125
Immunoselected
hepatitis
B virus mutant
SIR,-We have reported unusual serological data for hepatitis B virus (HBV) markers in a 66-year-old Japanese man with chronic hepatitis.’He had HBV DNA and HB e antigen in the presence of antibody to hepatitis B surface antigen and lacked both pre-S2 and antibody to pre-S2. Here we provide molecular evidence that the infecting virus had deletions in pre-S2 and aminoacid changes in the a determinant. This may be another instance of virus multiplication in the presence of immunity to HBsAg.2 DNA from serum was amplified by polymerase chain reaction (PCR)3,4 with HBV-specific primers. All the amplified fragments were smaller than expected and also smaller than those of the positive control. The fragments were cloned into pUC19 and the 4 positive colonies were sequenced by the dideoxy chain-termination method with some modification.’ The same serum was analysed in independent laboratories using different PCR primers (sequences available from K. M.) and the nucelotide sequences were found to be identical, suggesting the predominance of a single variant-type virus. The aminoacid deletions and changes consequent upon the altered DNA sequences were in positions aa9-22 (in the hydrophilic epitope), aa3, and aa8 of pre-S2, and at positions aal26, aa131, and aal33 in the first loop of the a determinanrö of the S product (figure).
The gene encoding the d and r determinants’ was unaltered. The same results were obtained on sera obtained in 1984 and 1989. The defective pre-S2 gene may explain why this patient with chronic hepatitis B lacked S products in his serum. These changes in surface determinants may have emerged through immunoselective pressure, although the process seems slower than it is in vivo in experimental murine retrovirus8 and lymphocytic choriomeningitis virus9 infections. Another possibility is that the mutant is infectious and was transmitted from another individual. These findings should be borne in mind when considering vaccine strategy. Department of Virology and First Department of Internal Medicine,
Kyushu University, Fukuoka 812, Japan, Institut Pasteur de Kyoto, Research Laboratory for Genetic Information, Kyushu University, Hamanomachi Hospital, Fukuoka, and Clinical Laboratory, Kyushu University Hospital 1.
K. MORIYAMA E. NAKAJIMA H. HOHJOH R. ASAYAMA K. OKOCHI
Moriyama K, Ishibashi H, Kashiwagi S, Asayama R. Presence of HBV DNA and HBeAg in serum of an anti-HBs-positive individual. Gastroenterology 1989; 97: 1068-69.
2. Carman
WF, Zanetti AR, Karayiannis P, et al. Vaccine-induced escape mutant of hepatitis B virus. Lancet 1990; 336: 325-29. 3. Thiers V, Nakajima E, Kresdorf D, et al. Transmission of hepatitis B from hepatitis seronegative subjects. Lancet 1987; ii: 1273-76. 4. Paterlini P, Gerken G, Nakajima E, et al. Polymerase chain reaction to detect hepatitis B virus DNA and RNA sequence m primary liver cancers from patients negative for hepatitis B surface antigen. N Engl JMed 1990; 323: 80-85. 5. Hattori M, Sakaki Y. Dideoxy sequencing methods using denatured plasmid templates. Analyt Biochem 1986; 152: 232-38 6. Brown SE, Howard CR, Zuckerman AJ, Steward MW. Affinity of antibody responses m man to hepatitis B vaccine determined with synthetic peptides. Lancet 1984; ii: 184-87. 7. Okamoto H, Imai
M, Tsuda F, et al. Point mutation in the S gene of hepatitis B virus d/y or w/r subtypic change in two blood donors carrying a surface antigen of compound subtype adyr or adwr. J Virol 1987; 61: 3030-34. 8. Pozsgay JM, Klysczek KK, Blank K. In vitro generation of antigenic variants of murine retroviruses. Virology 1989; 173: 330-34. for a
9. Pircher H,
Moskophidis D, Rohrer U, et al. Viral escape by selection of cytotoxic T cell-resistant virus variants in vivo. Nature 1990; 346: 629-33. 10. Ono Y, Onda H, Sasada R, et al. The complete nucleotide sequences of the cloned
hepatitis B virus DNA; subtype adr and adw
Nucl Acid Res 1983; 11: 1747-57.
