Serum Abs induced with M2e-MAP K2 exhibit cross-reactivity.

<p>(<b>A</b>) M2e amino acid sequence comparison of influenza virus A/PR8, A/FM and B/Lee. (<b>B</b>) Serum Abs of BALB/c mice (n = 5 mice) immunized i.n. or s.c. with K2 peptide and adjuvants were tested in an ELISA for binding to MDCK cells infected with influenza virus A/PR8 (square), A/FM (diamond) and B/Lee (circle). (<b>C</b>) M2e amino acid sequence from the pandemic influenza strain A/Texas/2009. (<b>D</b>) Sera with anti-M2e Abs induced by i.n. or s.c. immunization with K2 peptide and adjuvants or adjuvants alone were tested for binding to a synthetic peptide corresponding to the A/Texas/2009 M2e sequence as depicted in (<b>C</b>).</p

Viral titers in lungs and nose of X31-challenged mice following vaccination.

<p>BALB/c (n = 21), C3H (n = 5), C57BL/6 (n = 3), CD1/ICR (n = 7) and Swiss Webster (n = 5) mice were immunized three times i.n. with adjuvants CpG 1826 and CT alone or K2 and adjuvants and tested for their protection against viral challenge with X31 virus (1000 TCID₅₀ in 5 µL). Virus titers in lungs and noses were determined 5 days after challenge. Results are displayed as mean ± SD.</p

Immunization with M2e-MAP K2 protects against challenge with M2e-variant viruses.

<p>(<b>A</b>) Amino acid sequence comparison of M2e from influenza A/PR8 (P10) or M2e-escape mutant viruses (P10H and P10L) as described in Ref. 9. (<b>B</b>) Sera from BALB/c mice immunized i.n. three times with K2 peptide and adjuvants were assayed in an ELISA on peptides with M2e sequences as shown in (<b>A</b>) or control peptide cysBB: cysteine back-bone. (<b>C</b>) Pooled sera (1∶250 dilution) from BALB/c mice (n = 5 mice) immunized as in (<b>B</b>) were assayed in an ELISA for binding to MDCK cells infected with influenza virus A/PR/8, the M2e-escape mutants P10H and P10L, A/FM and B/Lee. The monoclonal anti-M2e Ab 14C2 (1 µg/mL) is shown as control. (<b>D</b>) BALB/c mice (n = 5 mice/group) immunized i.n. with K2 peptide and adjuvants were challenged with 1000 TCID₅₀/50 µL influenza virus A/PR/8, P10H and P10L three weeks after the third vaccine administration. Infectious virus in the lungs was determined 5 days after challenge.</p

Anti-M2e Ab induction in various mouse strains following vaccination.

<p>BALB/c (n = 21), C3H (n = 5), C57BL/6 (n = 3), CD1/ICR (n = 7) and Swiss Webster (n = 5) mice were immunized i.n. with adjuvants CpG 1826 and CT alone or K2 and adjuvants. After the 3^rd immunization sera were tested for M2e-specific Abs (in µg/mL) by ELISA on M2e-peptide and HeLa-M2 cells as indicated. Results are displayed as mean ± SD or results are shown from pooled samples.</p

M2e-based multiple antigenic peptides induce high M2e-Ab titers and elicit protection against viral challenge.

<p>(<b>A</b>) Design of M2e-multiple antigenic peptides K2 and K3 depicting a branched construct of the peptide back-bone (lysine-glycine chain) with each four M2e residues and two T helper determinants (for sequences see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028445#pone-0028445-t001" target="_blank"><b>Table 1</b></a>). (<b>B–D</b>) BALB/c mice (n = 4–6/group) were immunized intranasally with M2e-MAP K2 or K3 mixed with adjuvants (Adj.) or adjuvants alone three times in 3–4 week intervals. (<b>B</b>) Sera were collected 3–4 weeks after the indicated number of immunization and measured in an ELISA against M2e-peptide or HeLa cells expressing full-length M2 (HeLa-M2). (<b>C and D</b>) 3–4 weeks after the third vaccination, mice were challenged with (<b>B</b>) 5 µL of 1000 TCID₅₀ influenza virus A/X31 (2.5 µL/nare) or (<b>D</b>) 50 µL of 1000 TCID₅₀ influenza virus A/PR8. Mice were euthanized 4 or 5 days after challenge as indicated and lungs assayed for infectious virus in MDCK cells.</p

Genetic impact on anti-M2e immunity.

<p>(<b>A and B</b>) BALB/c, CD1/ICR and Swiss Webster (SW) mice (n = 5–6 mice) received three consecutive infections, first with influenza virus A viruses PR8, secondly with PR8-SEQ14 and thirdly with X31. PR8-specific IgG (<b>A</b>) and M2e-specific IgG (<b>B</b>) in sera were determined after first and third infection, respectively. The results of sera from naive mice (n = 1/mouse strain) were pooled. (<b>C</b>) BALB/c, C57BL/6, and C3H mice were immunized s.c. with K2 peptide and adjuvants or adjuvants alone. The draining lymph node was harvested on day 8 and proliferation was assessed by incorporation of ^[3]H-thymidine during the fourth day of an <i>in vitro</i> culture in presence of K2 peptide. One out of two independent experiments shown. (<b>D–F</b>) BALB/cxC57BL/6 (F1) mice (n = 5 mice) were immunized i.n. with K2 and adjuvants or adjuvants alone. (<b>D</b>) M2e-specific Ab titers measured in an ELISA against M2e-peptide or HeLa-M2 cells in serum of BALB/cxC57BL/6 (F1) mice after third immunization. (<b>E</b>) M2e-specific serum Abs at a 1∶150 serum dilution were determined with allotype-specific reagents (IgG2a[a] and IgG1[a] for BALB/c and IgG2c and IgG1[b] for C57BL/6 origin). (<b>F</b>) After the third immunization, BALB/cxC57BL/6 (F1) mice were challenged with X31 (1000 TCID₅₀/5 µL) and infectious virus in the lungs was determined 5 days post challenge.</p

Route of immunization with M2e-MAPs determines systemic and respiratory tract responses.

<p>BALB/c mice were immunized with K2 and adjuvants or adjuvants alone either intranasally (i.n.) or into the tailbase (s.c.) as described above. (<b>A</b>) M2e-specific Abs in the bronchio-alveolar lavage (BAL) fluid after third vaccination were determined by ELISA on M2e-peptide (n = 3 mice/group). (<b>B</b>) Sera were collected 3–4 weeks after the indicated number of immunizations and measured by ELISA for binding to M2e-peptide and HeLa-M2 cells. (<b>C</b>) Mice were euthanized after third vaccination and cells from lungs and bone marrow (BM) seeded onto ELISPOT plates coated with M2e-peptide. Plates were developed with anti-mouse IgG or IgA. Results from 4–6 mice from 2 independent experiments are expressed as mean counts (± SEM). (<b>D</b>) Groups of mice were challenged with influenza virus A/X31 (1000 TCID₅₀/5 µL) and infectious virus in the lungs determined 5 days after challenge.</p

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