Figure:

NP-specific
CD8⁺ T-cell responses in lungs after influenza
challenge. BALB/c mice
were immunized i.m. twice, 8 weeks apart, with PBS, 0.1 μg of
self-amplifying mRNA (SAM®) vectors SAM(NP), SAM(M1),
SAM(M1-NP), or with 0.2 μg of SAM(NP)+SAM(M1). Four weeks after
the second immunization, mice were infected with PR8 virus.
NP-specific CD8 T cells recruited in the lungs after the
infection were characterized by flow cytometry. (a) Numbers of
NP-specific CD8⁺ T cells determined using

Pro5® MHC Pentamers. Data are from individual mice (depicted as dots), while solid lanes
indicate the mean±SD. (b) Cumulative frequency of Ag-specific,
cytokine-secreting CD8⁺ T cells, indicated as
absolute number per lung. The color code represents the
different combinations of cytokine produced by the respective
cells after in vitro stimulation with medium (m), NP_147-155
peptide (NP), or M1 peptide pool (M1), as indicated. (c)
Absolute number of NP-specific CD8⁺ T cells positive
(black bar) or not (grey bar) for CD107a. Data derived from two
independent and merged experiments. Statistical analyses were
performed using the Mann-Whitney U test. *p<0.05; **p<0.01
compared to the PBS-treated group.

http://dx.doi.org/10.1371/journal.pone.0161193.g006

Abbreviated abstract:

Current hemagglutinin (HA)-based seasonal influenza vaccines induce
vaccine strain-specific neutralizing antibodies that usually fail to
protect against mismatched circulating viruses.  Inclusion of
conserved proteins such as the nucleoprotein (NP) and the matrix
protein 1 (M1) can increase effectiveness by eliciting
cross-reactive T-cells. However, efficient priming of T-cell
responses requires the right delivery system. Here we show novel
self-amplifying mRNA (SAM^®) vectors expressing influenza
NP (SAM(NP)), M1 (SAM(M1)), and NP and M1 (SAM(M1-NP)) delivered
with lipid nanoparticles (LNP) induce robust polyfunctional CD4 T
helper 1 cells, while NP-containing SAM also induced cytotoxic CD8 T
cells. Robust expansions of central memory (T_CM) and
effector memory (T_EM) CD4 and CD8 T cells were also
measured.  An enhanced recruitment of NP-specific cytotoxic CD8
T cells was observed in the lungs of SAM(NP)-immunized mice after
influenza infection that paralleled with reduced lung viral titers
and pathology, and increased survival after homologous and
heterosubtypic influenza challenge. We show for the first time that
the co-administration of RNA (SAM(M1-NP)) and protein (monovalent
inactivated influenza vaccine (MIIV)) was feasible, induced
simultaneously NP-, M1- and HA-specific T cells and HA-specific
neutralizing antibodies, and enhanced MIIV efficacy against a
heterologous challenge.

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