The Relationship between Poliovirus Multiplication, the sIgA Antibody Response and the Serum Neutralizing Antibody Titers after Trivalent Oral Polio Vaccination

Abstract

The time kinetics of poliovirus and secretory IgA（sIgA）antibody titers were examined in fecal sam- ples from four vaccinees immunized with two doses of trivalent oral polio vaccine（TOPV）from 1996 to 2000. Poliovirus types 1 and 2 multiplied in the human intestine after the first vaccination, and poliovirus type 3 multiplied after the second vaccination. Additionally, poliovirus type 3 multiplied for several days in two cases after the first vaccination, and poliovirus types 1 and 2 multiplied for before and after a week in one case after the second vaccination. Poliovirus type 2 multiplied most efficiently in the human intes- tine and stimulated the sIgA antibody response. The sIgA antibody titers are required to be ≧1：4 for poliovirus types 1 and 3, and ≧1：8 for poliovirus type 2 in 10％ fecal suspension for adequate TOPV pro- tection against reinfection. Serum neutralizing antibody titers against poliovirus type 3 were 1：6 and 1：

8 for the shorter periods of 16 and 13 days, respectively, and 1：60 and 1：45 for 36 days in multiplication of poliovirus type 3 after the second vaccination. For double infection of poliovirus types 1 and 2, only the higher titers were obtained.

These results suggest that poliovirus multiplication induces the sIgA antibody in the human intes- tine and the induced sIgA antibody defends the subject from poliovirus infection, and that the duration and the amount of virus multiplication tend to correlate with the levels of the serum neutralizing anti- body titers.

〔J.J.A. Inf. D. 75：1030〜1039, 2001〕

Introduction

The WHO Western Pacific Region declared the eradication of poliomyelitis on October 29 2000^1）. However, wild poliovirus infection cases have been reported in many areas of Africa and Asia, and the vaccination for children remains an urgent priority^2）. Even after the eradication, we still have to maintain immunity against poliovirus at least for a certain period of time. At the same time, issues such as when

The Relationship between Poliovirus Multiplication, the sIgA Antibody Response and the Serum Neutralizing Antibody Titers after

Trivalent Oral Polio Vaccination

Noriko MORIMOTO

Japan Poliomyelitis Research Institute

（Received：July 23, 2001）

（Accepted：October 29, 2001）

Correspondence to：Noriko MORIMOTO

Japan Poliomyelitis Research Institute, 5―34―4, Kumegawa-cho, Higashimurayama-shi, Tokyo, 189―0003, Japan oral polio vaccine, poliovirus multiplication, sIgA antibody, neutraliz- ing antibody

Key words：

and how vaccination against poliovirus should be stopped have become important from both the public health and industrial perspectives^3）. At present, an inactivated poliovirus vaccine（Sabin strain）is being developed at the Japan Poliomyelitis Research Institute.

Poliovirus is usually transferred through the fecal-oral route. The host defense factors that eventu- ally eliminate the virus remain obscure. The administration of trivalent oral polio vaccine（TOPV）induces both mucosal and systemic immune responses. The circulating neutralizing antibodies have been shown to efficiently prevent paralytic disease^4）5）. Several reports have been made on the sIgA antibody re- sponses after vaccination with poliovirus^6）〜15）.

A case has already been reported in which three types of poliovirus multiplied in the human intestine after the first vaccination and only poliovirus type 1 multiplied for one week after the second vaccination, and that the isolated polioviruses were examined for the relationship between poliovirus multiplication and rct（reproductive capacity at different temperatures）39.5℃ character^17）. In the present study, I ex- amine the time kinetics of poliovirus and sIgA antibody titers against poliovirus using fecal samples from four vaccinees to determined the necessary titers for protection against TOPV reinfection, and give a rea- son for the levels of the serum neutralizing antibody titers.

Materials and Methods

1．Vaccine and Vaccinees

TOPV contains 10^6.0CCID50of poliovirus type 1, 10^5.0CCID50of poliovirus type 2 and 10^5.5CCID50of po- liovirus type 3 per dose. Infants administered twice with TOPV at an interval of 2 months（case 1）, 3 months（case 2）, 4 months（case 3）or 6 months（case 4）by mass vaccinations were the subjects of the study.

The purpose and the protocol of the study were clearly explained. Informed consent was obtained from the parents for the infants before blood and fecal specimen were collected.

2．Preparation of 10％ fecal suspensions

The fecal samples were diluted 10-fold with M199（GIBCO）containing 0.1％ bovine serum albumin and centrifuged at 1,400g for 30 min andor at 16,770g for 20 min at 4℃. The supernatants were stored at

−20℃until assay for viral and sIgA antibody titers.

