Monoclonal antibody against H1N1 influenza virus hemagglutinin cross reacts with hnRNPA1 and hnRNPA2/B1

Abstract

Following influenza A vaccination, certain individuals exhibit adverse reactions in the nervous system, which causes a problem with the safety of the influenza A vaccine. However, to the best of our knowledge, the underlying mechanism of this is unknown. The present study revealed that a monoclonal antibody (H1‑84mAb) against the H1N1 influenza virus hemagglutinin (HA) protein cross‑reacted with an antigen from brain tissue. Total brain tissue protein was immunoprecipitated with this cross‑reactive antibody, and mass spectrometry revealed that the bound antigens were heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and hnRNPA2/B1. Subsequently, the two proteins were expressed in bacteria and it was demonstrated that H1‑84mAb bound to hnRNPA1 and hnRNPA2/B1. These two proteins were expressed in three segments and the cross‑reactivity of H1‑84mAb with the glycine (Gly)‑rich domains of hnRNPA1 (195aa‑320aa) and hnRNPA2/B1 (202aa‑349aa) was determined using ELISA blocking experiments. It was concluded that the Gly‑rich domains of these two proteins are heterophilic antigens that cross‑react with influenza virus HA. The association between the heterophilic antigen Gly‑rich domains and the safety of influenza A vaccines remains to be investigated.

Figure 1.

Identification of H1-84mAb combined with rat brain tissue. (A) SP2/0 as a negative control did not respond to brain tissue cells. Magnification, ×400. (B) H1-84mAb was bound to rat brain tissue and parts of the cerebral cell nucleus were stained brownish yellow. Magnification, ×400. (C) H1-84mAb reacted with the protein components of brain tissue, and the molecular weight was ~35 kDa (lane 2); SP2/0 was used as negative control (lane 3).

Figure 2.

Immunoprecipitation and identification of hnRNPA2/B1 and hnRNPA1 protein expressed in bacteria. (A) Western blot analysis of brain tissue protein following immunoprecipitation. SP2/0 (lane 1) and H1-84 (lane 2) were used as the primary antibodies. The red arrow indicated specific banding, which was performed for mass spectrometry. Lane 3 and lane 4 show that the target antigen, which specifically reacted with H1-84mAb, was ~35 kDa. (B) Coomassie-stained gels revealed that hnRNPA2/B1 and hnRNPA1 expressed in bacteria were ~50 kDa (SUMO tag size was 15 kDa). (C) Western blot analysis demonstrated that H1-84mAb bound to the recombinant protein hnRNPA2/B1 and hnRNPA1.

Figure 3.

Fine localization of H1-84mAb bound to the hnRNPA1. (A) Antigenic analysis of hnRNPA1. (B) hnRNPA1 was expressed in stages, which were NO.1 (14–97aa), NO.2 (105-184aa), and NO.3 (195-320aa). All three proteins were correctly expressed. (C) Western blot analysis revealed that H1-84mAb bound to the NO.3 of the hnRNPA1. NO.1, stands for RRM1 domain; NO.2, stands for RRM2 domain; NO.3, stands for Gly-rich domain.

Figure 4.

Fine localization of H1-84mAb bound to the hnRNPA2/B1. (A) Antigenic analysis of hnRNPA2/B1. (B) hnRNPA2/B1 was expressed in stages, which were NO.1 (21-111aa), NO.2 (112-191aa) and NO.3 (202-349aa). All three proteins were correctly expressed. (C) Western blot analysis revealed that H1-84mAb bound to the NO.3 of the hnRNPA2/B1.

Figure 5.

Verification of H1-84mAb combined with brain tissue antigen. (A) Protein purification sample. (B) Separate samples of brain tissue proteins, ensuring the same amount of sample for each well. Pre-incubation of H1-84mAb with 0, 0.2 and 0.6 µg hnRNPA2/B1-NO.3 and hnRNPA1-NO.3, respectively. Each incubated sample was different and required separate western blot analysis with brain tissue proteins. The results showed that the NO.3 segments of these proteins were the fine localization of H1-84mAb on the brain tissue. β-actin was used as a loading control. *P<0.05 vs. 0 µg group.