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Correlation of HBV DNA and monoclonal reactivity to HBsAg in serum of patients with HBV infection
Journal of Virological Methods, Elsevier
14 (1986) 153-166
JVM 00521
Correlation of HBV DNA and monoclonal reactivity to HBsAg in serum of patients with HBV infection Jerome
B. Zeldis’.’ , Edna Ben-Porath2, Rafael Jack Wands’
Enat2, Katarina
Kirsch’,
‘Gastrointestinal Unit, Massachusetts General Hospital and Department of Medicine. Harvard Medical School, Boston, Massachusetts, USA, and ‘Section of Virology and Department of Medicine C, Rambam Medical Center, Haifa, Israel (Accepted
7 May 1986)
Hepatitis B virus (HBV) DNA hybridization assay. a monoclonal radioimmunoassay (M-RIA) for hepatitis B surface antigen (HBsAg) and conventional polyclonal immunoassays for HBV associated antigens were used to study sera from patients on dialysis and with acute hepatitis B. HBV DNA was detectable in hepatitis B e antigen (HBeAg) negative patients with acute hepatitis but not in HBsAg+ HBeAgdialysis patients. In acute hepatitis, HBsAg immunoreactivity by M-RIA could still be detected even though a commercial immunoassay for HBsAg, the AUSRIA II, and the HBV DNA assay were no longer positive. Unlike in acute HBV infection, serum HBV DNA was detectable in dialysis patients who were AUSRIA II negative but M-RIA positive. Serial determination of HBsAg by MRIA and HBV DNA revealed episodes of HBV DNA positivity months after both the HBsAg was no longer positive by polyclonal immunoassay. Thus. the M-RIA for HBsAg and the molecular hybridization technique for HBV DNA are sensitive and specific assays for the identification of potentially infectious individuals who would not have been characterized as such based on the results of conventional polyclonal immunoassays. hepatitis, dialysis
HBV DNA,
hybridization
assay,
monoclonal
antibody,
immunoassay,
hepatitis
B virus, renal
Introduction Assays for hepatitis B virus (HBV) related antigens have been useful in identifying individuals whose sera and secretions are potentially infectious. However, in some patients despite no serological evidence of active HBV infection based on conventional polyclonal assays for hepatitis B surface (HBsAg) and e (HBeAg) * Present address: Division MA 0221.5, USA. 0166-0934/86/$03.50
of Gastroenterology,
@ 1986 Elsevier
Science
Beth Israel Hospital,
Publishers
B.V.
330 Brookline
(Biomedical
Division)
Avenue,
Boston.
154
antigens, other markers for HBV infection can be detected. Some have in their livers detectable core antigen (HBcAg), HBsAg and replicative forms of HBV DNA. Furthermore, HBsAg associated epitopes may be found in serum by more sensitive monoclonal radioimmunoassays (M-RIA) (Brechot et al., 1982, 198.5; Figus et al., 1984). The infectivity of these patients’ sera and secretions are uncertain especially since a serum may be infectious yet lack any detectable HBV related antigens (Scotto et al., 1983; Tur-Kaspa et al., 1984). It has been shown that fewer than 16 HBV virions are needed to initiate infection in a susceptible chimpanzee (Berninger et al., 1982; Robinson, 1982; Feinman et al., 1984). Serum HBsAg is the best indirect marker for potentially infectious sera. Some HBsAg positive sera may not contain virions, hence be non-infectious. The antigen detected may be the product of a transcript of HBV DNA integrated into the hepatocytes’ genomes and infectious virions may not be produced (Brechot et al., 1981; Kam et al., 1982; Hadziyannis et al., 1983). Other sera may be infectious, yet may have a concentration of HBsAg below the sensitivities of polyclonal immunoassays. Thus, despite universal HBsAg screening, approximately 10 percent of post-transfusion hepatitis cases is due to HBV (Robinson, 1982). Although the number of HBV post-transfusion hepatites may decline with the adoption of more sensitive monoclonal immunoassays for HBsAg (Ben-Porath et al., 1984), some sera have concentrations of surface antigen even below the sensitivities of these assays and may be potentially infectious. It would therefore be of interest to determine if other means of detecting HBV infection such as HBV DNA are more sensitive than the M-RIA for HBsAg. The HBeAg assay is an indirect measure of viral replication. Even though the presence of HBeAg correlates well with the presence of Dane particles and intrahepatic viral replication, the absence of HBeAg in a given serum sample does not rule out its infectivity. For example, needlestick exposures from HBeAg negative individuals have been documented to be one-tenth as infectious as HBeAg positive exposures (Werner and Grady, 1982). Consistent with this, serum HBV DNA has been detected in some HBeAg negative individuals (Brechot et al., 1981; Berninger et al., 1982; Robinson, 1982; Matsuyama et al., 1985). Tests for serum HBV DNA and DNA polymerase are more direct markers for the presence of HBV virions. While DNA polymerase activity correlates well with infectivity, this assay is not as sensitive as the HBV DNA spot assay (Robinson, 1982; Scotto et al., 1983). Berninger et a1.(1982) and Feinman et al. (1984) demonstrated that the number of HBV DNA molecules per ml as determined by a spot assay is roughly equivalent to the infectious dose 50% per ml of chimpanzee sera. Investigators have shown that the concentration of serum HBV DNA and the HBeAg titer poorly correlate with each other (Lieberman et al., 1983; Tur-Kaspa et al., 1984); therefore, HBV DNA concentration may be a better index of infectivity than HBeAg status. We have employed a hybridization spot assay for HBV DNA in order to determine under what clinical conditions HBV DNA were found to coexist with monoclonal immunoreactive material. In this study, sera from patients with acute HBV infection and dialysis patients were examined. Using the HBV DNA spot assay,
155
we identified potentially infectious sera that would have only been characterized as such based on a M-RIA for HBsAg. Furthermore, the M-RIA for HBsAg was a more sensitive marker for HBV than the HBV DNA dot blot assay.
Materials
and Methods
Patient population
Patients with acute HBV infections and dialysis patients from the Rambam Medical Center, Haifa, Israel were studied. Fifty Israeli patients with acute HBV infection were tested in the Virology Laboratory of the Rambam Medical Center and were IgM anti-HBc positive. Twenty-two dialysis patients with acute and chronic HBV infection were tested. Other groups of patients studied were individuals whose blood was found to lack serological HBV markers or to be anti-HBs and anti-HBc positive by the blood bank of the Massachusetts General Hospital by routine screening using a polyclonal immunoassay for HBsAg, anti-HBs, and anti-HBc (Abbott Laboratories, North Chicago, IL). Sera from 52 anti-HBs+ Ethiopian Jews with no evidence of liver disease were also tested. Sera were stored at -20°C until assayed. Serology
Tests for HBsAg, HBeAg, anti-HBc, anti-HBs, anti-HBe, and IgM-anti-HBc were performed using commercial radioimmunoassays (Abbott Laboratories, North Chicago, IL). All assays were performed with an overnight incubation. The M-RIA kits for HBsAg were a gift from Centocor, Malvern, PA. IgM anti-HBc was tested by a modification of a method described by Schable and Maynard (1982). An aliquot of a dilution of test serum was absorbed on purified Staphylococcus aureus protein A bound to Seplxrose CL-4B (Pharmacia Fine Chemicals, Sweden). Post and pre-absorption aliquots were tested for anti-HBc by RIA (Corab, Abbott Laboratories, North Chicago, IL) and a ratio was calculated between the two samples (post/pre). A ratio value of less than 2.0 indicates the presence of IgM anti-HBc. DNA probes
PBR322 (Bolivar et al., 1977) and pHBV-1 were gifts of Dr. Brian Seed and Dr. Jesse Summers, respectively. pHBV-1 contains the entire genome of an adw hepatitis B virus inserted into PBR322 at its EcoRI restriction site. Both plasmid DNAs were obtained using standard bacteriological techniques (Maniatis et al., 1982). HBV DNA free of PBR322 vector DNA that was used as a hybridization probe was purified by preparative electrophoresis of an EcoRI digest of pHBV-1. The purity of the HBV DNA was checked by hybridization of the nick translated HBV DNA to EcoRI digested pHBV-1 that was Southern blotted to nitrocellulose (Schleicher & Schuell, Keene, New Hampshire) after electrophoresis. The 32P-labeled HBV DNA hybridized only to the HBV DNA (3.2 kb) band and not to the vector (4.5 kb) band. 32P-labeled PBR322 also did not bind to the purified HBV DNA,.
