [52][53][54][55]. The antigenic drift in influenza HA and NA needs for vaccines to be updated in a yearly based so that they antigenically match the influenza seasonal circulating strains [56]. IBV LAIV contain the six internal viral gene segments (PB2, PB1, PA, NP, M and NS) of the MDV B/Ann Arbor/1/1966 and the HA and NA viral genes of circulating seasonal IBV Yamagata and/or Victoria strains recommended by the WHO. Notably, the MDV of the US IAV (A/Ann Arbor/6/1960 H2N2) and IBV (B/Ann Arbor/1/1966). LAIV have been approved by the US FDA and used in the US since 2003 but the IAV and IBV  [57,58], LAIV have been shown to have low efficacy in protecting against seasonal influenza viruses. Based on this low vaccine effectiveness data, the advisory committee on immunization practices (ACIP) concluded that a preference for the quadrivalent LAIV over the IIV was no longer warranted. The cause(s) of reduced effectiveness of quadrivalent LAIV are still unknown but this could be, at least in part, because of the lack of sequence similarities in the internal viral proteins of the MDV LAIV and circulating seasonal strains. Therefore, MDV based on more recent IAV and IBV strains may represents a better option for their use as MDV for the development of LAIV because on their most conserved amino acid sequence with seasonal strains.
We and others have previously shown that introducing the mutations of the US IAV MDV A/Ann Arbor/6/1960 H2N2 LAIV (PB2 N265S and PB1 K391E, D581G, and A661T) [59,60] [62] or pH1N1 [63] containing these ts, ca and att mutations were able to protect against lethal challenge with WT forms of these viruses, demonstrating the feasibility of using these new ts, ca, and att PR8 [62] or pH1N1 [63] as MDV LAIV for IAV. Notably, we have previously shown how the mutations of the US IAV MDV LAIV A/Ann Arbor/6/1960 H2N2 PB1, together with an HA epitope tag in the C terminus of IBV PB1, were able to confer a ts phenotype in vitro and an att phenotype in vivo to B/Brisbane/60/2008, while protecting against challenge with IBV [70].
In this manuscript we evaluated whether the ts, ca and att mutations present in IBV MDV LAIV B/Ann Arbor/1/1966 would be able to confer the same phenotype to a most recent B/Brisbane/60/2008 strain and, importantly, if this new IBV MDV LAIV will be able to protect, using a single intranasal vaccination, against challenge with WT B/Brisbane/60/2008 in a mouse model of infection. Using reverse genetics approaches, we generated a recombinant B/Brisbane/60/2008 containing the mutations in PB2 (S630R), PA (V341M), NP (P410H and A509T; V114 was already present in B/Brisbane/60/2008 WT), and M1 (H159Q and M183V) previously identified to be responsible of the ts, ca and att phenotype of B/Ann Arbor/1/1966 MDV LAIV to generate a B/Brisbane/60/2008 MDV LAIV. Our results demonstrate that B/Brisbane/60/2008 containing B/Ann Arbor/1/1966 MDV LAIV mutations has a similar ts and ca phenotype in vitro and att phenotype in vivo. Importantly, a single intranasal vaccination with our novel IBV MDV LAIV was able to protect against a challenge with WT B/Brisbane/60/2008. Altogether, these results demonstrate the feasibility of updating the current IBV MDV for the development of IBV LAIV based on the use of a more recent B/Brisbane/60/2008 strain.

Virus Rescue
Viral rescues were conducted as previously described [71]. Briefly, MDCK and 293T cells (6-welll plate format, 10 6 cells/well, triplicates) were transiently co-transfected, using Lipofectamine 2000, with pDP plasmids encoding the 8 viral gene segments of B/Brisbane/60/2008 WT or MDV LAIV. At 8 h, transfection media was replaced with postinfection media and cells were incubated at 33 • C. After 3 days, tissue culture supernatants were collected and used to infect fresh monolayers of MDCK cells (6-welll plate format, 10 6 cells/well, triplicates). Four days after infection, presence of B/Brisbane/60/2008 WT or MDV LAIV was determined by HA assay.