Combined HIV-1/2 assay kits SIR,—The lack of information about combined tests for HIV-1I and HIV-2 prompted us, like Dr Abiola and her colleagues (Dec 1, p 1386), to look at the performance of these kits.1 Using kits from four manufacturers, we came to similar conclusions: almost all kits have equal sensitivity in detecting the anti-HIV positive sera and anti-HIV-2 positive specimens from France and West Africa. Although we do not quibble with Abiola’s main conclusion, the only data she and her colleagues present are endpoint dilutions and this could give rise to misunderstandings about the performance of some of the kits. Having, like Abiola et al, diluted 4 HIV-2 positive serum samples, we tested four combined and one HIV-1 kit. At a 1/16 dilution all four combi-tests were positive for all 4 samples. The HIV-1kit (Wellcome), not unexpectedly, scored only 25% (1 in 4 samples) while the Wellcome combi-test had a 100% score (4 out of 4). However, at a 1/256 dilution the combi-kit scores (%) are: Pharmacia 0, Organon 25, Wellcome 75, and Pasteur 100. We found the same difference in endpoint scores with combi-tests for dilutions of HIV-1 positive sera, but recently seroconverted patients and donors were correctly identified by all four combi kits.’1 Nevertheless, the presentation of data in this way may be misleading because such comparative titrations are valid only if the test materials and reagents are similar. However, the present generation of tests differ in the choice of coating antigen (natural, recombinant, synthetic), in the technology of coupling to plastic, and in the choice of Nucleotide and deduced aminoacid sequences compared with reported sequences of HBV subtype adr. ’0 Left (and upper) preS2 Right (and lower) a determinant of S.
secondary enhancer-and every step, simultaneously or independently, could affect the dominant epitope to which specific antibody would react, rather than reflecting the spectrum of antibody present in the serum sample. Thus, at higher dilutions the endpoint score might reflect specific antibody directed against
Immunoselected
hepatitis
B virus mutant
SIR,-We have reported unusual serological data for hepatitis B virus (HBV) markers in a 66-year-old Japanese man with chronic hepatitis.’He had HBV DNA and HB e antigen in the presence of antibody to hepatitis B surface antigen and lacked both pre-S2 and antibody to pre-S2. Here we provide molecular evidence that the infecting virus had deletions in pre-S2 and aminoacid changes in the a determinant. This may be another instance of virus multiplication in the presence of immunity to HBsAg.2 DNA from serum was amplified by polymerase chain reaction (PCR)3,4 with HBV-specific primers. All the amplified fragments were smaller than expected and also smaller than those of the positive control. The fragments were cloned into pUC19 and the 4 positive colonies were sequenced by the dideoxy chain-termination method with some modification.’ The same serum was analysed in independent laboratories using different PCR primers (sequences available from K. M.) and the nucelotide sequences were found to be identical, suggesting the predominance of a single variant-type virus. The aminoacid deletions and changes consequent upon the altered DNA sequences were in positions aa9-22 (in the hydrophilic epitope), aa3, and aa8 of pre-S2, and at positions aal26, aa131, and aal33 in the first loop of the a determinanrö of the S product (figure).
The gene encoding the d and r determinants’ was unaltered. The same results were obtained on sera obtained in 1984 and 1989. The defective pre-S2 gene may explain why this patient with chronic hepatitis B lacked S products in his serum. These changes in surface determinants may have emerged through immunoselective pressure, although the process seems slower than it is in vivo in experimental murine retrovirus8 and lymphocytic choriomeningitis virus9 infections. Another possibility is that the mutant is infectious and was transmitted from another individual. These findings should be borne in mind when considering vaccine strategy. Department of Virology and First Department of Internal Medicine,
Kyushu University, Fukuoka 812, Japan, Institut Pasteur de Kyoto, Research Laboratory for Genetic Information, Kyushu University, Hamanomachi Hospital, Fukuoka, and Clinical Laboratory, Kyushu University Hospital 1.