3．Assay for virus titers

Viral titers were assayed by the tube method for GMK-2 cells（cases 2 and 4）or by the microplate method for HEp-2C cells（cases 1 and 3）．

In preliminary experiments, the poliovirus type 2 and 3 titers were 0.2〜0.3 Log10higher by the mi- croplate method than by the tube method but the poliovirus type 1 titers were the same.

4．ELISA for sIgA antibody titers in feces

ELISA plates（Nunc）were coated with 3µgml of each type of poliovirus（Sabin strain）by an over- night incubation at 4℃. The plates were then incubated with a blocking reagent（1％ bovine albumin in PBS with 0.05％ Tween 20［0.05％ T-PBS］）at 37℃ for 30 min. After washing 3 times, 10％ fecal samples were diluted serially 2-fold with 0.05％ T-PBS containing 10％ fetal calf serum and then shaken for 2 hr.

After washing 4 times , the plates were incubated for 1 hr with a 1 ： 2000 dilution of peroxidase- conjugated goat anti-human IgA（Bio Source International Camarillo, CA, USA）.After washing 5 times, 100µl of ortho-phenylenediamine and hydrogen peroxide were added to each well and incubated at room

temperature for 15 min in the dark. The reaction was stopped by adding 50µl of 4N sulfuric acid, and the optical density（OD）was measured at 492 nm with an Auto Reader. The sIgA antibody titers were calcu- lated at the highest reciprocal dilution when the difference in OD value between the test and control lanes exceeded 0.2. Positive and negative control fecal samples were included in all sIgA assays.

5．Serum neutralizing antibody titers

Reinformed consent prior to collect blood specimen was obtained from the mothers of the infants.

Neutralizing antibody titers were assayed using blood samples obtained from the infants earlobes with blood sampling paper（Toyo Roshi Kaisha Ltd.）. The blood absorbent area of the paper was extracted in PBS（pH 7.2）for 1 hr at room temperature and then for 1.5 hrs at 4℃. The sample was centrifuged at 1,400 g for 30 min and the supernatant was heated at 56℃ for 30 min. The neutralizing antibody titers were de- termined by the micro-neutralization method as follows：2-fold serial dilution of each sample（25µl）was prepared in microplate wells, to which 25µl of 100 CCID50of each type poliovirus（Sabin strain）were added, and the mixtures were incubated at 36℃ for 2 hr. Then, 100µl of GMK-2 cell or HEp-2C cell sus- pension（4×10⁵cellsml）was added to these wells and the wells were incubated at 36℃ in a 5％ CO2in- cubator. The neutralizing antibody titers were calculated by the Reed-Muench method.

Results

1．Poliovirus titers in feces

The virus titers in 10％ fecal suspensions from the four vaccinees showed that poliovirus type 1 mul- tiplied for 32, 36, 34 and 48 days, poliovirus type 2 multiplied for 33, 50, 25 and 48 days after the first vacci- nation（Fig. 1 A-1, Fig. 2 C-1, Fig. 3 E-1 and Fig. 4 G-1）, and poliovirus type 3 multiplied for 16, 36, 13 and 36 days after the second vaccination（Fig. 1 A-2, Fig. 2 C-2, Fig. 3 E-3 and Fig. 4 G-2）.Additionally, we ob- served that poliovirus type 3 multiplied for 5 days（Fig. 1 A-1）and 4 days（Fig. 3 E-1）after the first vac- cination, and that poliovirus type 1 multiplied for 8 days and poliovirus type 2 for 5 days after the second vaccination（Fig. 4 G-1）. The highest poliovirus titers in feces were about 10⁷CCID50g.

2．Poliovirus specific sIgA antibody titers after vaccination

Poliovirus type 1 specific sIgA antibody（type 1 sIgA antibody）and poliovirus type 2 specific sIgA antibody（type 2 sIgA antibody）titers rose from between the first and the second weeks, days 7 to 13 for type 1 and days 7 to 10 for type 2, after the first vaccination. The highest titers were 1：1024, 1：128 and 1：1024 for type 1, and 1：1024 for type 2（Fig. 1 B-1, Fig. 2 D-1 and Fig. 3 F-1, respectively）.

Poliovirus type 3 specific sIgA antibody（type 3 sIgA antibody）titers rose from the first week（day 6 or 8）after the second vaccination. The highest titers were 1：128（Fig. 1 B-2）, 1：256（Fig. 2 D-2）and 1：1024（Fig. 3 F-3）.At the same time, type 1 and 2 sIgA antibody responses also developed booster ef- fects after the second vaccination. These titers were increased from 1：4 to 1：128, from 1：8 to 1：512 and from 1：16 to 1：1024 for type 1 sIgA antibody, and from 1：8 to 1：512, from 1：64 to 1：1024 and from 1：32 to 1：4096 for type 2 sIgA antibody（Fig. 1 B-2, Fig. 2 D-2 and Fig. 3 F-3, respectively）.

