Development of dual-function ELISA for effective antigen and antibody detection against H7 avian influenza virus

Abstract Background Outbreaks in poultry involving influenza virus from H7 subtype have resulted in human infections, thus causing a major concern for public health, as well as for the poultry industry. Currently, no efficient rapid test is available for large-scale detection of either antigen or antibody of H7 avian influenza viruses. Results In the present study, a dual function ELISA was developed for the effective detection of antigen and antibody against H7 AIVs. The test was established based on antigen-capture-ELISA and epitope blocking ELISA. The two Mabs 62 and 98 which were exploited in the assay were identified to recognize two conformational neutralizing epitopes on H7 HA1. Both of the epitopes exist in all of the human H7 strains, including the recent H7N9 strain from China and &gt; 96.6% of avian H7 strains. The dual ELISA was able to detect all of the five H7 antigens tested without any cross reaction to other influenza subtypes. The antigen detection limit was less than 1 HA unit of H7. For antibody detection, the sensitivity and specificity of the dual ELISA was evaluated and compared to HI and microneutralization using immunized animal sera to different H7 strains and different subtypes of AIVs. Results indicated that antibodies to H7 were readily detected in immunized animal sera by the dual ELISA whereas specimens with antibodies to other AIVs yielded negative results. Conclusions This is the first dual-function ELISA reported for either antigen or antibody detection against H7 AIVs. The assay was highly sensitive and 100% specific in both functions rendering it effective for H7 diagnosis. </jats:sec

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

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

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

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

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

Confirmation and characterization of baculovirus surface displayed HA (Bac-HA) of H7N7 influenza virus by (A) immunofluorescence assay and (B) Western blot.

<p>(A) The Sf9 cells were infected with baculovirus expressed HA from H7N7 (Bac-HA) or wild type baculovirus (Bac-wt) at a MOI of 0.5. 48 h post infection the cells were fixed and analyzed by mAb specific to H7N7 influenza virus to determine the structural and antigenic confirmation of baculovirus expressed HA. (B) Western blot analysis of Bac-HA showing the cleavage of HA using anti-mouse HA polyclonal antibody. Lane M: broad range protein molecular weight marker; Lane 1: Bac-HA infected supernatant; Lane 2: H7N7 reassortant influenza virus, Lane 3: Bac-wt infected supernatant.</p