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

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

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

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