Therapeutic Activity of an Anti-Idiotypic Antibody-Derived Killer Peptide against Influenza A Virus Experimental Infection

Abstract

The in vitro and in vivo activities of a killer decapeptide (KP) against influenza A virus is described, and the mechanisms of action are suggested. KP represents the functional internal image of a yeast killer toxin that proved to exert antimicrobial and anti-human immunodeficiency virus type 1 (HIV-1) activities. Treatment with KP demonstrated a significant inhibitory activity on the replication of two strains of influenza A virus in different cell lines, as evaluated by hemagglutination, hemadsorption, and plaque assays. The complete inhibition of virus particle production and a marked reduction of the synthesis of viral proteins (membrane protein and hemagglutinin, in particular) were observed at a KP concentration of 4 μg/ml. Moreover, KP administered intraperitoneally at a dose of 100 μg/mice once a day for 10 days to influenza A/NWS/33 (H1N1) virus-infected mice improved the survival of the animals by 40% and significantly decreased the viral titers in their lungs. Overall, KP appears to be the first anti-idiotypic antibody-derived peptide that displays inhibitory activity and that has a potential therapeutic effect against pathogenic microorganisms, HIV-1, and influenza A virus by different mechanisms of action.

Influenza A viruses continue to be major causes of morbidity and mortality as seasonal or pandemic infectious agents worldwide. They are responsible for hospitalization and death especially among infants and elderly individuals with underlying chronic diseases. Although annual vaccination can be an effective strategy for preventing annual influenza, in the last few years serious concerns, brought about by the outbreaks of avian influenza A virus (H5N1) and its transfer to humans, about the measures of influenza prevention available have been raised. In 2005, the risk of a new pandemic prompted the World Health Organization (WHO) to update its global influenza preparedness plan, which describes measures that should be taken on a national level before and during influenza pandemics (38). Other than vaccination, which would be required to prevent and/or contain such a pandemic, great emphasis has been placed on the use of antiviral drugs before global disease spread and for the prophylaxis and treatment of exposed and infected people who may be vulnerable (9, 21). Two classes of antiviral compounds, M2 ion channel-blocking drugs (amantadine, rimantadine) and neuraminidase (NA) inhibitors (zanamivir, oseltamivir), have been approved for use for the treatment and prophylaxis of influenza, and WHO encourages their use during this interpandemic period to increase familiarity with their effective application (7, 27, 38). However, toxicity (which often limits the long-term efficacy of antiviral therapy) and, most importantly, the emergence of partially and fully resistant strains highlight the need for and interest in discovering new effective antiviral drugs (13, 20).

The objective of the present study was to assess the in vitro and in vivo inhibitory activities against influenza A virus of a previously described anti-idiotypic antibody (Ab)-derived killer decapeptide (KP). KP represents the functional internal image of a yeast (Pichia anomala) killer toxin and has microbicidal activity against wide pathogenic eukaryotic and prokaryotic microorganisms (5, 11, 22, 29, 32, 36). KP was therapeutic in experimental models of vaginal and/or systemic fungal infections (5, 29, 36), and on the basis of its sequence homology with critical segments in human immunodeficiency virus type 1 (HIV-1) gp160 loops, it was able to inhibit HIV-1 replication in peripheral blood mononuclear cells infected ex vivo and in vitro by downregulation of the CCR5 coreceptor and/or physical blockage of the gp120-receptor interaction. Conversely, the peptide showed no in vitro activity against many other DNA and RNA viruses (4). KP, moreover, was able to modulate the expression of costimulatory and major histocompatibility complex molecules on murine dendritic cells, improving their capacity to induce lymphocyte proliferation (6). A striking sequence homology has been observed between KP (AKVTMTCSAS) and the light-chain variable region between residues 17 and 26 (EKVTLTCSAS) of an Ab (HC63) that prevents the hemagglutinin (HA) low-pH fusogenic transition (2). On the basis of this observation, KP has been tested against two different strains of influenza A virus. Although its antiviral activity was based on a different mode of action, KP has demonstrated significant in vitro and in vivo antiviral activities. KP seems to be the first anti-idiotypic Ab-derived peptide that displays inhibitory activity and a potential therapeutic effect against pathogenic microorganisms, HIV-1, and influenza A virus by different mechanisms of action.

