Amino Acid Substitutions Improve the Immunogenicity of H7N7HA Protein and Protect Mice against Lethal H7N7 Viral Challenge.

Avian influenza A H7N7/NL/219/03 virus creates a serious pandemic threat to human health because it can transmit directly from domestic poultry to humans and from human to human. Our previous vaccine study reported that mice when immunized intranasally (i.n) with live Bac-HA were protected from lethal H7N7/NL/219/03 challenge, whereas incomplete protection was obtained when administered subcutaneously (s.c) due to the fact that H7N7 is a poor inducer of neutralizing antibodies. Interestingly, our recent vaccine studies reported that mice when vaccinated subcutaneously with Bac-HA (H7N9) was protected against both H7N9 (A/Sh2/2013) and H7N7 virus challenge. HA1 region of both H7N7 and H7N9 viruses are differ at 15 amino acid positions. Among those, we selected three amino acid positions (T143, T198 and I211) in HA1 region of H7N7. These amino acids are located within or near the receptor binding site. Following the selection, we substituted the amino acid at these three positions with amino acids found on H7N9HA wild-type. In this study, we evaluate the impact of amino acid substitutions in the H7N7 HA-protein on the immunogenicity. We generated six mutant constructs from wild-type influenza H7N7HA cDNA by site directed mutagenesis, and individually expressed mutant HA protein on the surface of baculovirus (Bac-HAm) and compared their protective efficacy of the vaccines with Bac-H7N7HA wild-type (Bac-HA) by lethal H7N7 viral challenge in a mouse model. We found that mice immunized subcutaneously with Bac-HAm constructs T143A or T198A-I211V or I211V-T143A serum showed significantly higher hemagglutination inhibition and neutralization titer against H7N7 and H7N9 viruses when compared to Bac-HA vaccinated mice groups. We also observed low level of lung viral titer, negligible weight loss and complete protection against lethal H7N7 viral challenge. Our results indicated that amino acid substitution at position 143 or 211 improve immunogenicity of H7N7HA vaccine against H7N7/NL/219/03 virus

Confirmation of baculovirus displayed H7N7HA and H7N7HA mutant in insects cells by immunofluorescence assay.

<p>The Sf9 cells were infected with Bac-HA_m constructs; a) T143A or b) T198A, or c) I211V or d) T143A-T198A or e) T198A-I211V or f) I211V-T143A or g) Bac-HA or h) wild-type baculovirus (wt-Bac), or i) Control Sf9 cells. 48 h post infection the cells were fixed and analyzed by mAb specific to H7N7 influenza virus.</p

Predicted T cell and B cell epitopes in HA molecules of H7N7, H7N9 influenza viruses.

<p>The differences between HA amino acid sequence of H7N9 and H7N7 influenza have been underlined.</p><p>Predicted T cell and B cell epitopes in HA molecules of H7N7, H7N9 influenza viruses.</p

Histopathological changes in lung sections of mice challenged with a lethal dose of H7N7 virus at day 8 after subcutaneous or intranasal immunizations.

<p>(A) Intranasal immunization with live Bac-HA. The mice had minimal or no lesions in the examined lung section. (B)–(F) Subcutaneous immunizations with live Bac-HA (B); inactivated IV+Adj (C); inactivated Bac-HA+Adj (D); live Bac-wt (E); PBS (F). (B)–(D) Mice present with moderate alveolar collapse throughout the lung tissue. (E)–(F) Both groups display severe alveolar collapse throughout the lungs tissue.</p

Western blot analysis of baculovirus displayed H7N7HA and H7N7HA mutant protein.

<p>All the samples were reduced with loading dye containing β-mercaptoethanol reducing agent. Lane 1: H7N7HA wild-type; Lane 2:T143A; Lane 3:T198A; Lane 4:I211V; Lane 5:T143A-T198A; Lane 6:T198A-I211V; Lane 7: I211V-T143A; Lane 8, wt-Bac.</p

Measurement of systemic and mucosal immune responses of mice sera obtained from intranasal immunizations on days 14 and 42.

<p>Groups of mice (n = 10) were intranasally immunized with live Bac-HA, inactivated IV, Bac-wt, inactivated Bac-HA or PBS controls on day 0 and 28. A constant dosage of 100 μl of 2⁸ HA titer of Bac-HA or inactivated IV suspended in PBS was administrated. The sera were used to determine the HA-specific IgG antibody titer by indirect ELISA (A), the serum hemagglutination inhibition (HI) titer (B), and the HA-specific IgA titer by indirect ELISA (C). Each point represents the arithmetic mean value (n = 5) ± SD. **, P<0.001; ***, P<0.0001.</p

Microneutralization titers of vaccinated mouse sera against 100TCID₅₀ of RG-H7N7 and RG-H7N9 viruses.

<p>Groups of mice (n = 10) were subcutaneously immunized with a constant dosage of 100 μl containing 2.5 μg of live Bac-HA, or Bac-HA_m constructs T143A or T198A or I211V or T143A-T198A or T198A-I211V or I211V-T143A or wild- type baculovirus (PFU 1X10⁸), controls on day 0 and 28. Serum samples were collected on day 42 from each experimental mice group (Five mice/Group) for measuring the serum neutralization antibody titers against H7N7 (A/Netherlands/219/03) or H7N9 (A/Shanghai/2/2013) viruses. Neutralizing titers are arithmetic mean of the highest dilution of serum which yielded a 50% reduction in virus infectivity. a-when compared with wild-type H7N7 Bac-HA vaccine group. Each point represents the arithmetic mean value (n = 5) ± SD (**<i>P</i><0.001).</p

Protection of mice from lethal H7N7 influenza virus challenge.

<p>Mice (n = 10/groups) were immunized subcutaneously on day 0 and 28. All groups were challenged intranasally with 5 MLD₅₀ of mouse-adapted H7N7 virus on day 49. Mice were monitored for survival for 14 days and the results were expressed in percent survival (5A). Weight loss of the mice groups was also monitored throughout the 14 day observation period and the results were expressed in percent body weight compared to the beginning of the trial (5B). Each point represents the arithmetic mean (n = 6) value ± SD (***<i>P</i><0.0001, when compared to Bac-HA vaccinated mice group); # represents ≥33.3 survival in the group.</p

H7N7 influenza viral titers in lungs at 8 day post challenge following subcutaneous (A) or intranasal (B) immunization.

<p>Viral titers were measured by quantitative real time RT-PCR using influenza virus M2 gene primers. The results were expressed in terms of log TCID50/g ± SD.</p

Protection of mice from lethal H7N7 influenza virus challenge.

<p><b>Mice (n = 6/groups) were immunized intranasally on day 0 and 28.</b> All groups were challenged intranasally with 5 MLD50 of mouse-adapted H7N7 virus on day 49. Mice were monitored for survival for 16 days and the results were expressed in percent survival (A). Weight loss of the mice groups was also monitored throughout the 16 day observation period and the results were expressed in percent body weight compared to the beginning of the trial (B).</p