Development of a novel Francisella tularensis Live Vaccine Strain expressing ovalbumin provides insight into Francisella tularensis-specific CD8+ T cell responses

Progress towards a safe and effective vaccine for the prevention of tularemia has been hindered by a lack of knowledge regarding the correlates of protective adaptive immunity and a lack of tools to generate this knowledge. CD8+ T cells are essential for protective immunity against virulent strains of Francisella tularensis, but to-date, it has not been possible to study these cells in a pathogen-specific manner. Here, we report the development of a tool for expression of the model antigen ovalbumin (OVA) in F. tularensis, which allows for the study of CD8+ T cell responses to the bacterium. We demonstrate that in response to intranasal infection with the F. tularensis Live Vaccine Strain, pathogen-specific CD8+ T cells expand after the first week and produce IFN-γ but not IL-17. Effector and central memory subsets develop with disparate kinetics in the lungs, draining lymph node and spleen. Notably, F. tularensis-specific cells are poorly retained in the lungs after clearance of infection. We also show that intranasal vaccination leads to more pathogen-specific CD8+ T cells in the lung-draining lymph node compared to scarification vaccination, but that an intranasal booster overcomes this difference. Together, our data show that this novel tool can be used to study multiple aspects of the CD8+ T cell response to F. tularensis. Use of this tool will enhance our understanding of immunity to this deadly pathogen.


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The Gram-negative bacterium, Francisella tularensis, is the etiological agent of the disease sequencing by the Penn State Genomics Core Facility. The bacterioferritin promoter, vgrG, and 1 6 2 OVA 239-345 -6xHis fragments were ligated with T4 DNA ligase (NEB) and cloned into XbaI-1 6 3 digested pKK214 to generate pKK214-vgrG-OVA or pKK214-OVAgB. Electroporation was 1 6 4 used to introduce plasmids to E. coli and LVS. A summary of primers, plasmids, and strains used 1 6 5 can be found in Table 1 propensity for effector functions, including killing infected cells and producing cytokines. In the lungs, the highest number of effector memory OT-I cells were observed on day 7 (Fig 5B), which coincides with the peak bacterial burden in this assay ( Fig 3A). T EM remained at high to the lungs and MLN, T EM peaked in number on day 14 in the spleen (Fig 5B). This may be due 3 5 5 to the fact that bacteria reach the spleen and replicate later in the course of pneumonic tularemia 3 5 6 compared to the lungs (Fig 3A and [44,45]). Consistent with the data for total OT-I cells (Fig 3), Francisella-specific T EM are retained well in the spleen and poorly in the lungs after bacterial 3 5 8 clearance.  As expected, the number of T CM in the lung was much lower than the number of T EM during peaked on day 14, then exhibited a trend towards gradual decrease over time. In the spleen, T CM 3 7 0 increased in number between day 7 and 21, and decreased on day 28 ( Fig 5C). These data show To demonstrate one potential application of LVS-OVA, we studied CD8 + T cell responses in 3 7 8 mice primed either intranasally or via scarification, both followed by an intranasal booster.

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Responses were assessed on day 28 after primary vaccination, and also three and five days after weight loss before recovering, while mice primed via scarification did not lose weight ( Fig 6A).
The intranasal booster was well tolerated by both groups (Fig 6B and Fig S1). On day 28 post- MLNs of scarification-primed mice, such that they matched intranasally-primed mice.

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Additionally, the number of Francisella-specific cells in the lungs of scarification-primed mice 3 8 8 increased after the booster, reaching significantly higher levels than in intranasally-primed and within the lungs after these vaccination schemes (Fig 7). For all animals, very few CD69 + 3 9 3 CD103 + cells were detected in the lungs, possibly reflecting the poor development of resident 3 9 4 memory cells in this model. Altogether, these data suggest that: 1) Intranasal LVS vaccination 3 9 5 leads to more Francisella-specific CD8 + T cells in the MLN compared to scarification 3 9 6 vaccination; 2) an intranasal booster increases the number of Francisella-specific CD8 + cells in 3 9 7 the MLN and lung of scarification-primed animals, while avoiding the weight loss associated Francisella-specific lung-resident CD8 + T cells. significance was determined by two-tailed, unpaired t-test at each timepoint * p<0.05 ** p<0.01.  OT-I cells. n=3 per group, means +/-SD are plotted. No statistically significant differences 4 1 5 according to two-tailed, unpaired t-tests. There is currently no approved vaccine for the prevention of tularemia, due to concerns immunity, in part due to a lack of tools to identify these correlates. Here we report the 4 2 3 development of an ovalumbin expression system for F. tularensis, which is the first tool to allow 4 2 4 for the study of F. tularensis-specific CD8 + T cells. Our early attempts to express ovalbumin in 4 2 5 F. tularensis were unsuccessful. However, after codon optimizing a fragment of ovalbumin, and 4 2 6 expressing it as a C-terminal tag of the native VgrG protein did we achieve a robust expression adoptively-transferred CD8 + OT-I cells in infected mice. We first sought to study the initial expansion of F. tularensis-specific CD8 + T cells after innate pro-inflammatory pathways early in the course of infection [10,44,45,47]; these data 4 3 7 suggest this stealth strategy may also dramatically affect the kinetics of the T cell response. Our  tularensis-specific CD8 + T cells produced IFN-γ upon stimulation (Fig 4). We did not observe A major advantage of LVS-OVA is that it allows us to study pathogen-specific memory 4 4 7 development, particularly at times when cells of other specificities may be more abundant. We contraction and retention properties in various tissues following intranasal LVS infection (Fig 5).
Notably, peaks in the T EM population in each organ coincide with peaks in IFN-γ producing to provide a rapid response that contributes to protection from reinfection, including against 4 5 5 respiratory pathogens [48,49]. However, we detected very few F. tularensis-specific CD8 + T 4 5 6 cells expressing the resident memory markers CD69 and CD103 in the lungs of vaccinated mice.