156
Nick translation was performed using standard techniques (Matsuyama et al., 1985) employing DNA polymerase I (Boehringer-Mannheim, Indianapolis, Indiana), DNase 1 grade 1 (Boehringer-Mannheim), radioactive dATP and dGTP [(CY32P)-labeled, 800 Ci/nmol, New England Nuclear], dCTP and dlTP (P.L. Biochemicals, Milwaukee, WI). Specific activities were typically between 2 and 6 x lo8 cpm/kg DNA. HBV DNA spot assay
The preparation of the serum sample and the neutralization were done as described (Scotto et al., 1983) but Biodyne A nylon membranes (Pall-Ultrafine Corp, Glen Cove, NY) were used instead of nitrocellulose and the membranes were baked for 1 h at 80°C in vacua instead of 2 h. The apparatus used for filtering was a Hybridot manifold (Bethesda Research Laboratories), the upper tier of which had been washed in 3% hydrogen peroxide to eliminate any possible DNA contamination and rinsed with distilled water. The nylon membranes were wet in 6 x SSC (1 x SSC = 0.15 M NaCl, 0.015 sodium citrate, pH 7.0) before spotting and after baking. Prior to using this technique, we independently determined that serum HBV DNA could be immobilized on a nitrocellulose or nylon filter and detected by hybridization to 32P-labeled HBV DNA probes if the Dane particles were disrupted with alkaline treatment. In earlier studies, proteinase K treatment and phenol extraction of the serum samples were performed but these steps were later omitted for reasons explained below. The membranes were prehybridized for at least 2 h at 68°C in 0.2 ml per sq cm filter of 6 x SSC, 0.5% SDS, 5 X Denhardt’s solution [l x Denhardt’s solution: 0.02% Ficoll 400 (Pharmacia, Piscataway, NJ), 0.02% polyvinylpyrrolidone 360 (Sigma, St. Louis, MO)], 0.02% bovine serum albumin fraction V [Sigma], and 100 P&ml alkaline denatured salmon testes DNA (type III, Sigma). Hybridization with 32P-labeled DNA probes occurred overnight at 68°C in 0.05 ml per sq cm of filter in 6 x SSC, 0.5% SDS, 5 X Denhardt’s solution, 10 mM EDTA, 100 pg/ml alkaline denatured salmon testes DNA, and 100 ng of alkaline denatured 32P-labeled DNA probe for each 400 sq cm of nylon filter. The filters were washed at room temperature with 2 X SSC with 0.5% SDS for 5 min, then 2 X SSC with 0.1% SDS for 15 min, then twice for 1 h in 0.1 X SSC with 0.5% SDS at 68°C. The filters were air dried, labeled with an UltEmit pen (New England Nuclear), wrapped in Saran wrap and autoradiographed overnight and for seven days at -70°C using Kodak X-Omat XAR Film and a Cronex Lightning-Plus enhancing screen (DuPont, Wilmington, DE). Each serum sample was examined blind under code and was spotted in duplicate. One membrane was hybridized with nick translated PBR322 DNA to control for bacterial contamination and the other was annealed with 32P-labeled HBV DNA. With each membrane, a control nylon membrane was also included that contained 100, 10, 1, 0.1, and 0.01 pg spots of denatured PBR322 DNA and pHBV1 DNA. Each filter included a HBV DNA positive and negative control serum. The negative control sera were obtained from the M-RIA for HBsAg from Centocor. Most samples were tested more than once. In a typical experiment, 50 l.~l
157
of sera was tested and less than 0.1 pg of DNA (i.e. 2.8 x lo4 molecules of HBV DNA) could be detected after seven days autoradiography. Southern blot analysis of serum
Sera were prepared by digestion in 1.4 mg/ml proteinase K, 90 mM Tris, pH 8.0, 23 mM EDTA, 0.9% SDS at 37°C overnight and electrophoresed in 1% agarose. The method described by Southern in (Maniatis et al., 1982) for denaturing and blotting DNA to nitrocellulose membranes was employed. The membranes were wet in 6 x SSC, baked in vacua at 80°C for 1 to 2 h, wet in 6 x SSC and hybridized to 32P-labeled DNA probes as described above in the HBV DNA spot assay.
Results The dot blot assay
A number of procedures were tried to develop a rapid, reproducible hybridization assay for the detection of HBV DNA. Proteinase K digestion followed by phenol extraction, ethanol precipitation and spotting onto filters gave reproducible results; however, the method was tedious and not as sensative as directly spotting or filtering through a nitrocellulose or nylon filter. Hybridization of 32P-labeled DNA probes to alkaline denatured filters spotted with serum was determined to be more sensitive for detecting HBV DNA sequences than any other method of preparing the filters. Some authors (Lieberman et al., 1983) have recommended proteinase K treatment of the spotted filter, however, we found that such enzyme digestion disproportionately decreased the hybridization signal of serum HBV DNA compared to that of spotted plasmid DNA. Figure 1 demonstrates the autoradiographic spots produced after hybridizing radioactive HBV DNA probes to filters either blotted with HBV DNA+ sera or plasmid HBV DNA after three different treatments after alkaline denaturation: incubation with albumin; incubation with
NO TREATMENT
ALBUMIN
PROTEINASE
K
SERUM
PLASMID HBV DNA
It
*
0
)
II
.
Fig. 1. Effect of proteinase K treatment on the HBV DNA dot blot assay. Sera and plasmid DNA were spotted and treated as described in Table 1.
158 TABLE Optical
Plasmid Serum
1 density
units of a 30 h autoradiogram
HBV DNA neat
l/10 dilution of serum
of plasmid
and serum
HBV DNA
Alkali denaturation
BSA treatment
Proteinase treatment
2.80
2.14
2.23
(100 pg/mU 2.26
(74) 2.24
(85) 0.18
(96) 0.58 (10.5)
(88) 0.27
(6.8) 0
K
(7)
2 )11of serial dilutions of plasmid DNA or a HBV DNA+ sera were spotted on three nylon filters. The filters were immersed in 0.5 M NaOH, 2.5 M NaCl for 1530 s then neutralized in 3 M sodium acetate, pH 5. Two filters were then rinsed in 1 M Tris pH 8 for 1 min then incubated at 50°C with either proteinase K 200 &ml or BSA 200 &ml in 0.5 M EDTA 0.5% Sarkosyl for 1 h. All three filters were rinsed briefly in 2 X SSC, air dried, baked in vacua at 80°C for 1 h and hybridized to a “2P-labeled HBV DNA probe as described in Materials and Methods. The optical density units were determined on a Tobins Associates Densitometer Model TBX. The numbers listed are units minus the background. The numbers in parentheses are the estimated amount of HBV DNA based on a curve of optical density units versus log HBV DNA concentration of an alkaline denatured filter containing concentrations of plasmid derived HBV DNA.
