Vol. 184, No. 3, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

May 15, 1992

Pages I152-I157

Gly 145 TO Arg SUBSTITUTION IN HBs ANTIGEN OF IMMUNE ESCAPE MUTANT OF HEPATITIS B VIRUS

H. Fujii 1, K. Moriyama 2., N. Sakamoto 3, T. Kondo 1, K. Yasuda 1, Y. Hiraizumi 1, M. Yamazaki 1, Y. Sakaki 3, K. Okochi 4, E. Nakajima 5

1Ogaki Municipal Hospital, Minaminokawa, Ogaki 503, J a p a n 2School of Medicine, 3Research Laboratory for Genetic Information and 4University Hospital, Kyushu University, Fukuoka 812, J a p a n 5Institut Pasteur de Kyoto, Tanakamonzen, Kyoto 606, Japan

Received March 25, 1992

A Japanese child born to an HBeAg-positive carrier mother received antiHBs immunoglobulins and a plasma-derived HBs vaccine with a poor antiHBs-antibody response. The child, who is now 3 years old, is p r e s e n t l y suffering from chronic hepatitis with unusual serological findings t h a t are positive for HBsAg, anti-HBs and HBeAg, since being infected with a measles virus at 12 months of age. The nucleotide sequences of the S region of HBV DNA obtained from the patient, the m o t h e r and an HBeAg-positive brother were completely identical except for one nucleotide at position 587 (mother and brother: guanosine, patient: adenosine), giving an amino acid change: Gly -> Arg at position 145 of the major HBs protein. ~ z992Aoad~mioP..... ~nc.

Since the use of vaccines against hepatitis B virus infection has spread worldwide, the ways of dealing with i n t r a u t e r i n e infection as well as improving the vaccine are growing problems. During a recent analysis of such infected patients, an escape m u t a n t arising through vaccine-induced i m m u n i t y was found in an Italian patient (1). Although it has not been d e t e r m i n e d w h e t h e r such a v a r i a n t is able to infect other anti-HBs

*To whom correspondence and reprint requests should be addressed. 0o06-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of.reproduction in any form reserved.

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seroconverted or vaccinated individuals, its presence may eventually raise future problems. In Japan, since the launch of a government-sponsored programme for mass protection against perinatal hepatitis B virus infection began in 1985, approximately 95% of infants born to HBeAg-positive carrier mothers (estimated number of neonates included in this programme: 5100 per year) have been protected, while the others have become carriers due either to intrauterine infection or a poor response to the vaccine (2). In Ogaki Municipal Hospital, out of 49 infants born to HBeAg-positive mothers between 1986 and 1990, 2 were diagnosed as contracting intrauterine infections by testing for HBsAg in cord blood serum, whereas 47 were seronegative for HBsAg and received high-titer anti-HBV immunoglobulin (HBIG) and HBs vaccine, resulting in 46 (98%) successful protections and 1 failure. Herein we describe the clinical course of this infected patient and the laboratory findings of serum tests.

M A T E R I A L S AND METHODS

Serological tests and vaccine ~ro~ramme: HBsAg, anti-HBs, HBeAg, anti-HBe and anti-HBc were determined by radioimmunoassay (Sorin Biomedica, Italy). HBIG (Nihon Seiyaku, Japan) and plasma-derived HBs vaccine (Midorijuji, Japan) were administered to children born to HBsAgand HBeAg-positive carrier mothers, and whose cord blood was negative for HBsAg. The programme consists of 200 U HBIG within 48 h of birth and again at 2 months of age, and three doses of 10 pg plasma-derived vaccine at ages 2, 3 and 5 months. PCR amplification: The preS1, preS2 and S regions of HBV DNA were amplified by the polymerase chain reaction (PCR) ( 3 ) u s i n g primers encompassing the preS and S regions (4, 5). The oligonucleotide primers were synthesized by an automatic DNA synthesizer (Applied Biosystems, Model 380A). DNA prepared from 200 ~1 of solution containing 5 ~tl serum and 195 ~1 distilled water was amplified in 100 ~tl of PCR mixture containing 0.2 mM deoxynucleotide triphosphates (dNTPs), 0.5 ~tM oIigonucleotide primers and the manufacturer's buffer and 2 U of Taq polymerase (Promega). Clonin~ of PCR products and sequencing: Termini of the amplified fragments were filled in by DNA polymerase 1 large fragment and dNTPs, 1153

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phosphorylated with polynucleotide kinase, cloned into plasmid vector pUC19 and sequenced by the dideoxy chain-termination method (6) using T7 polymerase (Pharmacia). The nucleotide sequences of 2 and 3 clones of each subject were determined for the whole of the preS and S regions and the partial nucleotides spanning the subtype determinants, respectively.

RESULTS

The patient was born by normal vaginal delivery in the 39th week of gestation with a birth weight of 3480 g. As shown in Fig. 1, the titer of antiHBs decreased from a m a x i m u m of 120 cut-off index (CI) at the time of passive immunization with HBIG down to 4.3 CI at 9 months after the last vaccination. He later developed a fever and cough at the age of 12 months. Both Koplik's spots on day 5 and skin eruptions over his entire body on day 6 followed, and thus a measles virus infection was diagnosed. Two months after contracting the measles, despite a lack of any clinical symptoms, both GPT and GOT increased to 215 KU and 130 KU, respectively. The anti-HBc antibody titer began to rise again, but HBsAg was found to be negative using a RIA kit. At 42 months of age, transaminases fell to the normal range with diminishing HBV-DNA polymerase activity.