Virus Growth Kinetics
Confluent monolayers of MDCK cells (12-well plates, 5 × 10 5 cells/well, triplicates) were infected (MOI of 0.01) for 1 h at room temperature. After 1 h virus absorption, virus was removed and replaced with post-infection media. Plates were incubated in 5% CO 2 incubators at either 33 • C or 37 • C. At the indicated h post-infection, tissue culture supernatants were collected and viral titers were determined by immunofocus assay (fluorescent forming units [FFU]/mL). Briefly, confluent monolayers of MDCK cells (96-well plates, 10 4 cells/cell, triplicates) were infected with 50 µL of virus from each time point (in triplicate) using 10-fold serial dilutions. Plates were placed at 33 • C for 12 h. After viral infection, cells were fixed and permeabilized with 4% formaldehyde and 0.5% Triton-X100 in 1× PBS. A primary monoclonal antibody against IBV NP (B017, AbCam Cat. Ab20711-100) was added to each well and incubated at 37 • C for 90 min. After 3× washes with 1× PBS, a secondary FITC-conjugated polyclonal rabbit anti-mouse antibody (Abcam Cat. ab6724) was added to each well and incubated at 37 • C for 45 min. After 3× washes with 1× PBS, plates were observed under an Olympus fluorescent microscope to quantify positive stained cells.

Plaque Assays
Confluent monolayers of MDCK cells (6-well plate format, 10 6 cells/well) were infected with B/Brisbane/60/2008 WT or MDV LAIV for 1 h at room temperature. After 1 h infection, cells were overlaid with DMEM/F-12 containing 2% Oxoid agar and incubated at 33 • C or 37 • C in 5% CO 2 . At 72 h post-infection, cells were fixed overnight with 2.5% formalin (in 1× PBS) at 4 • C. After overnight incubaton, agar overlays were removed and Viruses 2021, 13, 1278 5 of 18 cells were fixed and permeabilized using 4% formaldehyde with 0.01% Triton-X100 in 1× PBS at room temperature for 1 h. Immunostaining of viral plaques was performed using the IBV anti-NP monoclonal antibody B017 and vector kits (Vectastain ABC kit and DAB HRP Substrate Kit: Vector), according to manufacturer's specifications.

Animal Experiments
Six-week-old, female C57BL/6J mice were obtained from the National Cancer Institute (NCI) and maintain in the University of Rochester Animal Care Facilities under protocols outlined by the University Committee of Animal Reseources (UCAR). Mice were anesthetized through intraperitoneal injection of Avertin (240 mg/kg of body weight) and subsequently infected intranasally with 10 6 FFU of either B/Brisbane/60/2008 WT or MDV LAIV [74]. An additional cohort of mice (n = 6) was anesthetized as described above and treated with sterile 1× PBS as a negative control [74]. To determine viral titers in vivo, lung and nasal turbinates were harvest at days 2 (n = 3) and 4 (n = 3) post-infection [74]. Harvested tissues were homogenized and viral titers were determined by immunofocus assay (FFU/mL) [74]. Serum from infected mice were collected through submandibular bleeds 14 days post-infection [74]. On day 28 post-infection, mice were challenged with B/Brisbane/60/2008 WT (10 6 FFU). Mice were euthanized at day 2 and 4 post-challenge. Lungs were harvested and viral titers determined as described above [74].

Enzyme-Linked Immunosorbent Assay (ELISA)
To determine total IBV specific antibodies, serum collected at day 14 post-infection from infected mice was used in ELISA assays. Briefly, 96-well plates were coated at 250 ng/well with either B/Ohio/01/2005 (BEI Resources, NR-19243) or B/Malaysia/2506/ 2004 (BEI Resources, NR-51162) recombinant HA proteins and placed at 4 • C overnight. Plates were then blocked with 3% BSA in 1× PBS for 1 h at 37 • C. Serum diluted in 0.5% BSA in 1× PBS was then added to the plate using two-fold dilutions, starting 1:100 dilution, and incubated at room temperature for 2 h. Plates were washed 3× with ELISA wash buffer (1× PBS + 0.05% Tween) and then 50 µL of a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:2000; Southern Biotech) was added to each well for 1 h at 37 • C. Reactions were developed with tetramethylbenzidine (TMB) substrate (BioLegend) for 10 min at room temperature. Reaction was stopped with 2N H 2 SO 4 and read at 450 nm using the Vmax kinetic microplate reader (Molecular Devices).

Hemagglutination Inhibition (HAI) Assay
Serum from mice infected with either B/Brisbane/60/2008 WT or MDV LAIV at day 14 post-infection were RDE (receptor-destroying enzyme) treated overnight at 37 • C and then placed at 56 • C for 30 min for heat inactivation. To determine the presence of neutralizing antibodies, heat inactivated serum were diluted in 96-well V-bottom plates and incubated with 4 hemagglutinating units (HAU) of B/Brisbane/60/2008 WT and left at room temperature for 60 min. Then, 0.5% turkey red blood cells were added to each well and left on ice for 45 min. HAI antibody titers were calculated based on the last well in which hemagglutination did not occur, with the riciprocal of the corresponding dilution being the hemagglutination titer.

