Abstract
Human influenza A viruses have been the cause of enormous socio-economic losses worldwide. In order to combat such a notorious pathogen, hemagglutinin protein (HA) has been a preferred target for generation of neutralizing-antibodies as potent therapeutic/diagnostic agents. In the present study, recombinant anti-HA single chain variable fragment antibodies were constructed using the phage-display technology to aid in diagnosis and treatment of human influenza A virus infections. Spleen cells of mice hyper-immunized with A/New Caledonia/20/99 (H1N1) virus were used as the source for recombinant antibody (rAb) production. The antigen-binding phages were quantified after six rounds of bio-panning against A/New Caledonia/20/99 (H1N1), A/California/07/2009 (H1N1)-like, or A/Udorn/307/72(H3N2) viruses. The maximum phage yield was for the A/New Caledonia/20/99 (H1N1), however, considerable cross-reactivity was observed for the other virus strains as well. The HA-specific polyclonal rAb preparation was subjected to selection of single clones for identification of high reactive relatively conserved epitopes. The high-affinity rAbs were tested against certain known conserved HA epitopes by peptide ELISA. Three recombinant mAbs showed reactivity with both the H1N1 strains and one (C5) showed binding with all the three viral strains. The C5 antibody was thus used for development of an ELISA test for diagnosis of influenza virus infection. Based on the sample size in the current analysis, the ELISA test demonstrated 83.9% sensitivity and 100% specificity. Thus, the ELISA, developed in our study, may prove as a cheaper alternative to the presently used real time RT–PCR test for detection of human influenza A viruses in clinical specimens, which will be beneficial, especially in the developing countries.
Highlights
Flu is a respiratory illness, caused by influenza virus, with annual global attack rate of 5–10% in adults and 20–30% in children, causing significant levels of illness, hospitalization, and death [1]
We developed recombinant single chain variable fragment antibodies against the hemagglutinin protein (HA) antigen of A/New Caledonea/20/99 (H1N1) virus
A relatively high immune response was observed after booster doses of pandemic influenza A H1N1 (2009) virus as confirmed by Enzyme Linked Immunosorbent Assay (ELISA) with immunized mice sera
Summary
Flu is a respiratory illness, caused by influenza virus, with annual global attack rate of 5–10% in adults and 20–30% in children, causing significant levels of illness, hospitalization, and death [1]. Recombinant phage-scFv for flu diagnosis viruses to undergo antigenic shifts (major) and antigenic drifts (minor), which cause changes in the surface glycoproteins, HA and NA, and permit the virus to evade the adaptive immune response of the host cells. Such phenomena back the threat posed by influenza viruses for occurrence of frequent epidemics and pandemics [3], in spite of the available antivirals for treatment [4] or the vaccines for prevention and control of the disease [5]. Antibodies play important role in the course of natural protection against influenza virus infections; the HA antigen being the major target [7, 8]
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