Cooperativity Between CD8+ T Cells, Non-Neutralizing Antibodies, and Alveolar Macrophages Is Important for Heterosubtypic Influenza Virus Immunity

<div><p>Seasonal epidemics of influenza virus result in ∼36,000 deaths annually in the United States. Current vaccines against influenza virus elicit an antibody response specific for the envelope glycoproteins. However, high mutation rates result in the emergence of new viral serotypes, which elude neutralization by preexisting antibodies. T lymphocytes have been reported to be capable of mediating heterosubtypic protection through recognition of internal, more conserved, influenza virus proteins. Here, we demonstrate using a recombinant influenza virus expressing the LCMV GP33-41 epitope that influenza virus-specific CD8+ T cells and virus-specific non-neutralizing antibodies each are relatively ineffective at conferring heterosubtypic protective immunity alone. However, when combined virus-specific CD8 T cells and non-neutralizing antibodies cooperatively elicit robust protective immunity. This synergistic improvement in protective immunity is dependent, at least in part, on alveolar macrophages and/or other lung phagocytes. Overall, our studies suggest that an influenza vaccine capable of eliciting both CD8+ T cells and antibodies specific for highly conserved influenza proteins may be able to provide heterosubtypic protection in humans, and act as the basis for a potential “universal” vaccine.</p> </div

Alveolar macrophages are important for cooperative heterosubtypic protection.

<p>A) FcRγ-/- or IL-15-/- mice, which lack NK cells, were primed with LCMV Armstrong and given either naïve or X31-GP33 serum 1 day prior to rechallenge with PR8-GP33. Weight loss and lung function over time along with viral load at day 6 were determined. B) X31-GP33 immune mice were treated with clodronate i.n. to deplete alveolar macrophages, anti-NK1.1 (clone PK136) to deplete NK cells, or cobra venom factor to deplete complement. Additional mice were administered empty liposomes or PBS as controls. Mice were then rechallenged with PR8-GP33 and weight loss and lung function measured over time, and viral titer at day 6 determined. C) LCMV Armstrong immune mice were treated with clodronate liposomes or anti-NK1.1, and then given X31-GP33 serum one day prior to rechallenge. A non-treated LCMV Armstrong group was also given X31-GP33 serum. These mice were then rechallenged with PR8-GP33 and weight loss and lung function over time along with viral load at day 6 were determined. Data for all panels are representative 6–8 mice per group. Note, in some experiments where morbidity occurred, some animals where euthanized before the end of the experiment according to IACUC guidelines. Mice were anesthetized using avertin. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003207#s2" target="_blank">Results</a> are representative of two independent experiments.</p

<i>In vivo</i> primed CD8+ T cells alone are insufficient for optimal influenza rechallenge protection.

<p>To determine if CD8+ T cells alone can mediate influenza protection, mice were primed using different strategies, and rechallenged with 3 LD₅₀ PR8-GP33. Body weight, lung function, and viral load were determined following rechallenge. A) Mice were primed with the indicated viruses expressing GP33 or NP366. As a control, a virus expressing a non-influenza virus determinant was used (LCMV NP). Data are representative of 9 mice per group with three mice sacrificed at each time point for viral load determination. B) Mice were immunized using the indicated prime-boost strategy and rechallenged at either day 8 or day 30 following the boost. Additionally, mice were rechallenged at effector time point (d8) following primary infection with LCMV Arm i.n. Data are representative of 9 mice per group with three mice sacrificed at each time point for viral load determination. C) A prime-boost approach that elicited a CD8+ T cell response to either GP33 or a non-influenza epitope was also used. For the latter LCMV V35A, a variant virus in which the GP33 epitope is mutated was used following VV-LCMV NP priming to boost LCMV NP-specific T cells. Naïve mice infected with PR8-GP33 were used as a control in all experiments. Data are representative of 4–5 mice per group. Mice were anaesthetized using ketamine xylazine. The results are representative of three independent experiments.</p

Protection against influenza virus rechallenge appears to be independent of magnitude of virus-specific CD8+ T cell response.

<p>A) C57BL/6 mice were primed with either X31-GP33 i.n. or LCMV Armstrong i.n. These mice, as well as a naïve control group, were rechallenged with 3 LD₅₀ influenza PR8-GP33 virus on day 30+ following primary infection. Weight loss after rechallenge and lung function (as measured by pulse oximetry) were assessed. Mice were sacrificed on day 0, 4, 6, and 9 post rechallenge and viral titers were determined in the lungs. Data are representative of 9 mice per group with three mice sacrificed at each time point for viral load determination. B) The lung, spleen and BAL were collected on day 6 to assess the influenza virus-specific CD8+ T cell response. Following isolation, cells were stimulated using peptide pools from the influenza proteins HA, NA, NS1/2, PA, PB, NP, as well as the LCMV GP33 peptide. Intracellular staining (ICS) for IFNγin the lung, BAL, and spleen assessed the total virus-specific CD8+ T cell response. Mice were anaesthetized using avertin. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003207#s2" target="_blank">Results</a> are representative of two independent experiments.</p

Both influenza virus-specific CD8+ T cells and antibodies are needed for optimal heterosubtypic protection.

