IgA Tetramerization Improves Target Breadth but Not Peak Potency of Functionality of Anti-influenza Virus Broadly Neutralizing Antibody



Shinji Saito1,2, Kaori Sano1,3, Tadaki Suzuki1, Akira Ainai1, Yuki Taga4, Tomonori Ueno4, Koshiro Tabata1, Kumpei Saito1, Yuji Wada1, Yuki Ohara1, Haruko Takeyama5, Takato Odagiri2, Tsutomu Kageyama2, Kiyoko Ogawa-Goto4, Pretty Multihartina6, Vivi Setiawaty6, Krisna Nur Andriana Pangesti6, and Hideki Hasegawa1,3


  1. Department of Pathology, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan,
  2. Influenza Virus Research Center, National Institute of Infectious Diseases, Musashimurayama, Tokyo, Japan
  3. Division of Infectious Diseases Pathology, Department of Global Infectious Diseases, Tohoku Graduate School of Medicine, Sendai, Miyagi, Japan
  4. Nippi Research Institute of Biomatrix, Toride, Ibaraki, Japan
  5. Department of Life Science and Medical Bioscience, Waseda University, Shinjuku, Tokyo, Japan
  6. National Institute of Health Research and Development, Ministry of Health RI, Jakarta, Indonesia


Mucosal immunoglobulins comprise mainly secretory IgA antibodies (SIgAs), which are the major contributor to pathogen-specific immune responses in mucosal tissues. These SIgAs are highly heterogeneous in terms of their quaternary structure. A recent report shows that the polymerization status of SIgA defines their functionality in the human upper respiratory mucosa. Higher order polymerization of SIgA (i.e., tetramers) leads to a marked increase in neutralizing activity against influenza viruses. However, the precise molecular mechanisms underlying the effects of SIgA polymerization remain elusive. Here, we developed a method for generating recombinant tetrameric monoclonal SIgAs. We then compared the anti-viral activities of these tetrameric SIgAs, which possessed variable regions identical to that of a broadly neutralizing anti-influenza antibody F045-092 against influenza A viruses, with that of monomeric IgG or IgA. The tetrameric SIgA showed anti-viral inhibitory activity superior to that of other forms only when the antibody exhibits low-affinity binding to the target. By contrast, SIgA tetramerization did not substantially modify anti-viral activity against targets with high-affinity binding. Taken together, the data suggest that tetramerization of SIgA improved target breadth, but not peak potency of antiviral functions of the broadly neutralizing anti-influenza antibody. This phenomenon presumably represents one of the mechanisms by which SIgAs present in human respiratory mucosa prevent infection by antigen-drifted influenza viruses. Understanding the mechanisms involved in cross neutralization of viruses by SIgAs might facilitate the development of vaccine strategies against viral infection of mucosal tissues.

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CovalX has designed a detection system specifically for higher mass proteins and protein complexes. The HM1 and HM2 use an ion conversion detector which greatly increases the sensitivity of the mass spectrometer. Higher mass ions: incident ions collide with a conversion dynode array which creates smaller secondary ions. Then, the secondary ions are reaccelerated into a SEM and detected with higher sensitivity because of their higher velocity. The use of these detector systems for high mass protein ions helps to prevent detector saturation and allow for better sensitivity and profiling.

Experiments were performed by obtaining 12 μM thick tissue samples from the brains of adult male Wistar rats. The tissues were mounted onto conductive glass slides, frozen for an hour and washed. Following washing, the tissue sections were placed in a bath of cold acetone for 30 seconds, removed and a bath of cold 95% ethanol for another 30 seconds and finally, placed in chloroform for 1 minute. Samples were compared using different procedures (standard, HFIP, Leinweber, tween and H2O2) before 0.5 μL of the specific matrix was added using a micropipette or an automated sprayer.  Each sample preparation was analyzed using a mass spectrometer that had been modified with a CovalX HM1 detection system.

Standard sample preparation: chloroform and ethanol washes, mixed with matrix of 20 mg/ml sinapic acid in acetonitrile:0.1% TFA (&;3, v/v)

HFIP sample preparation: addition of 20 mg/ml sinapic acid in pure HFIP onto washed tissue and then addition of recrystallization solution (20 mg/mL sinapic acid in acetonitrile:0.1% TFA (7:3, v/v))

Leinweber sample preparation: placement of tissue samples onto a droplet of 20 mg/ml sinapic acid in 90% ethanol that contained 0.5 % Polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether and 0.1% TFA. The droplet was allowed to dry and a droplet of sonicated sinapic acid suspended in xylene was placed on top of the tissue sample before being dried in a vacuum desiccator. Finally, matrix droplets were applied again using 20 mg/mL sinapic acid solutions in 90% ethanol and 50% acetonitrile.

Tween sample preparation: the tissues were washed and a matrix solution (20 mg/ml sinapic acid in acetonitrile:0.1% TFA with a low concentration of polyethylene glycol sorbitan monolaurate) was added and allowed to dry.

H2O2 sample preparation: tissues were covered in a 3% solution of H2O2 before being incubated in a saturated vapor pressure chamber for 30 minutes and then dried in a vacuum desiccator. Finally, the samples were prepared using a matrix solution (20 mg/ml sinapic acid in acetonitrile:0.1% TFA (7:3, v/v)).

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Categories : Publications, HM5