3．sIgA antibody titer required to prevent TOPV reinfection

The sIgA antibody titers in the 10％ fecal suspensions on the date of the second dose（before the sec- ond vaccination）are shown in Table 1. Based on the data from cases 1 to 3, poliovirus types 1 and 2 were not detected after the second vaccination when the type 1 sIgA antibody titer was ≧1：4 and type 2 sIgA antibody titer was ≧1：8 in the 10％ fecal suspensions.

4．Serum neutralizing antibody titers

As shown in Table 2, neutralizing antibody titers against poliovirus were positive（≧1：4）in all four vaccinees after the second vaccination. The neutralizing antibody titers against poliovirus types 1 and 2 were high enough to protect the subjects from paralytic poliomyelitis. In cases 1 and 3, the neutralizing antibody titer against poliovirus type 3 was 1：6 or 1：8 after poliovirus type 3 multiplication for 13 or 16 days, whereas in cases 2 and 4, it was 1：45 or 1：60 after 36 days.

Fig. 1 Poliovirus titers and sIgA antibody titers in 10％ fecal samples from a vac- cinee administered twice with TOPV at an interval of 2 months（case 1）.

Discussion

Fecal samples were analyzed to acquire a basic knowledge of the relationships between poliovirus multiplication（Sabin strain）, sIgA antibody response and serum neutralizing antibody response.

Poliovirus type 2 predominated in virus multiplication and the sIgA antibody response in the intes- Fig. 2 Poliovirus titers and sIgA antibody titers in 10％ fecal samples from a vac-

cinee administered twice with TOPV at an interval of 3 months（case 2）.

Fig. 3 Poliovirus titers and sIgA antibody titers in 10％ fecal samples from a vac- cinee administered twice with TOPV at an interval of 4 months（case 3）.

tine, despite the 10％ viral titer of poliovirus type 1 in TOPV. Furthermore，≧1：4 neutralizing antibod- ies in 2 year old children were acquired in 96％ for poliovirus type 1, 98％ for poliovirus type 2 and 90％

for poliovirus type 3^16）. Poliovirus type 2 may also predominate in the acquisition rate of the neutralizing antibody.

Poliovirus types 1 and 2 were not detected after the second vaccination when the type 1 sIgA anti- body titer was ≧1：4 and type 2 sIgA antibody titer was ≧1：8 in 10％ fecal suspensions. Two other

Fig. 4 Poliovirus titers and sIgA antibody titers in 10％ fecal samples from a vac- cinee administered twice with TOPV at an interval of 6 months（case 4）.

Table  1 Relationship between poliovirus multiplication and sIgA antibody  titers in 10% fecal suspensions before or after the second vaccination

Virus detection after the  second vaccination sIgA antibody titer before the 

second vaccination Case No.

（interval）

Days Type

Type 3 Type 2

Type 1

16 3

＜ 1：2 1：8

1：4 1（2 months）

36 3

＜ 1：2 1：64

1：8 2（3 months）

13 3

＜ 1：2 1：32

1：16 3（4 months）

9 1

＜ 1：2 1：2

＜ 1：2

4（6 months） 2 5

36 3

Table  2 Neutralizing antibody titers after the first and second vaccinations or after  the second vaccination

Days after the first and second vaccinations  Neutralizing antibody titer

Case No.

or after the second vaccination Type 3

Type 2 Type 1

35 months after the second vaccination.

1：    6 1：     110

1：     320 1

58 days after the first vaccination.

65 days after the second vaccination.

＜ 1：10 1：60 1：     910

1：1,280 1：1,810

1：1,810 2

3 months after the first vaccination.

14 months after the second vaccination.

＜ 1：10 1：    8 1：     320

1：     450 1：     640

1：     640 3

54 months after the second vaccination.

1：45 1：       91

1：       91 4

cases were previously reported^17）and an additional case in which sIgA antibodies against poliovirus, at the date of the second dose, were titrated. Both had antibody titers <1：2 for type 1 sIgA, 1：8 for type 2 sIgA and 1：4 for type 3 sIgA, and poliovirus types 2 and 3 were not detected after the second vaccina- tion. These data suggest that sIgA antibody titers must be ≧1：4 for poliovirus types 1 and 3, and ≧1：8 for poliovirus type 2（I have no data indicating 1：4）in 10％ fecal suspensions for protection against TOPV reinfection.

