Neutralizing activity of intravenous immune globulin products against enterovirus D68 strains isolated in Japan

Background Enterovirus D68 (EV-D68), belonging to Enterovirus D, is a unique human enterovirus mainly associated with common respiratory diseases. However, EV-D68 can cause severe respiratory diseases, and EV-D68 endemic is epidemiologically linked to current global epidemic of acute flaccid myelitis. Methods In this study, we measured neutralizing antibody titers against six clinical EV-D68 isolates in nine intravenous immune globulin (IVIG) products commercially available in Japan to assess their potential as therapeutic options for severe EV-D68 infection. Results Seven IVIG products manufactured from Japanese donors contained high neutralizing antibody titers (IC50 = 0.22–85.01 µg/mL) against all six EV-D68 strains. Apparent differences in neutralizing titers among the six EV-D68 strains were observed for all IVIG products derived from Japanese and non-Japanese blood donors. Conclusions High levels of EV-D68–neutralizing antibodies in IVIG products manufactured from Japanese donors suggest that anti-EV-D68 antibodies are maintained in the Japanese donor population similarly as found in foreign blood donors. Apparent differences in neutralizing antibody titers against the six EV-D68 strains suggest distinct antigenicity among the strains used in this study regardless of the genetic similarity of EV-D68.

. However, in rare cases, EV-D68 can cause AFM, a polio-like neurological disease with an acute onset of flaccid limb weakness and spinal cord gray matter lesions on magnetic resonance imaging [8][9][10][11][12]. Previous reports indicated that EV-D68 was associated with the onset of AFM, as an EV-D68 epidemic was reported at same time when an increase in AFM cases was occurred [13][14][15].
From 1970 to 2005, 26 cases of EV-D68 infection were reported to the National Enterovirus Surveillance System in the United States [16], and after 2005, small clusters of EV-D68 epidemics occurred in North America, Asia, and Europe [17,18]. In 2014, a large outbreak of EV-D68 was reported in the United States, and 1,395 confirmed cases of respiratory illness were recorded from August 2014 to January 2015 by the Centers for Disease Control and Prevention [13,[19][20][21].
In Japan, an EV-D68 outbreak occurred from August to December in 2015 [22], and a clade B strain was detected around the time of the outbreak [23,24]. Additionally, the presence of clades A-C strains has expanded in recent years [5,6,25].
To treat EV-D68 infections, the anti-enterovirus activity of intravenous immune globulin (IVIG) products has been examined in vitro and in vivo. A previous report found that five IVIG products used in the United States and Europe have neutralizing activity against three EV-D68 strains derived from outbreaks in 2014 in Missouri, Illinois, and Kentucky [26]. In mice infected with an EV-D68 strain, IVIG products reduced paralysis symptoms and decreased spinal cord viral loads [27]. Additionally, monoclonal antibodies with neutralizing activity against an EV-D68 strain derived from Missouri in 2014 also reduced paralysis after paralysis onset in a mouse model with AFM caused by EV-D68 [28].
Currently, no vaccine against EV-D68 infection has been developed, and no therapeutic agents are available. Therefore, as the most realistic therapeutic option, IVIG products with neutralizing activity against EV-D68 strains will be used clinically. However, whether the IVIG products used in Japan contain neutralizing antibodies against domestic EV-D68 strains has not been clarified.
In this study, we examined whether IVIG products used in Japan have neutralizing activity against domestic EV-D68 strains. We used the EV-D68 strains isolated in Yamagata prefecture, Japan in 2010-2015.

Cell line
RD-A cells, a variant of the RD cell line (derived from human rhabdomyosarcoma), were gifted from the Centers for Disease Control and Prevention in United States, and were maintained in Eagle's Minimum Essential Medium (EMEM) (M4655; Sigma) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 µg/mL streptomycin (growth medium) or EMEM supplemented with 2% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin (maintenance medium). RD-A cells were passaged with growth medium once per week, and the medium was changed to maintenance medium 2 days after passage.

IVIG products
Nine IVIG products were obtained from five manufacturers in Japan and one each in the United States and Germany (Table 1). Seven products were produced using donated blood in Japan, the product manufactured in Germany was also produced using donated blood, and the product manufactured in the United States was derived from non-donated blood.

Neutralization assay
We performed a neutralization assay once as described previously for human parechoviruses with minor modifications [30]. RD-A cells (1.5 × 10 5 cells/mL) were seeded in 96-well plates, and 10 wells per dilution and per IVIG product were prepared. The next day, 4-fold serial dilutions of the IVIG products were added to growth medium containing each EV-D68 strain (final concentration, 100 CCID 50 /200 µL). The medium mixed with IVIG and EV-D68 was incubated for 1 h at 37 °C, and the supernatant of cultured RD-A cells was replaced with 200 µL/well mixed medium. RD-A cells were incubated in the mixed medium for 6 days at 35 °C, and cytopathic effects (CPEs) were observed. Additionally, we confirmed whether RD-A cells infected with each EV-D68 strain in all wells exhibited apparent CPE as the 100 CCID 50 /200 µL condition except that no IVIG product was added. The 50% inhibitory concentration (IC 50 ), which was the concentration of each IVIG product that neutralized EV-D68 by 50%, was calculated from the observation of CPEs using the Kärber formula with positive wells as neutralized ones. Ten wells per dilution and per IVIG product were used for calculation of IC 50 .