MATERIALS AND METHODS

Synthetic peptide.

The selection of KP (AKVTMTCSAS; molecular weight, 998.2) and procedures for its synthesis have been described previously (29).

Cell cultures and virus strains.

Monolayers of continuous monkey kidney epithelial (LLC-MK2) cells and Madin-Darby canine kidney (MDCK) cells were cultured in complete Earle's minimal essential medium supplemented with 10% fetal bovine serum (FBS). Influenza A/parrot/Ulster/73 (H7N1) (Ulster 73), a subtype of low pathogenicity obtained from the Istituto Zooprofilattico delle Tre Venezie, Padua, Italy, and A/NWS/33 (H1N1) (NWS 33) (ATCC VR-219), a neurotropic strain of human influenza virus derived from the WS strain by intracerebral inoculation of mice, were propagated in the allantoic cavities of hen eggs that had been embryonated for 11 days. After 28 h of growth, the allantoic fluid was harvested and centrifuged (2,000 × g, 30 min) for clarification, the virus titer was determined by hemagglutination with human group 0 Rh-positive red blood cells (RBCs), the virus particles were quantified by plaque assay, and the allantoic fluid was stored at −80°C until it was used as the inoculum.

Confluent cell monolayers were overlaid with infected allantoic fluid diluted to give the desired multiplicity of infection (MOI). After 40 min of adsorption at room temperature (RT), the infected cells were washed with 0.9% NaCl, overlaid with Earle's minimal essential medium containing 2% FBS (maintenance medium [MM]), and incubated at 37°C. Virus production was determined by measuring the numbers of hemagglutination units (HAUs) and PFU in the supernatants of the infected monolayers at different times until 72 h postinfection (p.i.) by standard procedures (3).

Effect of KP treatment on virus production.

Different amounts of KP (from 0 to 10 μg/ml) in MM were added to the virus suspensions before infection (1 h or 2 h) and left during the adsorption period or were added to the infected cells at various times after removal of the viral inoculum and left for 24 h (unless otherwise specified). KP diluent was the control. The supernatant was collected and clarified (2,000 × g), and the virus particles were quantified by hemagglutination and plaque assays (3). Each assay was carried out in duplicate. The 50% effective concentration was calculated by nonlinear regression analysis with GraphPad Prism (version 4.01) software (San Diego, CA). The effect of the treatment was further assessed by purifying the virus particles from supernatants of [³⁵S]methionine-labeled infected cells. For virus purification, cell monolayers were infected at an MOI of 2 and were labeled at 1 h p.i. with [³⁵S]methionine (20 μCi/ml) in MM. The culture supernatants were collected 24 h later and clarified (10,000 × g, 30 min), and the viral particles were pelleted (40,000 × g) and purified first onto 15 to 60% (wt/vol) discontinuous sucrose (65,000 × g, 90 min) linear gradients and then onto 20 to 45% (wt/vol) potassium tartrate (180,000 × g, 16 h) linear gradients. The virus-containing fractions were pooled, diluted, pelleted (180,000 × g, 30 min), and lysed with Laemmli's lysis buffer (14, 15); and the presence of virus proteins was determined by loading different sample amounts onto 15% sodium dodecyl sulfate (SDS)-polyacrylamide slab gels for electrophoresis and autoradiography on Kodak X-ray film.

Hemadsorption assay with infected cells.

According to the hemadsorption assay of Finter (10), infected cell monolayers either untreated or treated with KP (4 μg/ml) were washed at 48 h p.i. with PBS and then incubated for 5 min with 2 ml 0.4% (vol/vol) human RBCs in phosphate-buffered saline (PBS) at RT. After the removal of the unbound RBCs and washing of the RBCs with 2 ml of calcium- and magnesium-free PBS at 4°C, distilled water was added and the mixture was left at RT for 10 min to lyse the hemadsorbed RBCs. The decanted supernatants containing hemoglobin were removed after 5 min at 37°C and were evaluated by spectrophotometry at 410 nm.

Effect of KP treatment on uninfected cells.