proteinase K, or no further treatment. Table 1 summarizes the optical density readings of this experiment. Both proteinase K and albumin treated plasmid HBV DNA had approximately the same optical density which was less than untreated plasmid DNA. Proteinase K treatment of serum resulted in a markedly smaller signal than either the untreated or albumin exposed filter. Based on optical density, the proteinase K treated serum would have been estimated to contain less than one-tenth the HBV DNA concentration as the same serum either treated with albumin or with alkaline dena:;r,:ion only. Based on experiments like these, it was found that serum could either be spotted directly onto nitrocellulose then denatured, or denatured then passed through the filter with equal results. By filtering denatured serum, a larger volume of sample could be used, thereby increasing the sensitivity of the assay. Nylon filters were used in the experiments described below because they immobilized DNA as well as nitrocellulose filters and were more durable. In order to prove that positive spots were due to HBV DNA, 14 dot blot positive serum samples were digested with proteinase K, electrophoresed in agarose, Southern blotted onto nitrocellulose, and hybridized to 32P-labeled HBV DNA or PBR322. As illustrated in Fig. 2, samples that gave a moderately intense spot in the HBV DNA assay produced a detectable smear that was 3.2 kb and smaller. This pattern is consistent with the molecular forms of HBV DNA molecules in serum. Other samples that had less intense spots on the assay were indirectly demonstrated to contain HBV DNA by the loss of a positive signal after depurination by low pH treatment (data not shown). One hundred and ninety-nine control sera were found to have no detectable HEW DNA by the dot blot assay (Table 2). These sera included 129 blood donor sera
159
12345678
123456789
9
7.2
r)
5.7 4.8 4-3 3.6
:
2.3 1.9
1.4
kb HlGl-i
HBV STRINGENCY
WASH
LOW
PBR 322 STRINGENCY
WASH
Fig. 2. Southern blot analysis of HBV DNA fot blot positive sera. Fifty ~1 of sera were prepared as directed in Materials and Methods. The Southern blotted NC filter was hybridized to 32P-labeled HBV or PBR322 DNA probes. The autoradiogram shown was developed after a 7 day exposure to the NC filter. The intensity of the spots on HBV dot blot assay was most intense for samples 2 and 9. Samples 5 and 8 gave faint spots after 7 days autoradiography. kb = kilobase pairs of DNA.
that were negative for anti-HBs, anti-HBc, and HBsAg; 63 anti-HBs+ anti-HBc+ patients with normal liver function tests; 2 patients with acute HAV infections; 3 patients with chronic active hepatitis due to non-A, non-B hepatitis; and 1 patient each with acquired immune deficiency syndrome and Epstein-Barr viral hepatitis. Each of the 199 samples had no detectable HBsAg by both the polyclonal AUSRIA II assay and the M-RIA. Acute HBV hepatitis
Serum samples from 50 patients who had acute HBV hepatitis were assayed for the presence of HBV DNA. All patients had an acute rise in serum aminotransferase and were IgM anti-HBc and HBsAg positive. While 55% (12 of 22) of pa-
160 TABLE
2
Results
of serum
AUSRIA and M-RIA
HBV DNA in patients
II Anti-HBs
_
_
with no evidence
of current
HBV infection
Anti-HBc
HBV DNA
Comments
-
O/129
Blood donors on patients with no evidence active disease 52 Ethiopian Jews, 11 MGH blood donors EBV hepatitis Non-A, non-B CAH Acute HAV AIDS
0163 Oil o/3 012 O/l Total:
O/199 were HBV DNA
of
positive
tients who were HBeAg+ were HBV DNA positive, only 17% (3 of 18) of the HBeAg- persons were HBV DNA positive. Thirty-six percent of all 50 HBsAg+ patients were HBV DNA positive at time of serological diagnosis. Of note, 29% (2 of 7) of HBeAg- anti-HBe+ patients with acute HBV infection had detectable HBV DNA. Serial serum samples were obtained from 13 of the 50 patients with acute HBV hepatitis. Table 3 lists the data for 5 patients. In all cases, the M-RIA for HBsAg was more sensitive than the HBV DNA assay in detecting HBV-related determinants. No example of a M-RIA negative serum that was HBV DNA positive was found in this group of patients with acute hepatitis B. HBV DNA was not detected in any patient who was HBsAg- by AUSRIA II and anti-HBe+ (data not shown). Figure 3 plots the course of an Israeli who developed acute HBV infection. Although he resolved clinically, he later developed chronic lobular hepatitis. During
Cllnlcol ALTZ
2x
IgM
course
23
_I_
Normal
+
-
-
++
++.
HBeAg
+++
HBsAg
.++
+
AntI-HBc
+ . +
1
+a
HBV
+ - -
+*
l
DNA
, J
l
I, I, / I I FMAMJJASONDJ 1982
I
/
t
l
I
I
I
/
++
1
I
++*
+
+ + ++
-
I
l
_ --
l
l
t++
I,
I1
I,
I
i 983
1
c 14
FMAMJJASONDJ I
f
++ - -
+
1
6
2
4
L
I
FMAMJ 1
I
/
1 I
I
L
JASON
1984
Fig. 3. Clinical, biochemical, serological, and histological course of a 22 year old man who developed acute hepatitis B that progressed to chronic lobular hepatitis. Clinical course notes: 1, acute viral hepatitis; 2, nausea, anorexia, fatigue; 3, liver biopsy-nodular hepatitis; 4, asymptomatic; 5, nausea, fatigue; 6, liver biopsy-chronic lobular hepatitis; 7, asymptomatic.
161 TABLE
3
Serial determination Patient 1
no.
of serum
HBV DNA” in patients
Date
AUSRIA
319181 4112 4120 5/20
+ + _ _
2/l/81 215 2119
+ + -
l/30/81 2114 2119
+ + +
9/19/81 10127
+ _
3/26/81 417 4116 4/28,5/10 5/20,5/31 619 6116 7127
+ + + + + + _
II
with acute HBV
M-RIAb
HBeAg/anti-HBe
DNA
+
-I -I
+
_
+ fl
_ -
+ -I+
_ -
+i+
+ _
+ +
+ _ _ _
a +, a positive spot was seen after autoradiography for 7 days; this implies an HBV DNA concentration greater than 2.8 x 105 molecules/ml. -, no spot was detected. b M-RIA has been found to be positive if AUSRIA II is positive; therefore, M-RIA was usually not performed if AUSRIA II was positive.
the 34 months in which he was followed, he remained HBsAg and HBeAg positive but the IgM anti-HBc antibody converted from positive to negative. The serum HBV DNA became negative following the initial clinical episode of acute hepatitis, only to become detectable again with the recrudescence of his liver disease. Dialysis patients
Multiple serum samples from 22 dialysis patients who were HBsAg+ were studied. Sixty-eight percent (Z/22) of serum samples were HBV DNA positive. Nine of 10 (90%) HBsAg+ HBeAg+ sera were HBV DNA positive. This is in contrast to 55% HBV DNA+ patients with acute HBV (vide supra). No patient who was HBsAg+ HBeAg- had detectable serum HBV DNA. Serial serum samples were obtained from 12 HBsAg+ dialysis patients. Data from 8 patients are listed in Table 4. In seven of the 8 dialysis patients who were HBeAg+ , HBV DNA was detectable at the time the antigen was detectable. However, the absence of HBeAg positivity did not exclude the possibility that a patient would later become HBV DNA positive. For example, over a 28 month period, dialysis patient no. 5 was
162 TABLE
4
Serial determinations
on eight HBsAg+
dialysis
patients HBsA.g
Patient 1
no.
Date
HBeianti-HBe
115182 212182 312182 4182 9182
5
12/81,1/82,3182 4182,6/82 9182 12/81,1/83 2183
6
l/81 5182,7182 8/82,9/82 10/82,11/82 12182
L
ii t +
+/
_
_
+
6180 10/30/80 1011181 1 llll81 1211181 115182,2/2182,511182
4
HBV DNA II M-RIA*
+
9/80 10180 l/81 2181 6181 3182
9/8/80 l/6/81 515181 6124181 914181 312182 513182
AUSRIA
_ + t _
_
t t
+ + + t
fl -/
+I-
_
_ t _
_ + +
_ + t
t + + t + t + + + +
+I ii
10/80,6/81 8/81,12/82
ti
+ +
+ +
815182 912182 1113 l/4183
ii
+ + + -
+ t + _
* M-RIA is positive RIA II was positive.
if AUSRIA
21
II is positive,
therefore,
M-RIA
_ was not always
performed
if AUS-
163
repeatedly HBsAg+ by AUSRIA II and HBV DNA negative. One serum sample (dated 9/82) was HBeAg negative. On the 29th month of surveillance, the patient was both HBsAg and HBV DNA positive. Dialysis patients no. 3 and no. 4 probably represent examples of either reactivation of the HBV infection or increasing severity of infection with HBV DNA being detectable again months after the patients were serum HBV DNA-. It should be noted that at all times the M-RIA for HBsAg was positive. Dialysis patients no. 2 and no. 3 are examples that AUSRIA II negative patients may be infectious for HBV. Fourteen months after initial surveillance the patient no. 2 was no longer HBsAg positive by the AUSRIA II assay but positive by the .M-RIA for at least 20 months longer. More importantly, HBV DNA was detectable during a two-month period (November-December 1981), 13 months after being HBsAg negative by AUSRIA II. Similarly, patient no. 3 was HBV DNA positive 9 months after becoming HBsAg negative by the AUSRIA II assay. Thus patients no. 2 and no. 3 would not have been considered potentially infectious based on conventional HBV serology, but would be identified as infectious based on the more sensitive M-RIA and HBV DNA assays. The data on dialysis patients no. 2, no. 3, no. 4, and no. 5 illustrate that a negative test for HBV DNA does not preclude the possibility that the patient will become HBV DNA positive at a later time. Dialysis patient no. 8 is an example of a resolving acute HBV infection in a dialysis patient.