<p>A) Mice were primed with LCMV Armstrong and then given serum from X31-GP33 immune mice, naïve mice, or PR8-GP33 immune mice 30 days later. Other groups were given X31-GP33 serum that had been depleted of IgG and IgA or an equal volume PBS. Mice were then rechallenged with 3 LD₅₀ PR8-GP33 and weight loss and lung function were measured over time with the viral titer at day 6 determined. The viral load data represents the combined results from three independent experiments. B) X31-GP33, PR8-GP33, and naïve serum was also transferred into a separate group of naïve mice and weight loss and lung function were measured over time with viral titer at day 6 determined. C) LCMV Armstrong immune mice were given X31-GP33, naïve serum or PBS and challenged with PR8-WT to determine protection upon rechallenge with a virus lacking the GP33 epitope. Weight loss and lung function over time along with viral load at day 6 were determined. D) VVNP366 immune mice were given X31 or naïve serum and then challenged with SW/33 to evaluate cooperativity-based protection in non-recombinant influenza model system. Weight loss and lung function over time along with viral load at day 6 were determined. Data for all panels are representative of 6–8 mice per group. Mice were anaesthetized using avertin. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003207#s2" target="_blank">Results</a> are representative of two independent experiments.</p

Adoptive transfer of <i>in vivo</i> or <i>in vitro</i> activated CD8+ T cells and protection from influenza virus challenge.

<p>A) Ly5.1+ mice were immunized with LM-GP33 and then boosted with LCMV Arm. On day 8 post boost the mice were sacrificed and their spleens pooled. The CD8+ T cells were then isolated using MACS columns and 1.6×10⁶ or 0.8×10⁶ GP33+ CD8+ T cells were transferred into Ly5.2+ mice. A third group was given an equal volume of PBS. All three groups were then challenged with 3 LD₅₀ PR8-GP33 one day later. Weight loss after rechallenge was monitored until day 6 post rechallenge at which point the mice were sacrificed. Data are representative of at least 5 mice per group. B) Viral load in the lungs was analyzed on day 6 post challenge. C) Flow cytometric analysis of infiltrating GP33-specific CD8+ T cell in the lung was performed on day 6-post challenge. D) 20×10⁶, 10×10⁶, or 2×10⁶<i>in vitro</i> activated GP33-specific P14 CD8+ T cells were transferred into separate groups of mice, which were then challenged with 3 LD₅₀ PR8-GP33 the following day. The weight, and lung function of these mice was determined at different points following rechallenge. Viral loads were determined in the lungs at day 6 post rechallenge. Data are representative of at least 7 mice per group. Mice were anaesthetized using avertin. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003207#s2" target="_blank">Results</a> are representative of two independent experiments.</p

Heterosubtypic protection in B cell transgenic X31-GP33 primed mice is rescued through X31-GP33 serum transfer.

<p>To establish whether antibodies are responsible for protection in X31-GP33 primed mice, B cell transgenic (MD4) X31-GP33 immune mice were given either X31-GP33 serum or an equal volume PBS one day prior to rechallenge with PR8-GP33. Weight loss and lung function were determined at the indicated time points following rechallenge. Viral load was determined in the lungs at day 6 post rechallenge. Data are representative of at 4–8 mice per group. Mice were anaesthetized using ketamine xylazine. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003207#s2" target="_blank">Results</a> are representative of two independent experiments.</p

X31-GP33 mediated heterosubtypic protection is dependent on B cells.

<p>A) To determine the role of CD8+ and CD4+ T cells in X31-GP33 mediated protection, mice were primed with X31-GP33 and then rechallenged with PR8-GP33 30 days later. On days -3, -1, 1, 3, and 5 post rechallenge mice were treated with anti-CD4 (GK1.5), anti-CD8 (53.6), or anti-Thy1.2 to deplete both CD4+ and CD8+ T cells. Body weight, lung function, and viral load were determined at different time points post rechallenge. Data are representative of at least 7 mice per group. B) To elucidate the role of B cells in X31-GP33 mediated virus protection mice either lacking B cells (µMT) or mice with B cells specific for hen egg lysozyme (MD4) were used. These mice were primed with X31-GP33 and rechallenged 30 days later with 3 LD₅₀ PR8-GP33. Weight loss, and lung function were examined. Viral titers were determined on day 6. Lung function and viral load were not determined for the uMT group. Data are representative of at least 4 mice per group. C) Flow cytometric analysis was performed on the lungs on day 6 to compare the GP33 and NP366-specific CD8+ T cell response in B6 and B cell transgenic mice. Mice were anaesthetized using ketamine xylazine. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003207#s2" target="_blank">Results</a> are representative of two independent experiments.</p