Confluent monolayers of uninfected cells either untreated or treated with KP (4 μg/ml) were evaluated for viability by the dye exclusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (33). Furthermore, cells were labeled for 48 h with 10 μCi/ml of [³H]thymidine or [³H]uridine for DNA and RNA synthesis, respectively, and the radioactivity incorporated into material insoluble in trichloroacetic acid was determined by a previously reported procedure (31).

Effect of KP treatment on viral protein synthesis.

Confluent cell monolayers in tiny dishes were infected at an MOI of 2 and treated with KP (4 μg/ml) or diluent (untreated). At different times p.i., the cells were labeled with [³⁵S]methionine (25 μCi/ml) or [¹⁴C]mannose (20 μCi/ml) in methionine- or mannose-free medium with 2% FBS after a 30-min preincubation in those media for a starvation period. After 30 min of labeling, cells from two tiny dishes were washed in 0.9% NaCl, scraped off, pelleted (500 × g, 5 min), and then lysed with Laemmli's lysis buffer (18) before electrophoresis on 15% polyacrylamide gels at 50 V overnight. Virus-induced polypeptides were visualized by autoradiography. Densitometer analysis was performed with an SI personal densitometer, and the data were analyzed with ImageQuant software (Molecular Dynamics, GE Healthcare).

Fractionation of cell homogenates.

Fractionation of cell homogenates was performed by a previously described procedure (17). Briefly, infected, KP-treated (4 μg/ml) or untreated, [³⁵S]methionine-labeled LLC-MK2 cell monolayers were removed at 48 h p.i. and suspended in a buffer containing 0.01 M KCl, 0.02 M Tris (pH 7.5), 0.1% 2-mercaptoethanol, 1 mM MgCl₂, and 1 mM phenylmethylsulfonyl fluoride. After 10 min on ice, the cells were disrupted with 50 strokes in a Dounce homogenizer and centrifuged (1,000 × g, 10 min). The supernatant was then centrifuged again (100,000 × g, 40 min) to fractionate the membrane (pellet) and soluble cytoplasmic (supernatant) components. Both fractions were separately dissolved in radio immunoprecipitation assay (RIPA) buffer containing 0.3 M NaCl, 0.1 M Tris (pH 7.4), 1% Triton X-100, 1% deoxycholate, 0.1% SDS, and 1 mM phenylmethylsulfonyl fluoride. Membrane-containing fractions were immunoprecipitated with a polyclonal antiserum directed to Ulster 73 virus HA by a previously described procedure (35). After 3 h at 4°C, protein A-Sepharose beads (1:1) in 0.1 M sodium phosphate, pH 7.0, were added; and the immune complexes were washed with RIPA buffer, suspended in sample buffer, and analyzed (equal volumes) by 12.5% SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography.

Animals and treatments.

The antiviral activity of KP in vivo was evaluated in a well-established murine model of influenza virus infection (28, 30). Four-week-old BALB/c male mice (average weight, 20 g; Charles River, Calco, Italy) were housed and studied under Institutional Animal Care and Use Committee-approved protocols.

For the determination of the lethal dose, each mouse was infected intranasally (i.n.) with 1, 5, or 10 HAU of influenza A Ulster 73 and NWS 33 strains diluted in 25 μl 0.9% NaCl while the mouse was under light ether anesthesia. For determination of survival rates and pulmonary viral titers, each animal was infected with 5 HAU of strain NWS 33, as described above. Thirty minutes after virus inoculation, randomly divided groups of 10 animals each received intraperitoneal (i.p.) injections of either KP at a dose of 50 μg/mouse (2.5 mg/kg of body weight) or 100 μg/mouse (5.0 mg/kg) once a day or placebo (KP diluent) in 100 μl 0.9% NaCl. Treatments were repeated daily for the next 10 days. Similarly, as a positive control, a group (n = 10) of infected mice was treated i.p. with amantadine (AMN) at a dose of 2 mg/mouse (100 mg/kg) once a day (25). The mice were monitored daily for clinical signs of infection, including body temperature, motor activity, and weight loss. Survival was assessed in all groups for 30 days p.i., and mice that survived to day 30 were considered cured. Uninfected control groups received identical KP and placebo treatments, and animals were observed daily for clinical signs of toxicity (survival, motor activity, weight loss).