Discussion
Berninger et al. (1982) and Feinman et al. (1984) have estimated the concentration of HBV DNA molecules in serum by dot blot assays. These investigators then demonstrated that the DNA concentration approximates the concentration of infectious virions estimated from the serum ID,, for chimpanzees. Since the ability to perform these types of infectivity studies on chimpanzees is limited, it is not known if every serum that is HBV DNA positive contains intact virions and is, therefore, infectious. A safe assumption based on the work of Berninger et al. (1982) and Feinman et al. (1984) is that HBV DNA positive sera are potentially infectious. Furthermore, the identification of HBsAg by a sensitive M-RIA for HBsAg in sera that were HBsAg- by AUSRIA II but HBV DNA positive further supports the contention that these sera are potentially infectious. Our results have confirmed other investigators’ observations (Brechot et al., 1981; Berninger et al., 1982; Robinson, 1982; Weller et al., 1982; Matsuyama et al., 1985) that HBeAg positivity does not always correlate well with the presence of HBV DNA. A negative test for HBeAg does not rule out the serum’s potential infectivity. The manner in which the DNA hybridization assay was performed enabled sera to be detected as positive if there were a minimum of 5.6 x 105 DNA molecules per ml. It is not surprising that 45% of HBeAg+ patients with acute HBV hepatitis were HBV DNA-. These HBeAg+ sera are probably infectious since fewer than 5.6 x lo4 HBV virions are needed to initiate infection. Sera can be
164
identified that are HBsAg-t by M-RIA, but negative for HBeAg, HBV DNA and HBsAg by AUSRIA II. Since at this time no rapid, inexpensive assay can determine if the HBsAg detected by M-RIA is virion associated or produced from chromosomally integrated HBV DNA, such sera should be considered potentially infectious. Neither the assays for HBeAg and HBV DNA are more sensitive than M-RIA for HBsAg, therefore, the HBeAg and HBV DNA tests can only be used as further evidence that a particular serum sample that is HBsAg+ by M-RIA may be infectious. A negative HBV DNA or HBeAg test does not exclude the potential infectivity of that particular serum sample. Dialysis patients were studied because compared to the general population they are relatively immunosuppressed. HBV infections are often prolonged in this group. Since medical personnel are frequently exposed to their blood, it is important to determine how potentially infectious these patients are. Our results demonstrated that a higher percentage of HBeAg+ and HBsAg+ dialysis patients have detectable serum HBV DNA than patients with acute HBV hepatitis. The concentration of HBV DNA in dialysis patients in general was greater than patients with acute HBV hepatitis (data not presented). Serial studies on these patients demonstrated that the absence of HBV DNA does not preclude the possibility that a patient will later become HBV DNA positive, even though HBsAg may remain undetectable by the AUSRIA II assay. This point is especially of relevance to dialysis patients who often have transient rises in their aminotransferases that are attributable to non-A non-B hepatitis because they are HBsAg negative by AUSRIA II. The detection of HBsAg by M-RIA and HBV DNA in these AUSRIA II negative patients is consistent with the abnormal liver function tests being due to HBV, not some other agent. Our results have confirmed the findings of Scotto et al. (1983) that sera only need to be alkaline denatured and not treated with a protein digestion step to obtain a sensitive and specific HBV DNA assay. Proteinase K treatment decreases the sensitivity of detecting serum HBV DNA, by greater than lo-fold, but affects the hybridization signal of plasmid DNA only by about 30%. This suggests that an assay that includes a proteinase K treatment may have a higher rate of false negativity than an assay that omits this step, even though the sensitivity of both assays may be identical based on plasmid DNA standards. A possible reason for the decrease in hybridization signal after proteinase K treatment is that both nitrocellulose and nylon filters bind denatured (single stranded) DNA and protein. It is possible that the protein covalently bound to the 5’ end of the ‘long’ strand of the HBV DNA molecule helps to anchor it to the filter and that proteinase K treatment removes most of this protein. Alkaline treatment alone is sufficient to denature the intact virion and double stranded DNA so that it can be bound by the filter. A sufficient amount of the HBV genome is exposed to the radioactive probe to allow detection despite the excess denatured virion and serum protein also bound to the filter. In conclusion, the results of this study consolidate and extend the experience of others that assays for HBV DNA are sensitive and specific for identifying potentially infectious individuals. The sensitivity of the assay can be increased by omitting proteinase K treatment of sera prior to DNA hybridization. While the com-
165
mercial polyclonal RIA for HBsAg is an excellent screening test, these are a significant number of sera that contain HBsAg that are not detected by this assay and are infectious for HBV. The monoclonal antibody immunoassay for HBsAg is clearly the most sensitive immunoassay for HBV related antigens. The serum HBV DNA assay by measuring the presence of the genome can directly determine whether a particular serum is potentially infectious. At this time, its sensitivity is not greater than the monoclonal immunoassay for HBsAg; therefore, the HBV DNA assay’s place in the diagnostic armamentarium may be only to confirm, but not disprove, the hypothesis that an individual has circulating Dane particles and does not just have polymeric HBsAg in his serum.
Acknowledgements This work was supported in part by NIH Grants no. AA-02226 and no. CA35711, and NIH Research Career Development Award no. AA-00048 to J.W. J.B.Z. is a Pfizer Post-Doctoral Fellow.
References Ben-Porath, E., J. Wands, M. Gruia and K. Isselbacher, 1984, Clinical significance of enhanced detection of HBsAg by a monoclonal radioimmunoassay. Hepatology 4, 1269-1273. Berninger, M., M. Hammer, B. Hoyer and J.L. Gerin, 1982, An assay for the detection of the DNA genome of hepatitis B virus in serum. J. Med. Virol. 9, 57-6X. Bolivar, F., R.L. Rodriguez, P.J. Greene, et al., 1977, Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2, 95. Brechot, C., J. Scotto, P. Charnay, et al., 1981. Detection of hepatitis B virus DNA in liver and serum: A direct appraisal of the chronic carrier state. Lancet 2, 767-768. Brechot, C., F. Degos, E.S. Zafrani, D. Franc0 and P. Berthelot, 1982, Demonstration of latent HBV infection by the presence of HBV DNA in the liver. Hepatology (abstract) 2, 747. Brechot, C., F. Degos, C. Lugassy, et al., 1985, Hepatitis virus DNA in patients with chronic liver desease and negative tests for hepatitis B surface antigen. N. Engl. J. Med. 312, 270-276. Feinman, S.V., B. Berris, A. Guha, et al., 1984, DNA:DNA hybridization method for the diagnosis of hepatitis B infection. J. Viral. Methods 8, 199-206. Figus, A., H.E. Blum, G.N. Vyas, et al., 1984, Hepatitis B viral nucleotide sequences in non-A, nonB or hepatitis B virus related chronic liver desease. Hepatol. 4, 36%368. Hadziyannis, S.J., H.M. Lieberman, G.G. Karvountzis and D.A. Shafritz, 1983, Analysis of liver disease, nuclear HBcAg, viral replication, and hepatitis B virus DNA in liver and serum of HBeAg vs. anti-HBe positive carriers of hepatitis B virus. Hepatol. 3, 656-662. Kam, W., L.B. Rail, E.A. Smuckler, R. Schmidt and W.J. Rutter, 1982, Hepatitis B viral DNA in liver and serum of asymptomatic carriers. Proc. Natl. Acad. Sci. USA 79, 7522-7526. Lieberman, H.M., D.R. LaBrecque, M.C. Kew, S.J. Hadziyannis and D.A. Shafritz, 1983, Detection of hepatitis B virus DNA directly in human serum by a simplified molecular hybridization test: comparison to HBeAg/anti-HBe status in HBsAg carriers. Hepatol. 3, 285-291. Maniatis, ‘I., E.F. Fritsch and J. Sambrook, 1982, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Matsuyama, Y., M. Omata, 0. Yokosuka, F. Imazeki, Y. Ito and K. Okuda, 1985, Discordance of hepatitis B e antigen/antibody and hepatitis B virus deoxyribonucleic acid in serum: Analysis of 1063 specimens. Gastro 89, 1104-1108.
