Heterosubtypic anti-avian H5N1 influenza antibodies in intravenous immunoglobulins from globally separate populations protect against H5N1 infection in cell culture

Abstract

With antigenically novel epidemic and pandemic influenza strains persistently on the horizon it is of fundamental importance that we understand whether heterosubtypic antibodies gained from exposures to circulating human influenzas exist and can protect against emerging novel strains. Our studies of IVIG obtained from an infection-naive population (Australian) enabled us to reveal heterosubtypic influenza antibodies that cross react with H5N1. We now expand those findings for an Australian donor population to include IVIG formulations from a variety of northern hemisphere populations. Examination of IVIGs from European and South East-Asian (Malaysian) blood donor populations further reveal heterosubtypic antibodies to H5N1 in humans from different global regions. Importantly these protect against highly pathogenic avian H5N1 infection in vitro, albeit at low titres of inhibition. Although there were qualitative and quantitative differences in binding and protection between globally different formulations, the heterosubtypic antibody activities for the respective IVIGs were in general quite similar. Of particular note because of the relative geographic proximity to the epicentre of H5N1 and the majority of human infections, was the similarity in the antibody binding responses between IVIGs from the Malayan peninsula, Europe and Australia. These findings highlight the value of employing IVIGs for the study of herd immunity, and particularly heterosubtypic antibody responses to viral antigens such as those conserved between circulating human influenzas and emerging influenza strains such as H5N1. They also open a window into a somewhat ill defined arena of antibody immunity, namely heterosubtypic immunity.

Keywords: H1N1; H3N2; H5N1; Heterosubtypic; IVIG; antibody; influenza.

Figure 1.

2D gel electrophoretic profile of a representative IVIG preparation, with IPG isoelectric fractionation over 4-7 pI range. The separation of protein molecular weight standard markers in the acrylamide separation phase is shown on the left hand side of the 2D-separated immunoglobulin fractions. Note, the respective IVIG formulations used in this study (Table 1) have been de-identified, but each assigned a label A-G. The formulation used for 2D analysis was product D.

Figure 2.

Anti-human influenza binding antibodies in IVIGs: Assays for detection of Ab binding to conformational/ discontinuous influenza epitopes. A. Multiplex assay for anti-influenza IVIG Ab binding showing a comparison of the antibody binding activities of 4 different batches of a single IVIG formulation. IVIG antibody binding to Influenza A: H3N2 A/Shandong/9/1993 and Influenza B: B/Hong Kong/5/1972 antigens displayed on microbeads and performed at the Rules Based Medicine biomarker testing laboratory using multianalyte bead Fluorescence (www.rulesbasedmedicine.com). Binding to herpes simplex virus (HSV), human pappilloma virus (HPV), Influenza B (Infl B) respiratory syncytial virus (RSV) and varicella zoster (V. Zoster) are also included for comparison. The Y-axis values show the amount of IVIG antibody that binds to the respective antigens coated on beads and expressed as the mean ratio of total antibody binding to the respective viral antigens compared to highly-validated RBM internal-reference control standards. B. Microarray slides spotted with viral dilutions (1-0.125mg/ml) of H3N2 (A/Sydney/5/1997), H1N1 (A/Johannesburg/82/1996), tetanus toxoid (Tet Tox) or BSA. IVIG-antibody (D-3) bind robotically arrayed whole inactivated influenza and tetanus toxoid. The BSA is negative. Spots are 10nl and 0.8mm apart. [Note: antigens were bound in duplicate arrays with (top array panel of each slide) and without (bottom array panel on each slide) 0.005% CHAPS. CHAPS was tested as a means of improving liquid flow rates in the Piezorray, but as shown, had no influence on protein binding to the array substrate.]

Figure 3.

SDS-PAGE gel separations of influenza proteins showing coomassie-stained protein signatures of purified preparations of the human H1N1 A/Johannesbeg/82/1996 (β), and H3N2 strains A/Beijing/ 32/1992 (γ) and A/Sydney/5/97 (δ). The viruses used have been assigned a greek letters (Table 2). Relative molecular weights (Privalsky and Penhoet, 1978; Lynch et al, 2008) and approximate copies per virion (Lamb and Krug, 2001) for each of the influenza proteins are listed in the left panel.

Figure 4.

IVIG mmunoblotting profiles of human H1N1, H3N2 and avian H5N1 influenza proteins that bind human antibodies. Purified influenza samples were applied to 5-15% (w/v) SDS-PAGE gels and immunoblotted with the European (Sando-globulin-Eur, Octagam-Eur), Australian (Intragam P-Aust) or Malaysian (Intragam P-Malay) IVIGs. The Greek alphabets denote IVIG products, while the numbers A-2, B-1, C-1 and D-1 represent the particular batch of the product. The H5N1 virus (i.e., purified A/Chicken/Vietnam /8/2004 and identified as H5), is compared with the human H1N1 strains A/Texas/36/1991 (α) and A/Johannesbeg /82/1996 (β), and the H3N2 strains A/Beijing/32/1992 (γ) and A/Sydney/5/97 (δ).

Figure 5.

Identification of Heterotypicheterosubtypic IVIG antibody binding to the proteins of avian viruses by immunoblotting. IVIG antibody binding H5N1 protein signatures have been observed for Intragam P-Aust, Sandoglobulin-Eur, and Octagam-Eur IVIGs, or Intragam P-Malay. Two different IVIG formulations reactive to the proteins of H5N1 (A/Chicken/ Vietnam/8/2004), H7N7 (A/Chicken/Victoria/1985) and H9N2 (A/Duck/Mellacca /2003) avian strains are shown, and for comparison a human H1N1 (A/Johannesburg/82/1996) is shown at a 1/10 protein loading compared to the avian viruses.