Pulmonary viral titers.

Four groups of NWS 33-infected mice (n = 5/group) treated daily with KP (50 or 100 μg/mouse), AMN (2 mg/mouse), or placebo were killed at 6 days p.i. The day of killing was chosen according to data from our preliminary experiments that showed that the virus titers in the lungs of influenza NWS 33 virus-infected animals reached the maximum on day 6 after infection. Similarly, Sidwell and Smee (34) recovered on day 6 NWS virus from the lungs of infected mice treated with oseltamivir or not treated. Each lung was removed, weighed, and homogenized in RPMI 1640 medium; and the viral titer was determined by the 50% tissue culture infective dose (TCID₅₀) assay with MDCK cells. Briefly, confluent cell monolayers in 96-well plates were inoculated with 10-fold dilutions of the homogenate samples (eight wells per dilution) and incubated for 3 days. The number of wells showing a positive cytopathic effect was scored, and the titer (TCID₅₀) per gram of lung tissue was calculated by standard procedures (12).

Statistical analysis.

Each assay was carried out in triplicate, unless otherwise specified. Data are expressed as means ± standard deviations (SDs). The statistical significance of the data was determined by using Student's t test or analysis of variance (significance level, P < 0.05).

Survival curves were compared by the log-rank test, and the rates of mortality in the four groups were compared by using the Cox regression. A P value of <0.05 was considered significant.

RESULTS

Effect of KP on virus production in cell lines.

As evaluated either in the hemagglutination assay or in the plaque assay, treatment with KP of both influenza A virus suspensions before infection did not affect viral production in any of the cell lines. On the contrary, when KP was added p.i. to both virus-host cell systems, it proved to inhibit viral production in a dose- and time-dependent way. The results of the hemagglutination assay are presented for the avian strain in LLC-MK2 host cells, unless otherwise specified.

On the basis of the results of preliminary experiments, an MOI of 2, corresponding to an allantoic fluid dilution of 1:10, was chosen for all the in vitro experiments. Compared to the results for infected untreated control cells, KP was able to completely suppress viral production starting at a concentration of 4 μg/ml when it was added immediately p.i. to the infected cells and maintained in the medium for 24 h (Fig. 1A). The 50% effective concentration was 2.686 × 10⁻⁶ mol/liter (95% confidence interval, 2.516 × 10⁻⁶ to 2.868 × 10⁻⁶). No infective virus particle, moreover, was recovered by the plaque assay in the presence of KP at 4 μg/ml. KP at 4 μg/ml could completely inhibit virus production when it was added within 4 h p.i. and maintained in the medium up to 24 h (Fig. 1B). The inhibitory effect was abolished by the removal of KP within 4 h p.i. (Fig. 1C). The complete inhibition of virus production was also observed at 48 and 72 h in the continuous presence of KP (4 μg/ml). The same result was obtained after replacement of the KP-containing medium with fresh medium devoid of KP at 24 and 48 h.

FIG. 1.

Effect of KP treatment on influenza A/parrot/Ulster/73 (H7N1) virus replication in LLC-MK2 cells. (A) Different concentrations of KP were added to the cell culture medium immediately p.i. and were maintained in the culture medium for 24 h. (B) KP (4 μg/ml) was added to the cell culture medium at different time points (1, 2, 4, 5, 6, 8, 10 h) p.i. and was maintained in the culture medium up to 24 h. (C) KP (4 μg/ml) was added to cell cultures immediately p.i. and removed at different time points (1, 2, 4, 5, 6, 8, 10 h) p.i. The viral yield was quantified by hemagglutination assay. The viral titer in the absence of peptide (100% viral yield) was 1,024 HAU. Each assay was carried out in duplicate. Mean values + standard errors of the means are presented.

The ability of KP to block virus production was also demonstrated by the lack of viral proteins in KP-treated cells but the presence of proteins in the control cells, as observed by 15% SDS-PAGE and autoradiography performed with supernatants from [³⁵S]methionine-labeled infected cells (Fig. 2).

FIG. 2.