166 Robinson, W., 1982, The biology of human hepatitis viruses, in: Hepatology: A Textbook of Liver Disease, eds. D. Zakim and T.D. Boyer (W.B. Saunders, Philadelphia) pp. 863-898. Schable, C.A. and J.E. Maynard, 1982, Serodiagnosis of acute hepatitis B virus infection by a modified competitive binding radioimmunoassay. J. Clin. Microbial. 16, 973-975. Scotto, J., M. Hadchovel, C. Hery, J. Yuart, P. Tiollais and C. Brechot, 1983, Detection of hepatitis B virus DNA in serum by simple spot hybridization technique: Comparison with results for other viral markers. Hepatol. 3, 279-284. Tur-Kaspa, R., F. Keshet, M. Eliakim and D. Shouval, 1984, Detection and characterization of hepatitis B virus DNA in serum of HBeAntigen-Negative HBsAg carriers. J. Med. Viral. 14, 17-26. Weller, J.V.D., M.J.F. Fowler, J. Monjardino and H.C. Thomas, 1982, The detection of HBV-DNA in serum by molecular hybridization: A more sensitive method for the detection of complete HBV particles. J. Med. Viral. 9, 272-280. Werner, B.G. and G.F. Grady, 1982, Accidental hepatitis B surfaced antigen positive innoculations: Use of e antigen to estimate infectivity. Ann. Intern. Med. 97, 367-369.
14 (1986) 153-166
JVM 00521
Correlation of HBV DNA and monoclonal reactivity to HBsAg in serum of patients with HBV infection Jerome
B. Zeldis’.’ , Edna Ben-Porath2, Rafael Jack Wands’
Enat2, Katarina
Kirsch’,
‘Gastrointestinal Unit, Massachusetts General Hospital and Department of Medicine. Harvard Medical School, Boston, Massachusetts, USA, and ‘Section of Virology and Department of Medicine C, Rambam Medical Center, Haifa, Israel (Accepted
7 May 1986)
Hepatitis B virus (HBV) DNA hybridization assay. a monoclonal radioimmunoassay (M-RIA) for hepatitis B surface antigen (HBsAg) and conventional polyclonal immunoassays for HBV associated antigens were used to study sera from patients on dialysis and with acute hepatitis B. HBV DNA was detectable in hepatitis B e antigen (HBeAg) negative patients with acute hepatitis but not in HBsAg+ HBeAgdialysis patients. In acute hepatitis, HBsAg immunoreactivity by M-RIA could still be detected even though a commercial immunoassay for HBsAg, the AUSRIA II, and the HBV DNA assay were no longer positive. Unlike in acute HBV infection, serum HBV DNA was detectable in dialysis patients who were AUSRIA II negative but M-RIA positive. Serial determination of HBsAg by MRIA and HBV DNA revealed episodes of HBV DNA positivity months after both the HBsAg was no longer positive by polyclonal immunoassay. Thus. the M-RIA for HBsAg and the molecular hybridization technique for HBV DNA are sensitive and specific assays for the identification of potentially infectious individuals who would not have been characterized as such based on the results of conventional polyclonal immunoassays. hepatitis, dialysis
HBV DNA,
hybridization
assay,
monoclonal
antibody,
immunoassay,
hepatitis
B virus, renal
Introduction Assays for hepatitis B virus (HBV) related antigens have been useful in identifying individuals whose sera and secretions are potentially infectious. However, in some patients despite no serological evidence of active HBV infection based on conventional polyclonal assays for hepatitis B surface (HBsAg) and e (HBeAg) * Present address: Division MA 0221.5, USA. 0166-0934/86/$03.50
of Gastroenterology,
@ 1986 Elsevier
Science
Beth Israel Hospital,
Publishers
B.V.
330 Brookline
(Biomedical
Division)
Avenue,
Boston.
154
antigens, other markers for HBV infection can be detected. Some have in their livers detectable core antigen (HBcAg), HBsAg and replicative forms of HBV DNA. Furthermore, HBsAg associated epitopes may be found in serum by more sensitive monoclonal radioimmunoassays (M-RIA) (Brechot et al., 1982, 198.5; Figus et al., 1984). The infectivity of these patients’ sera and secretions are uncertain especially since a serum may be infectious yet lack any detectable HBV related antigens (Scotto et al., 1983; Tur-Kaspa et al., 1984). It has been shown that fewer than 16 HBV virions are needed to initiate infection in a susceptible chimpanzee (Berninger et al., 1982; Robinson, 1982; Feinman et al., 1984). Serum HBsAg is the best indirect marker for potentially infectious sera. Some HBsAg positive sera may not contain virions, hence be non-infectious. The antigen detected may be the product of a transcript of HBV DNA integrated into the hepatocytes’ genomes and infectious virions may not be produced (Brechot et al., 1981; Kam et al., 1982; Hadziyannis et al., 1983). Other sera may be infectious, yet may have a concentration of HBsAg below the sensitivities of polyclonal immunoassays. Thus, despite universal HBsAg screening, approximately 10 percent of post-transfusion hepatitis cases is due to HBV (Robinson, 1982). Although the number of HBV post-transfusion hepatites may decline with the adoption of more sensitive monoclonal immunoassays for HBsAg (Ben-Porath et al., 1984), some sera have concentrations of surface antigen even below the sensitivities of these assays and may be potentially infectious. It would therefore be of interest to determine if other means of detecting HBV infection such as HBV DNA are more sensitive than the M-RIA for HBsAg. The HBeAg assay is an indirect measure of viral replication. Even though the presence of HBeAg correlates well with the presence of Dane particles and intrahepatic viral replication, the absence of HBeAg in a given serum sample does not rule out its infectivity. For example, needlestick exposures from HBeAg negative individuals have been documented to be one-tenth as infectious as HBeAg positive exposures (Werner and Grady, 1982). Consistent with this, serum HBV DNA has been detected in some HBeAg negative individuals (Brechot et al., 1981; Berninger et al., 1982; Robinson, 1982; Matsuyama et al., 1985). Tests for serum HBV DNA and DNA polymerase are more direct markers for the presence of HBV virions. While DNA polymerase activity correlates well with infectivity, this assay is not as sensitive as the HBV DNA spot assay (Robinson, 1982; Scotto et al., 1983). Berninger et a1.(1982) and Feinman et al. (1984) demonstrated that the number of HBV DNA molecules per ml as determined by a spot assay is roughly equivalent to the infectious dose 50% per ml of chimpanzee sera. Investigators have shown that the concentration of serum HBV DNA and the HBeAg titer poorly correlate with each other (Lieberman et al., 1983; Tur-Kaspa et al., 1984); therefore, HBV DNA concentration may be a better index of infectivity than HBeAg status. We have employed a hybridization spot assay for HBV DNA in order to determine under what clinical conditions HBV DNA were found to coexist with monoclonal immunoreactive material. In this study, sera from patients with acute HBV infection and dialysis patients were examined. Using the HBV DNA spot assay,
155
we identified potentially infectious sera that would have only been characterized as such based on a M-RIA for HBsAg. Furthermore, the M-RIA for HBsAg was a more sensitive marker for HBV than the HBV DNA dot blot assay.