Nine IVIG products neutralized the EV-D68 strains isolated in Japan
We performed a neutralization assay to examine whether IVIG products used in Japan have neutralizing activity against EV-D68 strains (Fig. 1). Using RD-A cells infected with EVs, all nine IVIG products neutralized EV-D68 strains isolated in Japan, which included three lineages (clades A-C µL condition without each IVIG product. Apparent differences in neutralizing antibody titers against the six EV-D68 strains were observed for the IVIG products derived from Japanese and foreign blood donors (see the next section), suggesting distinct antigenicity among the strains used in this study regardless of the genetic similarity of EV-D68. Although the two clade C strains (2150-Yamagata-2010 and 2192-Yamagata-2010) were closely related phylogenetically, the neutralizing activities of the IVIG products were apparently different. The neutralization assay indicated that IVIG products used in Japan can neutralize EV-D68 strains prevalent in Japan.

IC 50 determination
We calculated the IC 50 from the CPEs observed in the neutralization assay (

Discussion
In this study, we found that all nine IVIG products used in Japan neutralized six EV-D68 strains isolated in Yamagata prefecture Japan in 2010-2015, which were categorized into three clades (clades A-C). Additionally, apparent differences in neutralizing titers among the six strains were observed for all IVIG products, and although the two clade C strains (2150-Yamagata-2010 and 2192-Yamagata-2010) were phylogenetically related [5,6], different neutralizing titers were measured for each IVIG product, suggesting that the antigenicity among the strains used in this study was not related to the genetic similarity of EV-D68.
A previous report indicated that antisera against clade A-C stains have neutralizing activity only against strains in the same clade as the antigen used to obtain the antisera [31], whereas the IVIG products used in this study exhibited neutralizing activity against strains from clades A-C. This was because all of the IVIG products used  [5,6].
in this study contained antibodies against clade A-C strains, and the extent of the neutralizing activity against the EV-D68 strains was different for each IVIG product. Although molecular detection and identification are currently common for the laboratory diagnosis of EV-D68 infection, conventional virus isolation approaches might permit identification of the predominant EV-D68 strains for neutralization assays and seroprevalence studies [5,6].
Vaccines and therapeutic agents against EV-D68 infection have not been introduced. Therefore, IVIG products represent a realistic and feasible option for the treatment of severe EV-D68-associated diseases. Whereas differences in neutralizing activity against different strains were revealed for the IVIG products investigated in this study, all IVIG products contained neutralizing antibodies; therefore, they are expected to have therapeutic efficacy against the three examined EV-D68 lineages. From the experience with polio and EV-A71 vaccines, there is a consensus regarding the importance of serum neutralizing antibodies for the prevention of clinical symptoms during the course of polio/enterovirus infections [32,33]. Although previous research observed effective neutralizing antibodies against EV-D68 in animal experiments, the Fig. 1 Neutralization of the EV-D68 strains by IVIG products. The neutralizing activity was performed once using RD-A cells (1.5 × 10 5 cells/mL) seeded in 96-well plates, and 10 wells per dilution and per IVIG product were prepared. On the next day, 4-fold serial dilutions of the IVIG products were added to growth medium containing each EV-D68 strain (final concentration, 100 CCID 50 /200 µL). The medium containing IVIG and EV-D68 was incubated for 1 h at 37 °C, and the supernatant of cultured RD-A cells was replaced with 200 µL/well mixed medium. RD-A cells in the mixed medium were incubated for 6 days at 35 °C, and CPEs were observed. A-I represents products 1-9, respectively. Diamond   [27,28]. AFM has been identified as an EV-D68-neurological disease [34][35][36][37][38][39][40], and the possibility of anti-EV-D68 antibodies as preventive and therapeutic options for AFM was reported [41]. In Japan, a relationship between EV-D68 and AFM was suggested [12]. Therefore, IVIG products are expected to emerge as preventive or therapeutic options.
Seven IVIG products manufactured in Japan, in addition to products manufactured in Germany and the United States, exhibited neutralizing activity against EV-D68 in this study, indicating that the Japanese donor population maintains anti-EV-D68 antibodies similarly as foreign donors. In a recent seroepidemiological study, neutralizing antibodies were detected in individuals in the United States [42], Netherlands [43], China [44,45], Malaysia [46], and Taiwan [47]. These findings were consistent with those of the present study revealing high EV-D68 neutralizing antibody titers in IVIG products manufactured in the United States and Germany. This study indicated that the IVIG products used in Japan contain neutralizing antibodies against the endemic EV-D68 strains.
In this study, it was the limitation that the neutralization assay was performed once therefore statistical comparisons of IC 50 remains difficult. Additionally, this study provides only in vitro analysis using RD-A cells for measuring the neutralization activity of IVIG products, therefore further in vivo researches would be necessary for the evaluation of therapeutic treatment.

Conclusion
In this study, we demonstrated that nine IVIG products used in Japan, including seven products manufactured in Japan and one each manufactured in the United States and Germany, neutralized six domestic EV-D68 strains. Further research will be needed to assess the efficacy of the IVIG products against severe EV-D68 infections in clinical settings.    The IC 50 of the 2150 strain (product 4) was not available, therefore this difference was not calculated.