Microneutralization Assays
To evaluate neutralizing antibodies against B/Brisbane/60/2008 WT elicited by infection with either WT or MDV LAIV B/Brisbane/60/2008, serum from infected mice were collected at day 14 post-infection, treated as outlined above, and serially diluted in 96-well plates. B/Brisbane/60/2008 expressing mCherry (100 FFU/well) [73,75] was added to the 96-well plate and incubated at room temperature for 1 h Next, 50 µL of the serum/virus mixture was added to confluent monolayers of MDCK cells in 96-well plate replicas and left at room temperature for 1 h to allow viral infection. After 1 h viral infection, serum/virus media was replaced with post-infection media. At 48 h a fluorescent microplate reader was Viruses 2021, 13, 1278 6 of 18 used to quantify mCherry expression [73,75,76]. Neutralizing capacity was determined by normalizing to mCherry expression in the absence of sera [73,75,76].

The Mutations of B/Ann Arbor/1/1966 MDV LAIV Confer a ts and ca Phenotype to B/Brisbane/60/2008
The first step towards identifying a new IBV MDV LAIV was to identify the ideal viral candidate. B/Brisbane/60/2008 was selected as the potential MDV LAIV candidate because this Victoria lineage IBV circulated more recently (2008) within the human population and because its internal genes have increased percent identity and sequence homology with contemporary IBV strains circulating in humans, relatively to the current B/Ann Arbor/1/1966 MDV LAIV (Table 1, supplementary material, and data not shown). In order to generate a virus that only replicate efficiently at 33 • C, but not at 37 • C, for its safe implementation as a MDV for the development of IBV LAIV, we first evaluated if the mutations responsible for the ts, ca, and att phenotype of B/Ann Arbor/1/1966 MDV LAIV were able to confer a similar phenotype to B/Brisbane/60/2008 ( Figure 1). To that end, 6 of the 7 mutations previously described to be responsible of the ts, ca, and att phenotype of the B/Ann Arbor/1/1966 MDV LAIV [52][53][54][55] Figure 1C). These results indicate that the mutations responsible for the ts and ca phenotype of B/Ann Arbor/1/1966 MDV LAIV were able to confer a similar phenotype to the polymerase PB2, PA, and NP of B/Brisbane/60/2008. ase of B/Brisbane/60/2008. Importantly, B/Brisbane/60/2008 MDV LAIV polymerase and NP complex underperformed at 37 °C, with Fluc expression levels significantly reduced relative to those of B/Brisbane/60/2008 WT, suggesting that these mutations also conferred a ts phenotype to B/Brisbane/60/2008 polymerase and NP ( Figure 1B). Minigenome GFP expression levels were also evaluated to determine the polymerase PB2, PA, and NP activities of both WT and MDV LAIV B/Brisbane/60/2008 ( Figure 1C). At 33 °C, GFP expression levels were comparable between WT and MDV LAIV B/Brisbane/60/2008. Conversely, at 37 °C, GFP expression levels by B/Brisbane/60/2008LAIV were drastically reduced compared to those of B/Brisbane/60/2008 WT ( Figure 1C). These results indicate that the mutations responsible for the ts and ca phenotype of B/Ann Arbor/1/1966 MDV LAIV were able to confer a similar phenotype to the polymerase PB2, PA, and NP of B/Brisbane/60/2008.

Influenza B/Brisbane/60/2008 WT Replication
To determine if B/Brisbane/60/2008 WT would be a suitable MDV LAIV candidate, we first evaluate its ability to replicate at 33 • C and 37 • C in MDCK cells (Figure 2). B/Brisbane/60/2008 WT was able to efficiently replicate at both 33 • C and 37 • C, with viral titers reaching 10 8 FFU/mL and 10 7 FFU/mL, respectively, at 48 h post-infection ( Figure 2A). Next, we evaluated the ability of B/Brisbane/60/2008 WT to replicate at the same temperatures by plaque assay ( Figure 2B). We also conducted similar viral growth kinetics and plaque assays at 39 • C but we were not able to detect B/Brisbane/60/2008 WT in the tissue culture supernatants of infected cells, or viral plaques, respectively (data not shown).
we first evaluate its ability to replicate at 33 °C and 37 °C in MDCK cells (Figure 2). B/Brisbane/60/2008 WT was able to efficiently replicate at both 33 °C and 37 °C, with viral titers reaching 10 8 FFU/mL and 10 7 FFU/mL, respectively, at 48 h post-infection ( Figure 2A). Next, we evaluated the ability of B/Brisbane/60/2008 WT to replicate at the same temperatures by plaque assay ( Figure 2B). We also conducted similar viral growth kinetics and plaque assays at 39 °C but we were not able to detect B/Brisbane/60/2008 WT in the tissue culture supernatants of infected cells, or viral plaques, respectively (data not shown).