Materials
and Methods
Patient population
Patients with acute HBV infections and dialysis patients from the Rambam Medical Center, Haifa, Israel were studied. Fifty Israeli patients with acute HBV infection were tested in the Virology Laboratory of the Rambam Medical Center and were IgM anti-HBc positive. Twenty-two dialysis patients with acute and chronic HBV infection were tested. Other groups of patients studied were individuals whose blood was found to lack serological HBV markers or to be anti-HBs and anti-HBc positive by the blood bank of the Massachusetts General Hospital by routine screening using a polyclonal immunoassay for HBsAg, anti-HBs, and anti-HBc (Abbott Laboratories, North Chicago, IL). Sera from 52 anti-HBs+ Ethiopian Jews with no evidence of liver disease were also tested. Sera were stored at -20°C until assayed. Serology
Tests for HBsAg, HBeAg, anti-HBc, anti-HBs, anti-HBe, and IgM-anti-HBc were performed using commercial radioimmunoassays (Abbott Laboratories, North Chicago, IL). All assays were performed with an overnight incubation. The M-RIA kits for HBsAg were a gift from Centocor, Malvern, PA. IgM anti-HBc was tested by a modification of a method described by Schable and Maynard (1982). An aliquot of a dilution of test serum was absorbed on purified Staphylococcus aureus protein A bound to Seplxrose CL-4B (Pharmacia Fine Chemicals, Sweden). Post and pre-absorption aliquots were tested for anti-HBc by RIA (Corab, Abbott Laboratories, North Chicago, IL) and a ratio was calculated between the two samples (post/pre). A ratio value of less than 2.0 indicates the presence of IgM anti-HBc. DNA probes
PBR322 (Bolivar et al., 1977) and pHBV-1 were gifts of Dr. Brian Seed and Dr. Jesse Summers, respectively. pHBV-1 contains the entire genome of an adw hepatitis B virus inserted into PBR322 at its EcoRI restriction site. Both plasmid DNAs were obtained using standard bacteriological techniques (Maniatis et al., 1982). HBV DNA free of PBR322 vector DNA that was used as a hybridization probe was purified by preparative electrophoresis of an EcoRI digest of pHBV-1. The purity of the HBV DNA was checked by hybridization of the nick translated HBV DNA to EcoRI digested pHBV-1 that was Southern blotted to nitrocellulose (Schleicher & Schuell, Keene, New Hampshire) after electrophoresis. The 32P-labeled HBV DNA hybridized only to the HBV DNA (3.2 kb) band and not to the vector (4.5 kb) band. 32P-labeled PBR322 also did not bind to the purified HBV DNA,.
156
Nick translation was performed using standard techniques (Matsuyama et al., 1985) employing DNA polymerase I (Boehringer-Mannheim, Indianapolis, Indiana), DNase 1 grade 1 (Boehringer-Mannheim), radioactive dATP and dGTP [(CY32P)-labeled, 800 Ci/nmol, New England Nuclear], dCTP and dlTP (P.L. Biochemicals, Milwaukee, WI). Specific activities were typically between 2 and 6 x lo8 cpm/kg DNA. HBV DNA spot assay
The preparation of the serum sample and the neutralization were done as described (Scotto et al., 1983) but Biodyne A nylon membranes (Pall-Ultrafine Corp, Glen Cove, NY) were used instead of nitrocellulose and the membranes were baked for 1 h at 80°C in vacua instead of 2 h. The apparatus used for filtering was a Hybridot manifold (Bethesda Research Laboratories), the upper tier of which had been washed in 3% hydrogen peroxide to eliminate any possible DNA contamination and rinsed with distilled water. The nylon membranes were wet in 6 x SSC (1 x SSC = 0.15 M NaCl, 0.015 sodium citrate, pH 7.0) before spotting and after baking. Prior to using this technique, we independently determined that serum HBV DNA could be immobilized on a nitrocellulose or nylon filter and detected by hybridization to 32P-labeled HBV DNA probes if the Dane particles were disrupted with alkaline treatment. In earlier studies, proteinase K treatment and phenol extraction of the serum samples were performed but these steps were later omitted for reasons explained below. The membranes were prehybridized for at least 2 h at 68°C in 0.2 ml per sq cm filter of 6 x SSC, 0.5% SDS, 5 X Denhardt’s solution [l x Denhardt’s solution: 0.02% Ficoll 400 (Pharmacia, Piscataway, NJ), 0.02% polyvinylpyrrolidone 360 (Sigma, St. Louis, MO)], 0.02% bovine serum albumin fraction V [Sigma], and 100 P&ml alkaline denatured salmon testes DNA (type III, Sigma). Hybridization with 32P-labeled DNA probes occurred overnight at 68°C in 0.05 ml per sq cm of filter in 6 x SSC, 0.5% SDS, 5 X Denhardt’s solution, 10 mM EDTA, 100 pg/ml alkaline denatured salmon testes DNA, and 100 ng of alkaline denatured 32P-labeled DNA probe for each 400 sq cm of nylon filter. The filters were washed at room temperature with 2 X SSC with 0.5% SDS for 5 min, then 2 X SSC with 0.1% SDS for 15 min, then twice for 1 h in 0.1 X SSC with 0.5% SDS at 68°C. The filters were air dried, labeled with an UltEmit pen (New England Nuclear), wrapped in Saran wrap and autoradiographed overnight and for seven days at -70°C using Kodak X-Omat XAR Film and a Cronex Lightning-Plus enhancing screen (DuPont, Wilmington, DE). Each serum sample was examined blind under code and was spotted in duplicate. One membrane was hybridized with nick translated PBR322 DNA to control for bacterial contamination and the other was annealed with 32P-labeled HBV DNA. With each membrane, a control nylon membrane was also included that contained 100, 10, 1, 0.1, and 0.01 pg spots of denatured PBR322 DNA and pHBV1 DNA. Each filter included a HBV DNA positive and negative control serum. The negative control sera were obtained from the M-RIA for HBsAg from Centocor. Most samples were tested more than once. In a typical experiment, 50 l.~l
157
of sera was tested and less than 0.1 pg of DNA (i.e. 2.8 x lo4 molecules of HBV DNA) could be detected after seven days autoradiography. Southern blot analysis of serum
Sera were prepared by digestion in 1.4 mg/ml proteinase K, 90 mM Tris, pH 8.0, 23 mM EDTA, 0.9% SDS at 37°C overnight and electrophoresed in 1% agarose. The method described by Southern in (Maniatis et al., 1982) for denaturing and blotting DNA to nitrocellulose membranes was employed. The membranes were wet in 6 x SSC, baked in vacua at 80°C for 1 to 2 h, wet in 6 x SSC and hybridized to 32P-labeled DNA probes as described above in the HBV DNA spot assay.
Results The dot blot assay
A number of procedures were tried to develop a rapid, reproducible hybridization assay for the detection of HBV DNA. Proteinase K digestion followed by phenol extraction, ethanol precipitation and spotting onto filters gave reproducible results; however, the method was tedious and not as sensative as directly spotting or filtering through a nitrocellulose or nylon filter. Hybridization of 32P-labeled DNA probes to alkaline denatured filters spotted with serum was determined to be more sensitive for detecting HBV DNA sequences than any other method of preparing the filters. Some authors (Lieberman et al., 1983) have recommended proteinase K treatment of the spotted filter, however, we found that such enzyme digestion disproportionately decreased the hybridization signal of serum HBV DNA compared to that of spotted plasmid DNA. Figure 1 demonstrates the autoradiographic spots produced after hybridizing radioactive HBV DNA probes to filters either blotted with HBV DNA+ sera or plasmid HBV DNA after three different treatments after alkaline denaturation: incubation with albumin; incubation with
NO TREATMENT
ALBUMIN
PROTEINASE
K
SERUM
PLASMID HBV DNA
It
*
0
)
II
.
Fig. 1. Effect of proteinase K treatment on the HBV DNA dot blot assay. Sera and plasmid DNA were spotted and treated as described in Table 1.
