Influenza nucleoprotein delivered with aluminium salts protects mice from an influenza virus that expresses an altered nucleoprotein sequence

Influenza virus poses a difficult challenge for protective immunity. This virus is adept at altering its surface proteins, the proteins that are the targets of neutralizing antibody. Consequently, each year a new vaccine must be developed to combat the current recirculating strains. A universal influenza vaccine that primes specific memory cells that recognise conserved parts of the virus could prove to be effective against both annual influenza variants and newly emergent potentially pandemic strains. Such a vaccine will have to contain a safe and effective adjuvant that can be used in individuals of all ages. We examine protection from viral challenge in mice vaccinated with the nucleoprotein from the PR8 strain of influenza A, a protein that is highly conserved across viral subtypes. Vaccination with nucleoprotein delivered with a universally used and safe adjuvant, composed of insoluble aluminium salts, provides protection against viruses that either express the same or an altered version of nucleoprotein. This protection correlated with the presence of nucleoprotein specific CD8 T cells in the lungs of infected animals at early time points after infection. In contrast, immunization with NP delivered with alum and the detoxified LPS adjuvant, monophosphoryl lipid A, provided some protection to the homologous viral strain but no protection against infection by influenza expressing a variant nucleoprotein. Together, these data point towards a vaccine solution for all influenza A subtypes

NP protein delivered with alum provides optimal protection from influenza A infection.

<p>B6 mice were immunized i.m. in both hind-legs with PBS (closed triangles) or a total of 10 µg of NP protein delivered alone (closed diamonds), or with 10 µg of MPL (closed circles), 100 µg alum (closed squares), or both adjuvants (open squares). At least 70 days later, these animals were infected with PR8 influenza A i.n. Mice were weighed daily and the percent of original weight calculated (A) or the amount of virus present in one lung lobe examined 4–5 days after infection (B). Data are combined from two separate experiments with 4–5 mice per group. In A, significant differences between the PBS control mice infected with PR8 and the experimental groups are indicated by the following symbol on the indicated days post-infection. Significant differences in mice immunized with NP and alum are indicated by **(p<0.01) and ***(p<0.001). Significant difference in mice immunized with NP and MPL are indicated by ## (p<0.01). Significant differences in mice immunized with NP and alum and MPL are indicated by $(p<0.05),$$(p<0.01),$$$ (p<0.001). No significant difference between control PBS mice and those immunized with NP protein were found. In B, * = p<0.05.</p

NP protein delivered with alum primes a specific T and B cell immune response.

<p>B6 mice were immunized i.m. in both hind-legs with a total of 10 µg of NP protein delivered with or without 100 µg of alum. The percentages (A, B) or numbers (C, D) of D^b/NP_366–74 CD8 or IA^b/NP_311–25 CD4 T cells were examined in the spleen or the two popliteal lymph nodes (DLN) 9 days later. Cells are gated on CD8+dump negative live cells (A) or CD4+dump negative live cells (B) with the number in the plot indicating the percentage of CD8 or CD4 cells that are CD44^hi tetramer+ as indicated by the gate. In C and D each point represents a mouse and the line shows the mean of the group. The X axis is set at the level of detection, determined by staining cells from naïve animals with the MHC tetramers. Serum from these animals was used to examine the level of NP specific IgG1 antibody (E), with each line representing one animal. These data are combined from 2 independent experiments with 3 mice per group.</p

The NP amino acid sequence from PR8 and NY1682 influenza viruses differ in known epitopes.

<p>Sequence alignment for NP proteins from PR8 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061775#pone.0061775-Grimm1" target="_blank">[46]</a> and NY1682 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061775#pone.0061775-Zhou1" target="_blank">[20]</a>. The IA^b and D^b binding peptides are highlighted in red and green respectively.</p

NY1682 infection does not prime T cells specific for immunodominant epitopes from PR8’s NP.