Effect of KP treatment on mature viral particles production. Lane 1, pellet of a gradient-purified supernatant from untreated infected LLC-MK2 cells (50 μl); lanes 2, 3, and 4, pellet of a gradient-purified supernatant from KP-treated (4 μg/ml) infected LLC-MK2 cells (100, 50, and 25 μl, respectively). Viral structural proteins PB2, PB1, and PA are RNA-dependent RNA polymerase subunits. HA, hemagglutinin; NP, nucleoprotein; M1, membrane protein 1.

In the hemadsorption assay, KP treatment (4 μg/ml) caused significant decreases in the optical density values in comparison with those for the untreated infected cells for both cell line cultures (the optical densities at 410 nm were 0.110 ± 0.010 for KP-treated LLC-MK2 cells and 0.920 ± 0.039 for untreated cells, and they were 0.201 ± 0.013 for KP-treated MDCK cells and 0.817 ± 0.019 for untreated cells; P < 0.0001).

No significant effect of KP treatment (4 μg/ml) on either cell viability or DNA and RNA synthesis in uninfected cells was observed (data not shown).

Effect of KP on viral protein synthesis.

As shown in Fig. 3, which provides results representative of the similar results obtained in repeated assays, the time course of viral protein synthesis in KP-treated infected LLC-MK2 cells appeared to be quite different from that in the untreated infected cells. Whereas viral nucleoprotein (NP), HA, and membrane protein (M1)/nonstructural protein 1 (NS1) were effectively synthesized in untreated cells, KP treatment caused a marked reduction in their synthesis, in particular, the synthesis of M1 and HA (Fig. 3A; Table 1). Glycosylated viral (HA and NA) proteins (in particular, HA) were also reduced in the presence of KP (Fig. 3B; Table 2).

FIG. 3.

Effect of KP treatment on the synthesis of viral proteins in LLC-MK2 cell monolayers. (A) SDS-PAGE of cells pulse labeled with [³⁵S]-methionine: (left) untreated; (right) KP treated (4 μg/ml). Lanes 1 and 5, uninfected cells; lanes 2 and 6, infected cells at 2 h p.i.; lanes 3 and 7, infected cells at 6 h p.i.; lanes 4 and 8, infected cells at 8 h p.i. (B) SDS-PAGE of cells pulse labeled with [¹⁴C]mannose; Lane 1, infected KP-treated (4 μg/ml) cells at 4 h p.i.; lane 4, infected KP-treated (4 μg/ml) cells at 6 h p.i.; lane 2, infected untreated cells at 4 h p.i.; lane 3, infected untreated cells at 6 h p.i.; lane 5, infected untreated cells at 6 h p.i. pulse labeled with [³⁵S]methionine. HA, hemagglutinin; NP, nucleoprotein; M1, membrane protein 1; NS1, nonstructural protein 1.

TABLE 1.

Densitometer analysis of viral proteins synthesized in KP-treated infected LLC-MK2 cells

Viral protein	Densitometer values and % decrease for KP-treated (4 μg/ml) vs untreated cellsa at:
	2 h p.i.			6 h p.i.			8 h p.i.
	Densitometer value		% Decrease	Densitometer value		% Decrease	Densitometer value		% Decrease
	Without KP	With KP		Without KP	With KP		Without KP	With KP
HA	29.55	12.08	59.12	475.12	52.53	88.94	654.75	188.61	71.19
NP	355.6	13.06	96.33	1,497.43	346.49	76.86	1,885.12	1,007.54	46.55
M1	5.29	—b	—	351.2	—	—	570.36	36.38	93.62
NS1	231.19	—	—	1,115.82	155.27	86.08	1,087.54	796.66	26.75

^aQuantitative data from bands shown in Fig. 3A.

^b—, undeterminable.

TABLE 2.

Densitometer analysis of viral proteins synthesized in KP-treated infected LLC-MK2 cells

Viral glycoproteinb	Densitometer values and % decrease for KP-treated (4 μg/ml) vs untreated cellsa at:
	4 h p.i.			6 h p.i.
	Densitometer value		% Decrease	Densitometer value		% Decrease
	Without KP	With KP		Without KP	With KP
HA	121.05	45.46	62.44	112.37	39.13	65.18
NA	133.04	73.02	45.11	101.38	51.00	49.69

^aQuantitative data from bands shown in Fig. 3B.