158 TABLE Optical
Plasmid Serum
1 density
units of a 30 h autoradiogram
HBV DNA neat
l/10 dilution of serum
of plasmid
and serum
HBV DNA
Alkali denaturation
BSA treatment
Proteinase treatment
2.80
2.14
2.23
(100 pg/mU 2.26
(74) 2.24
(85) 0.18
(96) 0.58 (10.5)
(88) 0.27
(6.8) 0
K
(7)
2 )11of serial dilutions of plasmid DNA or a HBV DNA+ sera were spotted on three nylon filters. The filters were immersed in 0.5 M NaOH, 2.5 M NaCl for 1530 s then neutralized in 3 M sodium acetate, pH 5. Two filters were then rinsed in 1 M Tris pH 8 for 1 min then incubated at 50°C with either proteinase K 200 &ml or BSA 200 &ml in 0.5 M EDTA 0.5% Sarkosyl for 1 h. All three filters were rinsed briefly in 2 X SSC, air dried, baked in vacua at 80°C for 1 h and hybridized to a “2P-labeled HBV DNA probe as described in Materials and Methods. The optical density units were determined on a Tobins Associates Densitometer Model TBX. The numbers listed are units minus the background. The numbers in parentheses are the estimated amount of HBV DNA based on a curve of optical density units versus log HBV DNA concentration of an alkaline denatured filter containing concentrations of plasmid derived HBV DNA.
proteinase K, or no further treatment. Table 1 summarizes the optical density readings of this experiment. Both proteinase K and albumin treated plasmid HBV DNA had approximately the same optical density which was less than untreated plasmid DNA. Proteinase K treatment of serum resulted in a markedly smaller signal than either the untreated or albumin exposed filter. Based on optical density, the proteinase K treated serum would have been estimated to contain less than one-tenth the HBV DNA concentration as the same serum either treated with albumin or with alkaline dena:;r,:ion only. Based on experiments like these, it was found that serum could either be spotted directly onto nitrocellulose then denatured, or denatured then passed through the filter with equal results. By filtering denatured serum, a larger volume of sample could be used, thereby increasing the sensitivity of the assay. Nylon filters were used in the experiments described below because they immobilized DNA as well as nitrocellulose filters and were more durable. In order to prove that positive spots were due to HBV DNA, 14 dot blot positive serum samples were digested with proteinase K, electrophoresed in agarose, Southern blotted onto nitrocellulose, and hybridized to 32P-labeled HBV DNA or PBR322. As illustrated in Fig. 2, samples that gave a moderately intense spot in the HBV DNA assay produced a detectable smear that was 3.2 kb and smaller. This pattern is consistent with the molecular forms of HBV DNA molecules in serum. Other samples that had less intense spots on the assay were indirectly demonstrated to contain HBV DNA by the loss of a positive signal after depurination by low pH treatment (data not shown). One hundred and ninety-nine control sera were found to have no detectable HEW DNA by the dot blot assay (Table 2). These sera included 129 blood donor sera
159
12345678
123456789
9
7.2
r)
5.7 4.8 4-3 3.6
:
2.3 1.9
1.4
kb HlGl-i
HBV STRINGENCY
WASH
LOW
PBR 322 STRINGENCY
WASH
Fig. 2. Southern blot analysis of HBV DNA fot blot positive sera. Fifty ~1 of sera were prepared as directed in Materials and Methods. The Southern blotted NC filter was hybridized to 32P-labeled HBV or PBR322 DNA probes. The autoradiogram shown was developed after a 7 day exposure to the NC filter. The intensity of the spots on HBV dot blot assay was most intense for samples 2 and 9. Samples 5 and 8 gave faint spots after 7 days autoradiography. kb = kilobase pairs of DNA.
that were negative for anti-HBs, anti-HBc, and HBsAg; 63 anti-HBs+ anti-HBc+ patients with normal liver function tests; 2 patients with acute HAV infections; 3 patients with chronic active hepatitis due to non-A, non-B hepatitis; and 1 patient each with acquired immune deficiency syndrome and Epstein-Barr viral hepatitis. Each of the 199 samples had no detectable HBsAg by both the polyclonal AUSRIA II assay and the M-RIA. Acute HBV hepatitis
Serum samples from 50 patients who had acute HBV hepatitis were assayed for the presence of HBV DNA. All patients had an acute rise in serum aminotransferase and were IgM anti-HBc and HBsAg positive. While 55% (12 of 22) of pa-
160 TABLE
2
Results
of serum
AUSRIA and M-RIA
HBV DNA in patients
II Anti-HBs
_
_
with no evidence
of current
HBV infection
Anti-HBc
HBV DNA
Comments
-
O/129
Blood donors on patients with no evidence active disease 52 Ethiopian Jews, 11 MGH blood donors EBV hepatitis Non-A, non-B CAH Acute HAV AIDS
0163 Oil o/3 012 O/l Total:
O/199 were HBV DNA
of
positive
tients who were HBeAg+ were HBV DNA positive, only 17% (3 of 18) of the HBeAg- persons were HBV DNA positive. Thirty-six percent of all 50 HBsAg+ patients were HBV DNA positive at time of serological diagnosis. Of note, 29% (2 of 7) of HBeAg- anti-HBe+ patients with acute HBV infection had detectable HBV DNA. Serial serum samples were obtained from 13 of the 50 patients with acute HBV hepatitis. Table 3 lists the data for 5 patients. In all cases, the M-RIA for HBsAg was more sensitive than the HBV DNA assay in detecting HBV-related determinants. No example of a M-RIA negative serum that was HBV DNA positive was found in this group of patients with acute hepatitis B. HBV DNA was not detected in any patient who was HBsAg- by AUSRIA II and anti-HBe+ (data not shown). Figure 3 plots the course of an Israeli who developed acute HBV infection. Although he resolved clinically, he later developed chronic lobular hepatitis. During
Cllnlcol ALTZ
2x
IgM
course
23
_I_
Normal
+
-
-
++
++.
HBeAg
+++
HBsAg
.++
+
AntI-HBc
+ . +
1
+a
HBV
+ - -
+*
l
DNA
, J
l
I, I, / I I FMAMJJASONDJ 1982
I
/
t
l
I
I
I
/
++
1
I
++*
+
+ + ++
-
I
l
_ --
l
l
t++
I,
I1
I,
I
i 983
1
c 14
FMAMJJASONDJ I
f
++ - -
+
1
6
2
4
L
I
FMAMJ 1
I
/
1 I
I
L
JASON
1984
Fig. 3. Clinical, biochemical, serological, and histological course of a 22 year old man who developed acute hepatitis B that progressed to chronic lobular hepatitis. Clinical course notes: 1, acute viral hepatitis; 2, nausea, anorexia, fatigue; 3, liver biopsy-nodular hepatitis; 4, asymptomatic; 5, nausea, fatigue; 6, liver biopsy-chronic lobular hepatitis; 7, asymptomatic.
161 TABLE
3
Serial determination Patient 1
no.
of serum
HBV DNA” in patients
Date
AUSRIA
319181 4112 4120 5/20
+ + _ _
2/l/81 215 2119
+ + -
l/30/81 2114 2119
+ + +
9/19/81 10127
+ _
3/26/81 417 4116 4/28,5/10 5/20,5/31 619 6116 7127
+ + + + + + _
II
with acute HBV
M-RIAb
HBeAg/anti-HBe
DNA
+
-I -I
+
_
+ fl
_ -
+ -I+
_ -
+i+
+ _
+ +
+ _ _ _
a +, a positive spot was seen after autoradiography for 7 days; this implies an HBV DNA concentration greater than 2.8 x 105 molecules/ml. -, no spot was detected. b M-RIA has been found to be positive if AUSRIA II is positive; therefore, M-RIA was usually not performed if AUSRIA II was positive.
the 34 months in which he was followed, he remained HBsAg and HBeAg positive but the IgM anti-HBc antibody converted from positive to negative. The serum HBV DNA became negative following the initial clinical episode of acute hepatitis, only to become detectable again with the recrudescence of his liver disease. Dialysis patients
Multiple serum samples from 22 dialysis patients who were HBsAg+ were studied. Sixty-eight percent (Z/22) of serum samples were HBV DNA positive. Nine of 10 (90%) HBsAg+ HBeAg+ sera were HBV DNA positive. This is in contrast to 55% HBV DNA+ patients with acute HBV (vide supra). No patient who was HBsAg+ HBeAg- had detectable serum HBV DNA. Serial serum samples were obtained from 12 HBsAg+ dialysis patients. Data from 8 patients are listed in Table 4. In seven of the 8 dialysis patients who were HBeAg+ , HBV DNA was detectable at the time the antigen was detectable. However, the absence of HBeAg positivity did not exclude the possibility that a patient would later become HBV DNA positive. For example, over a 28 month period, dialysis patient no. 5 was
162 TABLE
4
Serial determinations
on eight HBsAg+
dialysis
patients HBsA.g
Patient 1
no.