<p>B6 mice were infected (bottom) or not (top) with NY1682 i.n. and 25 days later the percentages of D^b/PA_224–38, D^b/NP_366–74 CD8 T cells, or IA^b/NP_311–25 CD4 in the MLN were examined (A). The numbers are the percentages of tetramer+CD44^hi cells out of gated CD8+ or CD4+ live cells that were also dump negative. The serum from these animals was tested for the presence of IgG1 and IgG2c antibody that bound to recombinant PR8 NP with each line representing one mouse (B).</p

Immunization with PR8’s NP and alum primes protective immunity to NY1682.

<p>B6 mice were injected i.m. in both hind-legs with PBS (closed triangles) or a total of 10 µg of NP and 100 µg of alum with (open squares) or without (closed squares) 10 µg of MPL. At least 70 days later, these animals were infected with NY1682 i.n. The mice were weighed daily and the percent of original weight of each mouse calculated for each day. The data are combined from two experiments with 4–5 mice per group (A). These mice were bled one day prior to infection with NY1682 or 5 days following infection. The relative units of NP specific IgG1 and IgG2c present in the serum were determined using a NP specific ELISA (B). The percentages (C) or numbers (D) of D^b/NP_366–74 tetramer+ cells present in one lung lobe of these and naïve control animals were examined. In A, significant differences between the PBS control mice infected with NY1682 and those first immunized with PR8’s NP and alum are indicated with *(p<0.05) and **(p<0.01). No significant differences between PBS control mice and those first immunized with PR8’s NP and alum and MPL were found. In C, cells were gated on live CD8+ lymphocytes that were dump negative. Representative plots are shown from 1 experiment with 4 mice per group with numbers in the plot indicating the percentages of D^b/NP_366–74 tetramer+ out of gated live CD8+ cells. In D, each point represents a mouse and the line shows the mean of the group. The X-axis is set at the level of background staining.</p

The number of D^b/NP_366–74 CD8 T cells is increased in protected animals.

<p>B6 mice were immunized i.m. in both hind-legs with PBS or a total of 10 µg of NP protein delivered with 100 µg alum with or without 10 µg of MPL. At least 70 days later these animals were infected with PR8 influenza A i.n. and the numbers of IA^b/NP_311–25 CD4+ T cells (A and B) or D^b/NP_366–74 CD8+ (C and D) were examined in the medistinal lymph node (A and C) or in one lung lobe (B and D) 5 days after infection. Cells were gated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061775#pone-0061775-g001" target="_blank">Figure 1</a>. Each point represents a mouse and the line shows the mean of the group. The X axis is set at the level of detection. The relative amounts of NP specific IgG1 (E) and IgG2c (F) antibody in the serum of these animals were examined prior to and 5 days after infection, error bars show SEM. No NP specific antibodies could be detected at these time points in mice that had been infected but not immunized. Data are combined from two separate experiments with 4–5 mice per group. n.s.: not significant.</p

The NP specific memory T cell response is similar regardless of the adjuvant combination used.

<p>The numbers of memory NP specific memory CD8 (A, B) or CD4 (C, D) T cells were determined in the spleens (A, C) and popliteal lymph nodes (B, D) of B6 mice immunized with NP protein and alum and MPL or alum at least 70 days previously. Cells were gated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061775#pone-0061775-g001" target="_blank">Figure 1</a>. The data are combined from two independent experiments with 4 mice per group indicated by each point, the line shows the mean of the group. The X axis is set at the level of detection. n.s. not significant. The expression of memory markers on either NP specific CD8 (E) or CD4 (F) T cells was examined in the spleen (CD8 T cells) or popliteal lymph nodes (CD4 T cells) at least 70 days after immunization. Cells are gated on naive/CD44 low CD8 or CD4 T cells (filled histogram) or on D^b/NP_366–74 tetramer positive CD8 memory T cells or IA^b/NP_311–25 tetramer positive CD4 memory T cells from mice immunized with NP protein and alum (black dashed line) or alum and MPL (red line). Tetramer positive cells were gated on live CD8 or CD4 positive cells that were negative for B220, F4/80, MHC II and either CD4 or CD8 respectively. The data show representative plots from 1–2 experiments with 4 mice per group.</p