K. MORIYAMA E. NAKAJIMA H. HOHJOH R. ASAYAMA K. OKOCHI
Moriyama K, Ishibashi H, Kashiwagi S, Asayama R. Presence of HBV DNA and HBeAg in serum of an anti-HBs-positive individual. Gastroenterology 1989; 97: 1068-69.
2. Carman
WF, Zanetti AR, Karayiannis P, et al. Vaccine-induced escape mutant of hepatitis B virus. Lancet 1990; 336: 325-29. 3. Thiers V, Nakajima E, Kresdorf D, et al. Transmission of hepatitis B from hepatitis seronegative subjects. Lancet 1987; ii: 1273-76. 4. Paterlini P, Gerken G, Nakajima E, et al. Polymerase chain reaction to detect hepatitis B virus DNA and RNA sequence m primary liver cancers from patients negative for hepatitis B surface antigen. N Engl JMed 1990; 323: 80-85. 5. Hattori M, Sakaki Y. Dideoxy sequencing methods using denatured plasmid templates. Analyt Biochem 1986; 152: 232-38 6. Brown SE, Howard CR, Zuckerman AJ, Steward MW. Affinity of antibody responses m man to hepatitis B vaccine determined with synthetic peptides. Lancet 1984; ii: 184-87. 7. Okamoto H, Imai
M, Tsuda F, et al. Point mutation in the S gene of hepatitis B virus d/y or w/r subtypic change in two blood donors carrying a surface antigen of compound subtype adyr or adwr. J Virol 1987; 61: 3030-34. 8. Pozsgay JM, Klysczek KK, Blank K. In vitro generation of antigenic variants of murine retroviruses. Virology 1989; 173: 330-34. for a
9. Pircher H,
Moskophidis D, Rohrer U, et al. Viral escape by selection of cytotoxic T cell-resistant virus variants in vivo. Nature 1990; 346: 629-33. 10. Ono Y, Onda H, Sasada R, et al. The complete nucleotide sequences of the cloned
hepatitis B virus DNA; subtype adr and adw
Nucl Acid Res 1983; 11: 1747-57.
Combined HIV-1/2 assay kits SIR,—The lack of information about combined tests for HIV-1I and HIV-2 prompted us, like Dr Abiola and her colleagues (Dec 1, p 1386), to look at the performance of these kits.1 Using kits from four manufacturers, we came to similar conclusions: almost all kits have equal sensitivity in detecting the anti-HIV positive sera and anti-HIV-2 positive specimens from France and West Africa. Although we do not quibble with Abiola’s main conclusion, the only data she and her colleagues present are endpoint dilutions and this could give rise to misunderstandings about the performance of some of the kits. Having, like Abiola et al, diluted 4 HIV-2 positive serum samples, we tested four combined and one HIV-1 kit. At a 1/16 dilution all four combi-tests were positive for all 4 samples. The HIV-1kit (Wellcome), not unexpectedly, scored only 25% (1 in 4 samples) while the Wellcome combi-test had a 100% score (4 out of 4). However, at a 1/256 dilution the combi-kit scores (%) are: Pharmacia 0, Organon 25, Wellcome 75, and Pasteur 100. We found the same difference in endpoint scores with combi-tests for dilutions of HIV-1 positive sera, but recently seroconverted patients and donors were correctly identified by all four combi kits.’1 Nevertheless, the presentation of data in this way may be misleading because such comparative titrations are valid only if the test materials and reagents are similar. However, the present generation of tests differ in the choice of coating antigen (natural, recombinant, synthetic), in the technology of coupling to plastic, and in the choice of Nucleotide and deduced aminoacid sequences compared with reported sequences of HBV subtype adr. ’0 Left (and upper) preS2 Right (and lower) a determinant of S.
secondary enhancer-and every step, simultaneously or independently, could affect the dominant epitope to which specific antibody would react, rather than reflecting the spectrum of antibody present in the serum sample. Thus, at higher dilutions the endpoint score might reflect specific antibody directed against