Date
HBeianti-HBe
115182 212182 312182 4182 9182
5
12/81,1/82,3182 4182,6/82 9182 12/81,1/83 2183
6
l/81 5182,7182 8/82,9/82 10/82,11/82 12182
L
ii t +
+/
_
_
+
6180 10/30/80 1011181 1 llll81 1211181 115182,2/2182,511182
4
HBV DNA II M-RIA*
+
9/80 10180 l/81 2181 6181 3182
9/8/80 l/6/81 515181 6124181 914181 312182 513182
AUSRIA
_ + t _
_
t t
+ + + t
fl -/
+I-
_
_ t _
_ + +
_ + t
t + + t + t + + + +
+I ii
10/80,6/81 8/81,12/82
ti
+ +
+ +
815182 912182 1113 l/4183
ii
+ + + -
+ t + _
* M-RIA is positive RIA II was positive.
if AUSRIA
21
II is positive,
therefore,
M-RIA
_ was not always
performed
if AUS-
163
repeatedly HBsAg+ by AUSRIA II and HBV DNA negative. One serum sample (dated 9/82) was HBeAg negative. On the 29th month of surveillance, the patient was both HBsAg and HBV DNA positive. Dialysis patients no. 3 and no. 4 probably represent examples of either reactivation of the HBV infection or increasing severity of infection with HBV DNA being detectable again months after the patients were serum HBV DNA-. It should be noted that at all times the M-RIA for HBsAg was positive. Dialysis patients no. 2 and no. 3 are examples that AUSRIA II negative patients may be infectious for HBV. Fourteen months after initial surveillance the patient no. 2 was no longer HBsAg positive by the AUSRIA II assay but positive by the .M-RIA for at least 20 months longer. More importantly, HBV DNA was detectable during a two-month period (November-December 1981), 13 months after being HBsAg negative by AUSRIA II. Similarly, patient no. 3 was HBV DNA positive 9 months after becoming HBsAg negative by the AUSRIA II assay. Thus patients no. 2 and no. 3 would not have been considered potentially infectious based on conventional HBV serology, but would be identified as infectious based on the more sensitive M-RIA and HBV DNA assays. The data on dialysis patients no. 2, no. 3, no. 4, and no. 5 illustrate that a negative test for HBV DNA does not preclude the possibility that the patient will become HBV DNA positive at a later time. Dialysis patient no. 8 is an example of a resolving acute HBV infection in a dialysis patient.
Discussion
Berninger et al. (1982) and Feinman et al. (1984) have estimated the concentration of HBV DNA molecules in serum by dot blot assays. These investigators then demonstrated that the DNA concentration approximates the concentration of infectious virions estimated from the serum ID,, for chimpanzees. Since the ability to perform these types of infectivity studies on chimpanzees is limited, it is not known if every serum that is HBV DNA positive contains intact virions and is, therefore, infectious. A safe assumption based on the work of Berninger et al. (1982) and Feinman et al. (1984) is that HBV DNA positive sera are potentially infectious. Furthermore, the identification of HBsAg by a sensitive M-RIA for HBsAg in sera that were HBsAg- by AUSRIA II but HBV DNA positive further supports the contention that these sera are potentially infectious. Our results have confirmed other investigators’ observations (Brechot et al., 1981; Berninger et al., 1982; Robinson, 1982; Weller et al., 1982; Matsuyama et al., 1985) that HBeAg positivity does not always correlate well with the presence of HBV DNA. A negative test for HBeAg does not rule out the serum’s potential infectivity. The manner in which the DNA hybridization assay was performed enabled sera to be detected as positive if there were a minimum of 5.6 x 105 DNA molecules per ml. It is not surprising that 45% of HBeAg+ patients with acute HBV hepatitis were HBV DNA-. These HBeAg+ sera are probably infectious since fewer than 5.6 x lo4 HBV virions are needed to initiate infection. Sera can be
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identified that are HBsAg-t by M-RIA, but negative for HBeAg, HBV DNA and HBsAg by AUSRIA II. Since at this time no rapid, inexpensive assay can determine if the HBsAg detected by M-RIA is virion associated or produced from chromosomally integrated HBV DNA, such sera should be considered potentially infectious. Neither the assays for HBeAg and HBV DNA are more sensitive than M-RIA for HBsAg, therefore, the HBeAg and HBV DNA tests can only be used as further evidence that a particular serum sample that is HBsAg+ by M-RIA may be infectious. A negative HBV DNA or HBeAg test does not exclude the potential infectivity of that particular serum sample. Dialysis patients were studied because compared to the general population they are relatively immunosuppressed. HBV infections are often prolonged in this group. Since medical personnel are frequently exposed to their blood, it is important to determine how potentially infectious these patients are. Our results demonstrated that a higher percentage of HBeAg+ and HBsAg+ dialysis patients have detectable serum HBV DNA than patients with acute HBV hepatitis. The concentration of HBV DNA in dialysis patients in general was greater than patients with acute HBV hepatitis (data not presented). Serial studies on these patients demonstrated that the absence of HBV DNA does not preclude the possibility that a patient will later become HBV DNA positive, even though HBsAg may remain undetectable by the AUSRIA II assay. This point is especially of relevance to dialysis patients who often have transient rises in their aminotransferases that are attributable to non-A non-B hepatitis because they are HBsAg negative by AUSRIA II. The detection of HBsAg by M-RIA and HBV DNA in these AUSRIA II negative patients is consistent with the abnormal liver function tests being due to HBV, not some other agent. Our results have confirmed the findings of Scotto et al. (1983) that sera only need to be alkaline denatured and not treated with a protein digestion step to obtain a sensitive and specific HBV DNA assay. Proteinase K treatment decreases the sensitivity of detecting serum HBV DNA, by greater than lo-fold, but affects the hybridization signal of plasmid DNA only by about 30%. This suggests that an assay that includes a proteinase K treatment may have a higher rate of false negativity than an assay that omits this step, even though the sensitivity of both assays may be identical based on plasmid DNA standards. A possible reason for the decrease in hybridization signal after proteinase K treatment is that both nitrocellulose and nylon filters bind denatured (single stranded) DNA and protein. It is possible that the protein covalently bound to the 5’ end of the ‘long’ strand of the HBV DNA molecule helps to anchor it to the filter and that proteinase K treatment removes most of this protein. Alkaline treatment alone is sufficient to denature the intact virion and double stranded DNA so that it can be bound by the filter. A sufficient amount of the HBV genome is exposed to the radioactive probe to allow detection despite the excess denatured virion and serum protein also bound to the filter. In conclusion, the results of this study consolidate and extend the experience of others that assays for HBV DNA are sensitive and specific for identifying potentially infectious individuals. The sensitivity of the assay can be increased by omitting proteinase K treatment of sera prior to DNA hybridization. While the com-
165
mercial polyclonal RIA for HBsAg is an excellent screening test, these are a significant number of sera that contain HBsAg that are not detected by this assay and are infectious for HBV. The monoclonal antibody immunoassay for HBsAg is clearly the most sensitive immunoassay for HBV related antigens. The serum HBV DNA assay by measuring the presence of the genome can directly determine whether a particular serum is potentially infectious. At this time, its sensitivity is not greater than the monoclonal immunoassay for HBsAg; therefore, the HBV DNA assay’s place in the diagnostic armamentarium may be only to confirm, but not disprove, the hypothesis that an individual has circulating Dane particles and does not just have polymeric HBsAg in his serum.
Acknowledgements This work was supported in part by NIH Grants no. AA-02226 and no. CA35711, and NIH Research Career Development Award no. AA-00048 to J.W. J.B.Z. is a Pfizer Post-Doctoral Fellow.
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