Generation and Characterization of B/Brisbane/60/2008 MDV for the Development of IBV LAIV
Next, we used our previously described plasmid-based reverse genetics techniques [16,71,72]

Generation and Characterization of B/Brisbane/60/2008 MDV for the Development of IBV LAIV
Next, we used our previously described plasmid-based reverse genetics techniques [16,71,72]

Safety of B/Brisbane/60/2008 MDV LAIV
In order to implement B/Brisbane/60/2008 MDV as a safe intranasal LAIV, we first confirmed that replication of B/Brisbane/60/2008 MDV LAIV was restricted to the lower temperatures of the upper respiratory track. To that end, 6-week old, female C57BL/6J mice were infected with 10 6 FFU of B/Brisbane/60/2008 MDV LAIV or WT and viral replication in the upper (nasal turbinate) and lower (lungs) respiratory track were evaluated at days 2 and 4 post-infection (Figure 4). We were able to detect B/Brisbane/60/2008 WT in both the nasal turbinate ( Figure 4A) and the lungs ( Figure 4B) of infected animals. Viral titers of B/Brisbane/60/2008 MDV LAIV in the nasal turbinate were slighted, but significantly, reduced (~1 log) to those of B/Brisbane/60/2008 WT at day 2 post-infection ( Figure  4A). However, at day 4 post-infection, viral titers in the nasal turbinate of mice infected with B/Brisbane/60/2008 MDV LAIV were significantly higher than those of B/Brisbane/60/2008 WT ( Figure 4A). Notably, we were not able to detect B/Brisbane/60/2008 MDV LAIV in the lungs of infected mice at days 2 or 4 post-infection ( Figure 4B). These results suggest that B/Brisbane/60/2008 MDV LAIV is able to replicate efficiently in the URT but is completely impaired in viral replication in the LRT of infected mice, important for its implementation as a safe LAIV.
Here, we describe the development of a new IBV MDV LAIV candidate in the backbone of a recent B/Brisbane/60/2008 strain. Our results demonstrate how the ts, ca, and att mutations of the current US FDA-approved MDV LAIV B/Ann Arbor/1/1966 into B/Brisbane/60/2008 PB2, PB1, NP and M resulted in a ts and ca phenotype in vitro (Figure 1) able to replicate to high titers at permissive temperatures, important for vaccine production (Figure 3). Using a mouse model we also demonstrate that the new B/Brisbane/60/2008 MDV LAIV is only able to replicate in the URT but not in the LRT of infected mice ( Figure  4), important for its safe implementation as a MDV to generate IBV LAIV. Notably, a single intranasal immunization with this new B/Brisbane/60/2008 MDV LAIV was able to induce robust humoral immune responses, including HAI and neutralizing antibodies, against IBV ( Figure 5) and complete protection against challenge with B/Brisbane/60/2008 WT ( Figure 6). Based on these results, we hypothesize that this new MDV based on a more recent IBV seasonal strain could be used to generate LAIV containing the internal genes of the B/Brisbane/60/2008 MDV LAIV and the HA and NA glycoproteins of contemporary Yamagata and Victoria strains for the more efficient treatment of IBV infections in humans.
Since the last few years, there is a global interest in developing universal vaccine approaches that require fewer updates and provide long-lasting immunity for the treatment of influenza infections [100][101][102][103][104]. Since their initial development, LAIV are based on the use of the internal genes of MDV that contain mutations able to replicate efficiently at low
Here, we describe the development of a new IBV MDV LAIV candidate in the backbone of a recent B/Brisbane/60/2008 strain. Our results demonstrate how the ts, ca, and att mutations of the current US FDA-approved MDV LAIV B/Ann Arbor/1/1966 into B/Brisbane/60/2008 PB2, PB1, NP and M resulted in a ts and ca phenotype in vitro ( Figure 1) able to replicate to high titers at permissive temperatures, important for vaccine production (Figure 3). Using a mouse model we also demonstrate that the new B/Brisbane/60/2008 MDV LAIV is only able to replicate in the URT but not in the LRT of infected mice (Figure 4), important for its safe implementation as a MDV to generate IBV LAIV. Notably, a single intranasal immunization with this new B/Brisbane/60/2008 MDV LAIV was able to induce robust humoral immune responses, including HAI and neutralizing antibodies, against IBV ( Figure 5) and complete protection against challenge with B/Brisbane/60/2008 WT ( Figure 6). Based on these results, we hypothesize that this new MDV based on a more recent IBV seasonal strain could be used to generate LAIV containing the internal genes of the B/Brisbane/60/2008 MDV LAIV and the HA and NA glycoproteins of contemporary Yamagata and Victoria strains for the more efficient treatment of IBV infections in humans.
