M2e sequence of human influenza type A virus isolates. (A) Human epidemic strains. (B) Sporadic (nonepidemic) isolates believed to have been transmitted to humans directly from infected fowl. The top sequence in each alignment is from the strain A/Brevig Mission/1/1918(H1N1), which caused the “Spanish” flu pandemic of 1918. The M2e sequence is subdivided into three blocks to show the genetic relation between M2e and M1: nt 1 to 26 (codons 1 to 9) and nt 27 to 68 (codons 10 to 23) are bicistronic and encode M2e and M1 in the same or different reading frames, respectively. nt 69 to 72 are monocistronic for M2e. The columns underneath each subtype show the number of epidemic strains (identified in the database by unique geographic location and time of isolation) that exhibit the indicated M2e sequence. Dashes indicate identity to the sequence of A/Brevig Mission/1/1918. No dash indicates sequence data were unavailable. Sequence data are from the Influenza Sequence Database (flu.lanl.gov) (20).

Chronic treatment of infected SCID mice with M2e-specific MAb prolongs survival. SCID mice were infected i.n. with a 5-μl inoculum containing 10⁴ TCID₅₀ (A) or 10³ TCID₅₀ (B) of PR8. At 4 to 24 h p.i., mice were injected with 100 μg and at weekly intervals thereafter with 50 μg of purified MAbs specific for NP (□) or M2e: 14C2-S1-4 (•), M2-56 (▴), M2-80 (▾), or M2-15 (⧫). Mice were monitored for weight loss and killed when they were severely moribund and had lost ≥30% of their initial body weight, which is defined as survival endpoint. Panel A is a compilation of data from three independent experiments, comprising 16 mice (three experiments) in the control group, 8 mice (one experiment) in the 14C2-S1-4, 8 mice (two experiments) in the M2-56, and 5 mice (one experiment) in the M2-80 group. Panel B shows a single experiment with six mice in the control group and seven mice in each of the anti-M2e MAb treatment groups. In this experiment, the last Ab injection was given on day 42. Experiments were terminated 46 to 56 days p.i. All M2e-specific MAbs significantly increased survival compared to the control group (log rank test, P < 0.01).

Sequence analysis of virus isolates. Sequence chromatograms of the M2e region were generated directly from M2 PCR products by using the sequence primer M2-1 Rev (see Materials and Methods). Corresponding nucleotide and protein sequences are shown above each chromatogram. The arrow indicates nucleotide position 29 (amino acid position 10 of M2e). The three top chromatograms exemplify isolates that appeared to contain only wt virus and the P10H and P10L mutants, respectively. The bottom two chromatograms exemplify isolates that contained mixtures of wt and a mutant virus.

Pathogenicity of M2e escape mutants in BALB/c mice. BALB/c mice (three to four/group) were infected i.n. with 500 TCID₅₀ (A and C) or 1,500 TCID₅₀ (B and D) of wt PR8 (□), P10 isolate (•), P10H (•), or P10L (⧫). Infection was done under anesthesia with 50 μl of inoculum, which initiates a total respiratory tract infection. Mice were then observed for weight loss (A and B) and mortality (C and D). Panels A and C show the mean ± the SEM within each group. Since BALB/c mice generate an immune response and may recover after loss of ≥30% of body weight, natural disease-associated death was used as the endpoint in panels C and D.

Progression of a nasal PR8 virus infection in SCID mice. Anesthetized SCID mice were infected i.n. with 5 μl (2 to 3 μl/nare) of PR8 (10⁴ TCID₅₀). Mice were killed at various times p.i. and tested for the presence of infectious virus in nasal turbinates (A), trachea and extrapulmonary bronchi (B), and lungs (C). Tissue extracts were first titrated in MDCK cell microcultures and, if negative in this assay, injected undiluted (100 μl per two eggs) into the allantoic cavity of 10-day-old embryonated hen's eggs. The stipulated horizontal lines in panels A, B, and C show the threshold of virus detection. Each symbolshows the titer (log₁₀, TCID₅₀, or EID₅₀ per total tissue) from one mouse. All symbols below the stipulated line indicate absence of detectable infectious virus. The line shows the mean virus titer. All time points except days 1, 21, and 25 p.i. are from two or more independent experiments. (D) Survival. The curve was created by the method of Kaplan and Meier.

Antigenic analysis of virus isolates. Replicate MDCK cell monolayers in 96-well plates were infected with egg-grown lung virus isolates derived from mice treated with the M2e-specific MAb 14C2-S1-4 (isolate notation A) or with NP-specific (B isolates) or rat immunoglobulin-specific (C isolates) control MAbs. The virus isolates used for infection of the MDCK cells are indicated to the left of each bar. They were obtained from severely moribund mice (>30% weight loss) or at termination of experiments, ranging from days 33 to 46 (isolates A) and days 16 to 34 (isolates B and C). PR8 wt is the virus used for infection of the mice. After 8 h of incubation at 37°C, infected monolayers were fixed with formalin and tested by ELISA for reaction with the HA-specific MAb H36-4 and the M2e-specific MAb 14C2, both at 1 μg/ml, in three replicates per MAb. For each isolate, the average OD (above background) seen with the M2e-specific MAb was expressed as percentage of the OD seen with HA-specific MAb. Assay background was determined with the nonreactive MAb 14-4-4S. Each bar shows the mean percent ± 95% confidence interval of at least three assays performed with MDCK cell monolayers infected with different doses of virus isolate. The stipulated vertical lines show the 95% confidence interval seen with MDCK cell monolayers infected with wt PR8 virus.

Growth kinetics of mutants in MDCK cells in vitro and their antigenicity. (A) Six replicate MDCK cell microcultures were infected with wt PR8, P10, P10L, or P10H and then cultured for 5 days. Small samples a culture medium were harvested at 10- to 14-h intervals and pooled within replicate cultures. The samples were then tested for virus titer by HA test. The figure shows the results of a representative experiment in which the cells were infected with 10 TCID₅₀ of virus. (B) A pool of sera obtained from four BALB/c mice 2 weeks after the third i.n. inoculation with 5 μg of wt M2e-MAP, together with 3 μg of immunostimulatory ODN and 0.5 μg of cholera toxin as described previously (21), was tested in an ELISA for reaction with MDCK cell monolayers infected with the isolates P10, P10H, P10L, or B/Lee. ODs above B/Lee background (means and standard error of the mean [SEM] of triplicates) are shown. Similar results were obtained with sera from three other immunization experiments. (C) MDCK cell monolayers infected with the indicated viruses were tested in ELISA for recognition by purified HA- and M2e-specific MAbs. All MAbs were used at a saturating dose of 1 to 2 μg/ml. The columns show the mean ± the SEM of triplicates.

P10H and P10L isolates are effective escape mutants. SCID mice (three to four per group) were infected i.n. with 5 μl (10⁴ TCID₅₀) of PR8 (A), P10 (B), P10H (C), or P10L (D) and chronically treated with anti-NP MAb (open symbols) or anti-M2e MAb M2-56 (closed symbols) as described in the legend of Fig. 3. The mice were observed for body weight (panels on left) and survival (panels on right). The weight curves show the mean ± the SEM of the group. Mice were euthanized when their weight dropped below 70% of their initial body weight and this time point was taken as time of death. The statistical differences between survival curves were determined by log rank test and are indicated.