Mimotopes selected with neutralizing antibodies against multiple subtypes of influenza A

Abstract

Background: The mimotopes of viruses are considered as the good targets for vaccine design. We prepared mimotopes against multiple subtypes of influenza A and evaluate their immune responses in flu virus challenged Balb/c mice.

Methods: The mimotopes of influenza A including pandemic H1N1, H3N2, H2N2 and H1N1 swine-origin influenza virus were screened by peptide phage display libraries, respectively. These mimotopes were engineered in one protein as multi- epitopes in Escherichia coli (E. coli) and purified. Balb/c mice were immunized using the multi-mimotopes protein and specific antibody responses were analyzed using hemagglutination inhibition (HI) assay and enzyme-linked immunosorbent assay (ELISA). The lung inflammation level was evaluated by hematoxylin and eosin (HE).

Results: Linear heptopeptide and dodecapeptide mimotopes were obtained for these influenza virus. The recombinant multi-mimotopes protein was a 73 kDa fusion protein. Comparing immunized infected groups with unimmunized infected subsets, significant differences were observed in the body weight loss and survival rate. The antiserum contained higher HI Ab titer against H1N1 virus and the lung inflammation level were significantly decreased in immunized infected groups.

Conclusions: Phage-displayed mimotopes against multiple subtypes of influenza A were accessible to the mouse immune system and triggered a humoral response to above virus.

Figure 1

The ELISA results of top 12 phage clones with higher C179/BSA ELISA signal ratio from Ph.D. -7, Ph.D. -12 and Ph.D. -C7C peptide phage-display libraries. a: Phage clones from Ph.D. -7 peptide phage-display library. b: Phage clones from Ph.D. -12 peptide phage-display library; c: Phage clones from Ph.D. -C7C peptide phage-display library.

Figure 2

The purification of recombinant multi-mimotope of influenza A virus. a: The multi-mimotope was expressed in soluble form in bacteria and purified with affinity chromatography (lane 1: protein marker: 116.0, 66.2, 45.0, 35.0, 25.0, 18.4, 14.4KDa; lane 2: multi-mimotope gene was transferred to bacteria and induced with IPTG; lane 3: supernatant of ultrasound-broken bacteria; lane 4: pellete of ultrasound-broken bacteria; lane 5: flow-through of supernatant loaded on Ni²⁺-NTA-resin; lane 6: first 0.25 ml elution from 0.25 ml Ni²⁺-NTA-resin with 60 mM imidazole; lane 7: second 0.25 ml elution from Ni²⁺-NTA-resin with 60 mM imidazole; lane 8-9: elution from Ni²⁺-NTA-resin with 1 M imidazole). b: The optimized concentration gradient between 5 and 60 mM imidazole for affinity chromatography (lane 1: protein marker: 97.4, 66.4, 43.0KDa; lane 2-3: elution from Ni²⁺-NTA-resin with 5 mM imidazole; lane 4-5: elution from Ni²⁺-NTA-resin with 10 mM imidazole; lane 6-7: elution from Ni²⁺-NTA-resin with 30 mM imidazole; lane 8-9: elution from Ni²⁺-NTA-resin with 60 mM imidazole). c: The multi-mimotope was purified with repeat affinity chromatography (lane 1: protein marker: 116.0, 66.2, 45.0, 35.0, 25.0, 18.4, 14.4KDa; lane 2: supernatant of ultrasound-broken bacteria; lane 3: multi-mimotope purified by repeat affinity chromatography).

Figure 3

The in vitro binding activities of recombinant multi-mimotope.

Figure 4

The prophylactic effect of multi-mimotope agaist influenza virus challenge. a: The survival rate of mice after virus challenge following multi-mimotope vaccination. b: The body weight loss of mice after virus challenge following multi-mimotope vaccination.

Figure 5

Serum IgG Ab titers were detected by ELISA. Nine mice were immunised with 200 μg multi-mimotope. The mice were bled once (unimmunized serum), and were bled every 10 days after the booster immunization (immunized serum). Ten days after the booster injection, mice were challenged with H1N1 virus by intranasal inoculation of 50 μl per mouse. Two weeks after challenge, mice were sacrificed for the anti-H1N1 IgG Ab detection by ELISA (immunized infected serum). Triplicate ELISAs were performed to test each serum sample. All assays were carried out in triplicate and the error bars indicate standard deviation.

Figure 6

The lung tissue pathological changes. A: the unimmunized infected group (×200). The lung tissue pathological changes from the unimmunized infected groups died on day 6. Necrotizing interstitial pneumonia was found in all cases of death. The pulmonary tissue exhibited hyperemia,hemorrhage,and inflammatory exudation. B: the 200 μg multi-mimotope immunized infected group (×200). The lung tissue pathological changes from the 200 μg multi-mimotopes immunized infected group on day 14. Showing the lung inflammation level were significantly decreased compared with those of their matched unimmunized infected group.