^bHA, hemagglutinin; NA, neuraminidase.

Analysis by SDS-PAGE and autoradiography of membrane-containing fractions demonstrated the complete lack of immunoprecipitated HA from KP-treated infected cells (Fig. 4).

FIG. 4.

Effect of KP treatment on the occurrence of HA in membranes of infected LLC-MK2 cells. Immunoprecipitated membrane-containing fractions were from infected KP-treated (4 μg/ml) (lane 1) or untreated (lane 2) cells. Lane 3, infected untreated cells (8 h p.i.) used as a control for viral proteins. PB2, PB1, and PA are RNA-dependent RNA polymerase subunits. HA, hemagglutinin; NP, nucleoprotein; M1 and M2, membrane proteins 1 and 2; NS1 and NS2, nonstructural proteins 1 and 2.

Antiviral activity of KP in vivo.

In preliminary experiments, different doses (1, 5, or 10 HAU/mouse) of Ulster 73 and NWS 33 influenza virus strains were administered i.n. to determine their lethal doses. Due to the lack of pathogenicity of the avian strain (Ulster 73) for mice, in further experiments the animals were infected i.n. with 5 HAU/mouse of NWS 33, a dose that causes 90% mortality within 10 days. Under these experimental conditions, the peak viral titer in the lung was found at 6 days p.i.

Figure 5 shows the survival curves for animals treated with KP (50 or 100 μg/mouse), AMN (2 mg/mouse), or placebo. While 90% of the infected mice treated with placebo were dead by day 8 p.i., 50% of the animals treated with 100 μg KP survived (P = 0.02431 versus the results for placebo-infected animals). In the control group of animals treated with AMN, the rate of survival was increased by 50% compared with that for placebo-treated mice (P = 0.02011). No significant difference between KP (100 μg) and AMN treatments was detectable. Four different experiments, each performed with 10 mice per group, gave similar results (variability, <10%). None of the mice that survived to day 10 p.i. showed any signs of disease for the following 30 days and were considered cured. Moreover, in order to compare the rates of mortality between the four groups, the mortality rate ratios were calculated by using proportional hazards regression. The results indicated that the mortality rate for mice treated with KP (100 μg) was reduced by 72% compared with that for mice treated with the placebo (mortality rate for mice treated with KP at 100 μg/mortality rate for mice treated with placebo, 0.27529; 95% CI, 0.0888 to 0.8545), and this was statistically significant (P = 0.0258). Similarly, the mortality rate for mice treated with AMN was reduced by 77% compared with that for mice treated with placebo (mortality rate for mice treated with AMN/mortality rate mice treated with placebo, 0.22684; 95% CI, 0.0674 to 0.7638; P = 0.0166).

FIG. 5.

Effect of KP treatment on survival of influenza A virus-infected mice. BALB/c mice (n = 10/group) were infected i.n. with 5 HAU/mouse of NWS 33 strain; and KP (50 or 100 μg/mouse/once a day), AMN (2 mg/mouse/once a day), or placebo was administered i.p. 30 min after virus inoculation and daily for the next 10 days. The results are expressed as the percent survival evaluated daily for 30 days. Survival curves were compared by using the log-rank test. The results of one representative experiment of four experiments performed are shown.

No significant differences in the survival and the mortality rates were found between the animals treated with 50 μg KP and the animals treated with placebo.

Then, to evaluate whether the increased survival was associated with decreased pulmonary viral titers, the TCID₅₀s for other groups of mice were determined at 6 days p.i. The average of the results obtained with five separate homogenates of mice treated with 100 μg KP showed an approximately 2-log reduction in the titer in the lung compared with that obtained with five separate homogenates of the placebo-treated mice (mean ± SD, 3.6 × 10³ ± 6 × 10² and 2.2 × 10⁵ ± 4 × 10³ units/gram of lung tissue, respectively; P < 0.01 versus the results for the placebo-treated mice). Moreover, a significant reduction in the titer in the lungs was observed with five separate homogenates from mice treated with 50 μg KP (mean ± SD, 2.4 × 10⁴ ± 1.9 × 10³; P < 0.01 versus the results for the placebo-treated mice). As a further control, pulmonary viral titers were determined in five separate homogenates of AMN-treated mice (mean ± SD, 3.4 × 10³ ± 1 × 10³; P = 0.001 versus the results for the placebo-treated mice).