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This suggests that lung T RM development is poor in this model, and that strategies to boost F. shown to be more virulent [10]. It is interesting to speculate that the increased protectiveness of 4 6 2 the RML LVS strain may be due to increased generation of F. tularensis-specific T RM in the vaccine responses will help to define correlates protective adaptive immunity. An ideal vaccine would preferentially induce such protective responses without causing any 4 6 7 harm to the host. In this regard, vaccination with F. tularensis LVS by the respiratory route 4 6 8 appears to provide superior protection compared to other routes [12,19,50,51], but is also 4 6 9 associated with significant adverse effects [50]. We used LVS-OVA to compare the CD8 + T cell before recovering, while mice vaccinated via scarification did not lose weight (Fig 6). Intranasal animals, but they did not lose any weight and tolerated the booster similarly to intranasal-primed 4 7 7 animals. One possible implication of these data is that a scarification-prime, respiratory-boost 4 7 8 strategy may achieve the same benefits as respiratory vaccination, with enhanced safety. In summary, we have developed an ovalbumin expression system that allows for the    u  o  p  p  a  K  ,  F  o  r  s  b  e  r  g  Å  ,  N  o  r  q  v  i  s  t  A  .  C  o  n  s  t  r  u  c  t  i  o  n  o  f  a  r  e  p  o  r  t  e  r  p  l  a  s  m  i  d  f  o  r  s  c  r  e  e  n  i  n  g  i  n  v  i  v  o  5  7  2  p  r  o  m  o  t  e  r  a  c  t  i  v  i  t  y  i  n  F  r  a  n  c  i  s  e  l  l  a  t  u  l  a  r  e  n  s  i  s  .  F  E  M  S  M  i  c  r  o  b  i  o  l  L  e  t  t  .  2  0  0  1  ;  2  0  5  :  7  7  -8  1  .  5  7  3  d  o  i  :  1  0  .  1  1  1  1  /  j  .  1  5  7  4  -6  9  6  8  .  2  0  0  1  .  t  b  1  0  9  2  8  .  x  5  7  4   2  8  .  A  b  d  H  ,  J  o  h  a  n  s  s  o  n  T  ,  G  o  l  o  v  l  i  o  v  I  ,  S  a  n  d  s  t  r  ö  m  G  ,  F  o  r  s  m  a  n  M  .  S  u  r  v  i  v  a  l  a  n  d  G  r  o  w  t  h  o  f  F  r  a  n  c  i  s  e  l  l  a  5  7  5 I  A  I  .  0  0  2  0  3  -1  3  6  0  8  4  0  .  S  l  i  g  h  t  S  R  ,  M  o  n  i  n  L  ,  G  o  p  a  l  R  ,  A  v  e  r  y  L  ,  D  a  v  i  s  M  ,  C  l  e  v  e  l  a  n  d  H  ,  e  t  a  l  .  I  L  -1  0  r  e  s  t  r  a  i  n  s  I  L  -1  7  t  o  l  i  m  i  t  l  u  n  g  6  0  9  p  a  t  h  o  l  o  g  y  c  h  a  r  a  c  t  e  r  i  s  t  i  c  s  f  o  l  l  o  w  i  n  g  p  u  l  m  o  n  a  r  y  i  n  f  e  c  t  i  o  n  w  i  t  h  F  r  a  n  c  i  s  e  l  l  a  t  u  l  a  r  e  n  s  i  s  l  i  v  e  v  a  c  c  i  n  e  6  1  0  s  t  r  a  i  n  .  A  m  J  P  a  t  h  o  l  .  2  0  1  3  ;  1  8  3  :  1  3  9  7  -1  4  0  4  .  d  o  i  :  1  0  .  1  0  1  6  /  j  .  a  j  p  a  t  h  .  2  0  1  3  .  0  7  .  0  0  8  6  1  1   4  1  .  N  o  l  z  J  C  .  M  o  l  e  c  u  l  a  r  m  e  c  h  a  n  i  s  m  s  o  f  C  D  8  +  T  c  e  l  l  t  r  a  f  f  i  c  k  i  n  g  a  n  d  l  o  c  a  l  i  z  a  t  i  o