Since the last few years, there is a global interest in developing universal vaccine approaches that require fewer updates and provide long-lasting immunity for the treatment of influenza infections [100][101][102][103][104]. Since their initial development, LAIV are based on the use of the internal genes of MDV that contain mutations able to replicate efficiently at low temperatures (ts, ca) with a safety profile (att) and the HA and NA viral glycoproteins of seasonal circulating IAV or IBV. These include the US MDV A/Ann Arbor/6/60 H2N2 (IAV) and B/Ann Arbor/1/1966 (IBV). The ts, ca, and att A/Ann Arbor/6/60 H2N2 and B/Ann Arbor/1/1966 have been licensed for human use since 2003 and they have been used as MDV for the generation of both seasonal and potentially pandemic, in the case of IAV, human LAIV by creating reassortant viruses containing the six internal vRNA segments PB2, PB1, PA, NP, M and NS of the IAV or IBV MDV, and the viral glycoproteinencoding vRNAs HA and NA from viruses that antigenically match the strains predicted to circulate in the upcoming influenza season or potentially pandemic strains. However, since their implementation, these IAV or IBV MDV have not been updated.
The reason(s) why LAIV had low effectiveness since the 2013-2014 season remains unknown. One of the potential explanations for the lack of effectiveness of LAIV is the use of same IAV and IBV MDV backbones. If this is the case, updating the MDV of IAV (A/Ann Arbor/6/60 H2N2) and IBV (B/Ann Arbor/1/1966) to different and more contemporary strains backbones, could solve this problem and provide with more effective LAIV for the treatment of both IAV and IBV infections. We have previously shown how the ts, ca, and att mutations of the US MDV A/Ann Arbor/6/60 H2N2 were able to confer a similar phenotype to other IAV strains, including PR8 [61,62] and pH1N1 [63], A/canine/NY/dog23/2009 H3N8 [64][65][66], and A/equine/Ohio/1/2003 H3N8 [67,68] viruses. Likewise, we have also shown that the mutations responsible for the Russian MDV A/Leningrad/17/1957 H2N2 also conferred a similar ts, ca and att phenotype to PR8 [62] and pH1N1 [63] IAV. Moreover, we have shown how a LAIV elicits enhanced protection when the internal genes of the vaccine match those of the challenge virus [63]. Here, we demonstrate that the same experimental approach can be used for the development of an updated IBV MDV LAIV. Another reason is that vaccine strains must represent viruses currently in circulation. In this regard, we have demonstrated how incorporation of the ts, ca and att mutations of the IAV and IBV MDV in more recent strains represent a reasonable option to implement alternative and more protective LAIV. This could prevent potential outbreaks based on vaccine strains not matching circulating viruses. Importantly, these strategies will still use the same production and manufacturing currently used to generate LAIV and, therefore, will not need adaptation to what it is currently in use to develop LAIV. Moreover, the new IBV MDV LAIV is based on mutations that have been previously described to be responsible for the ts, ca and att phenotype of B/Ann Arbor/1/1966 MDV LAIV, which have a proven history of safety and stability. In addition, since this new IBV MDV LAIV based on the use of B/Brisbane/60/2008 was generated using plasmid-based reverse genetics approaches [16,71,72], it would be possible to easily update IBV LAIV by simply combining the internal viral genes of B/Brisbane/60/2008 MDV LAIV and the HA and NA of Yamagata or Victoria lineage new seasonal strains for the rapid development of updated IBV LAIV. Based on these multiple advantages over current approaches, our novel IBV MDV LAIV platform represents an excellent option for its implementation to prevent and control IBV infections. Future studies aimed to evaluate safety, immunogenicity and protection efficacy in a more relevant animal model would be needed for the application of this new MDV LAIV for the treatment of IBV infections in humans.