The treatment with KP at up to 100 μg/mouse seemed to be nontoxic for the animals, as determined by no deaths and a lack of differences in the overall health status, evaluated as motor activity and weight loss, between uninfected KP-treated mice and placebo-treated animals (data not shown).

DISCUSSION

This paper reports on the in vitro and in vivo activities of KP against influenza A viruses. The results indicate that KP was able to completely inhibit in vitro the production of an avian influenza A virus strain and a human neurotropic mouse-adapted avian influenza A virus strain and to exert a therapeutic effect in a mouse model of infection. Preliminary studies have suggested that another viral strain, A/ISS PR/6/07 (H3N2), obtained from the Istituto Superiore di Sanità, Rome, Italy, a clinical isolate characterized by a mutation in the M2 gene that leads to a corresponding amino acid substitution at position 31 in the M2 protein associated with AMN resistance, is also susceptible to KP (data not shown). Furthermore, by using a recently described rapid test based on the inhibition of viral cytopathogenicity (8), KP was also shown to exert inhibitory activity on the replication of a H7N3 avian virus in vitro (A. Cassone and A. Savarino, personal communication).

Even if the precise molecular mechanism by which KP can interfere with viral multiplication remains to be determined, our results suggest that adsorption and fusion are not affected, as reported from studies with monoclonal Ab HC63 (2, 37). According to the experimental conditions adopted, in the presence of KP, appreciable reductions in the levels of viral proteins, including glycosylated forms, have been observed. In particular, M1 and HA synthesis in the late phase of viral multiplication were mainly affected. These findings support the hypothesis that the marked reduction of M1 synthesis may be the major cause of the lack of production of mature viral particles, owing to the fundamental role played by this protein (26). On the basis of the immunoprecipitation and hemadsorption assay results, however, the possibility for the concomitant involvement of HA may not be excluded due to its incorrect or defective association and/or insertion into the plasma membrane.

The in vitro antiviral effects of KP were mirrored in a murine model of influenza. Treatment of strain NWS 33-infected mice markedly improved their survival, decreased the pulmonary virus titers, and caused no significant toxicity. The curative effect of KP was comparable to that of a recognized anti-influenza A virus compound, such as AMN. These findings are consistent with those of previous in vivo studies (5, 29, 36).

Notably, in further experiments that have not been reported in detail, KP-treated animals that survived a first sublethal challenge and that were then rechallenged with the lethal inoculum of the same influenza virus strain showed 50% survival. The same percentage of survival was observed in infected KP-untreated control mice (data not shown). Thus, KP treatment had no apparent effect on host resistance to a second viral infection.

Despite the purported immune modulatory activity, KP treatment did not affect the production of specific Abs, as demonstrated by the results of a hemagglutination inhibition test with mice immunized with UV-treated NWS 33 virus (unpublished data).

Taking into consideration the increasing appearance of influenza virus strains resistant to the existing classes of antiviral drugs and the risk of a pandemic outbreak caused by strains of avian origin, new effective molecules may be useful adjuncts to influenza vaccination, the main preventive measure used to combat novel epidemic or pandemic strains (1, 16, 19).

Antimicrobial and antiviral synthetic and natural peptides have been widely described and have been considered experimental tools, until enfuvirtide, a biomimetic synthetic peptide, was approved by FDA and the European Union as the first HIV-1 entry and fusion inhibitor for treatment-experienced patients, thus demonstrating the potential of the development of peptides into effective antiviral drugs (24).

KP appears to be the first Ab-derived peptide essentially devoid of antigenicity (L. Polonelli, unpublished observations) and toxicity that displays inhibitory activities and therapeutic effects against different pathogenic microorganisms and viruses by distinct mechanisms of action (4, 23). Thus, KP may have the potential for use for the structure-based design of a new class of broad-spectrum antimicrobial and antiviral drugs for the treatment of infectious diseases, including those caused by influenza A viruses.