GPT(Ku)

anti-HBs (c0

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125

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Fig. 1. Clinical course of the patient harboring a variant HBV. Horizontal scale

represents age. CI: cut-off index, KU: Karmen unit, Ig: administration of hightiter immunoglobulin, v: vaccination. *Serum was further examined for serological markers and HBsAg was detected by different kits. 1154

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IUInth~r

'~Y~ A G C T

Rroth~,r

A G C T

Patient

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Fig. 2. Nucleotide and deduced amino acid sequences of HBV DNA obtained from the mother's, brother's and the patient's serum, showing the Gly 145 -> Arg amino acid change in the HBs protein.

For molecular analysis, serum samples were obtained from the patient at 36 months of age. The nucleotide sequences of the preS2 and S regions of H B V DNA from 10 individual clones from each s e r u m sample were completely identical to each other, and those of the patient, his mother and his 6-year-old HBeAg-positive brother were also identical except for one nucleotide substitution from guanosine (mother and brother) to adenosine (patient) at position 587, which alters glycine to arginine at position 145 in the major HBs protein (Fig 2). No termination codon was found in the open reading frames for preS2 and S.

DISCUSSION The anti-HBc antibody (IgG) appearing initially is p r e s u m e d to be a m a t e r n a l transitional

antibody. The p a t i e n t also seems to be a non-

r e s p o n d e r w h e n we see the decline of anti-HBs t i t e r soon after t h e administrations with HBIG. In addition, the increase in titers of both antiHBs and anti-HBc as well as elevated transaminases 7 months after the last vaccination all seem to have been an immune response against the HBV infection, and not against the vaccine. In addition, we consider it likely that the measles virus played an indirect role in the development of hepatitis by altering the i m m u n e system to attack the infected liver, r a t h e r t h a n by acting as a directly causal agent of this episode. There has been a report of a child whose cord blood serum was negative for HBsAg by conventional RIA kits but positive for HBV-DNA by PCR, and 1155

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who did not respond to repeated vaccination, and became an HBsAg-positive HBV carrier at the age of 10 months (7). In addition, the use of a single serological test for HBsAg is known to be unreliable in diagnosing carrier status (8-10). In our present case, HBsAg was detected in the tested serum of 36 months of age by other RPHA and EIA kits (Kaketsuken, J a p a n and Abott, Japan, respectively) as well as b y monoclonal antibodies. Thus, since it is technically difficult in the PCR to avoid contamination of the cord blood with m a t e r n a l HBV, we believe t h a t more detailed serological examinations of cord blood m u s t be performed before d e t e r m i n i n g the suitability for vaccination. A variant possessing the same mutation as t h a t first found in an Italian child (1) occurred in a Japanese patient, and proliferated in the presence of anti-HBs. This suggests not only an accumulation of amino acid changes in this epitope in a determinant, but t h a t Arg 145 m a y play a key role in the viral proliferation of i n d i v i d u a l s who are seropositive for anti-HBs, a l t h o u g h d i f f e r e n t m u t a t i o n s are also possible (11). A l t h o u g h it is p r e m a t u r e to draw conclusions until d a t a can be obtained on infection experiments using a n i m a l models and until more such reports on the vaccine appear, it might be worth considering the addition of both such mutant-type HBs and other HBV antigens to conventional vaccines.

ACKNOWLEDGMENTS We thank Prof. T. Orii, Gifu University, and Prof. R. Mori, Prof. Y. Niho, Dr. B. T. Quinn and Dr. H. Ishibashi, Kyushu University, for encouraging this study, and Prof. M. Mayumi, Jichi Medical School, for his pertinent advice. Screening tests for HBV infection were done at Ogaki Municipal Hospital. The PCR, molecular clonings and nucleotide sequences were performed at Kyushu University.

REFERENCES 1. Carman, W,F., Zanetti, A.R., Karayiannis, P., Waters, J., Manzillo, G., Tanzi, E., Zuckerman, A.J., and Thomas, H.C. (1990) Lancet 336, 325-329. 2. Eto, T., and Shiraki, K. (1989) Acta Pediatr. Jpn. 31, 681-684. 3. Saiki, R.K., Gelfand, D.H., Stoffel, S., Sharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A. (1988) Science 239, 487-491. 1156

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4. Thiers, V., Nakajima, E., Kresdorf, D., Mack, D., Schellekens, H., Driss, F., Goudeau, A., Wands, J., Sninsky, J., and Tiollais, P. (1988) Lancet ii, 12731276. 5. Patelini, P., Gerken, G., Nakajima, E., Terre, S., D'Errico, A., Grigioni, W., Nalpas, B., Franco, D., Wands, J., Kew, M., Pisi, E., Tiollais, P., and Brechot, C. (1990) N. Engl. J. Med. 323, 80-85. 6. Hattori, M., and Sakaki, Y. (1986) Analyt. Biochem. 152, 232-238. 7. Shimizu, H., Mitsuda, T., Ookawa, N., Huzita, S., Ibe, M., and Yokota, S. (1990) Clin. Virol. 18, 66-70. 8. Tabor, E., Ziegler, J.L., and Gerety, R.J. (1980) J. Infect. Dis. 141, 289-292. 9. Cossart, Y.E., Kirsch, S., and lsmay, S.L. (1982) Lancet i, 208-213. 10. Moriyama, K., Ishibashi, H., Kashiwagi, S., and Asayama, R. (1989) Gastroenterology 97, 1068-1069. II. Moriyama, K., Nakajima, E., Hohjoh, H., Asayama, R., and Okochi, K. (1991) Lancet 337, 125.

1157

Gly145 to Arg substitution in HBs antigen of immune escape mutant of hepatitis B virus.

A Japanese child born to an HBeAg-positive carrier mother received anti-HBs immunoglobulins and a plasma-derived HBs vaccine with a poor anti-HBs-anti...
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