After the second vaccination, poliovirus type 1 multiplied at an interval of five months^17）, six months

（Fig. 4 G-2）or seven months（data not shown）, whereas it did not multiply at an interval of two months

（Fig. 1 A-2）, three months（Fig. 2 C-2）or four months（Fig. 3 E-3）. It may thus be optimal to administer TOPV twice at an interval of two to four months rather than an interval of half a year.

Geometric mean titers（GMTs）of serum neutralizing antibodies against poliovirus by the age of 2

−4 were 270 for type 1, 171 for type 2 and 45 for type 3^16）. GMTs measured by the paper method were 427, 277 and 23 for types 1−3, respectively. These GMTs were similar titers except for type 3 compared with those already reported.

In cases 1 and 3, poliovirus type 3 was isolated for 4 or 5 days after the first vaccination. It is specu- lated that for the slight immune response against poliovirus type 3（though undetectable）, poliovirus type 3 may multiply during the shorter period 13 or 16 days after the second vaccination. Neutralizing anti- body titers against poliovirus type 3 were ＜1：10 in the absence of poliovirus type 3 multiplication（case

2）after the first vaccination, and 1：6 or 1：8 in a shorter period 13（case 3）or 16（case 1）days after the second vaccination. However, they were 1：60, 1：45 and 1：60（cases 2, 4 and the reported case^17））in the presence of poliovirus type 3 multiplication for 36 days. A portion of the multiplying poliovirus in the in- testine is recognized by antigen presenting cells as viral antigen and, finally, the antibody against poliovi- rus is produced by B cells. For these reasons, the duration of poliovirus multiplication in the intestine seems to correlate with the levels of the serum neutralizing antibody titers.

Neutralizing antibody titers against poliovirus types 1 and 2 were high titers（1：91―1：1810 for type 1 and 1：91―1：1280 for type 2）,because viruses may multiply repeatedly for a longer period following the double andor contact infections（see Fig. 1―4）.These data are supported from the Annual Report 1997 that percentages of 1：4＜1：8 neutralizing antibody titers by the age group of 2―4 yrs were 0.8％

for type 1, 0％ for type 2 and 16％ for type 3^16）. The duration of plural virus multiplication may not always be correlated with the levels between the higher neutralizing antibody titers. But these results may sug- gest that the duration and the amount of virus multiplication tended to correlate with the levels of the neutralizing antibody titers.

These examinations showed that sIgA antibodies to TOPV protected the subject from poliovirus in- fection in the intestine and that the maintenance of protective levels of sIgA antibodies was short-lived.

Thus, vaccination with TOPV or TIPV（trivalent inactivated polio vaccine）may be difficult for the eradi- cation of poliovirus.

Acknowledgement

I am grateful to Dr. Shingo Takagaki（Hirayama Clinic：Hino, Tokyo）for collecting the blood sam- ples. I thank Ms. Toshiko Iijima（Matsudo, Chiba），Ms. Hideyo Ishida（Hino, Tokyo）,Ms. Michiko Mori

（Hino, Tokyo）, Ms. Yuko Sano（Hino, Tokyo）, Ms. Yuki Shinoda（Toshima, Tokyo）, Ms. Yumiko Tsutsui

（Machida, Tokyo）and Ms. Yuri Yoshioka（Hino Tokyo）for collecting the fecal samples（alphabetical or- der）.

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ポリオワクチン投与後のポリオウイルスの増殖，分泌型 IgA 抗体産生と 血中中和抗体価との関係

日本ポリオ研究所

森本 紀子

1996 年から 2000 年にポリオワクチンを二回投 与した四人の乳幼児の糞便から排泄ウイルス量と 分泌型 IgA（sIgA）抗体価を経日的に測定した．

腸管内で主に第一回ワクチン投与後 1 型と 2 型の ポリオウイルスが増殖し，第二回ワクチン投与後 3 型のポリオウイルスが増殖した．10％ 糞便中の sIgA 抗体価は，1 型と 3 型が 4 倍以上，2 型が 8 倍 以上の時投与したワクチン中のポリオウイルスの

増殖を抑制した．血中中和抗体価は，腸管内で 3 型ポリオウイルスが短期間増殖した時は低く長期 間増殖した時は高かったが，1 型と 2 型の混合感 染の時は高い抗体価のみ得られた．

ポリオウイルス型特異的 sIgA 抗体がポリオウ イルス感染を抑制し，ポリオウイルスの増殖期間 と産生ウイルス量が血中中和抗体価の高低と相関 する傾向にあるという結果であった．

〔感染症誌 75：1030〜1039，2001〕