AS04, a hepatitis B vaccine with a novel adjuvant

Vaccine 20 (2002) 2597–2602

Immune response of HLA DQ2 positive subjects, vaccinated with HBsAg/AS04, a hepatitis B vaccine with a novel adjuvant夽 Isabelle Desombere a , Marie Van der Wielen b , Pierre Van Damme b , Michel Stoffel c , Norbert De Clercq c , Christian Goilav c , Geert Leroux-Roels a,∗ b

a Centre for Vaccinology, Ghent University Hospital, De Pintelaan 185 9000, Ghent, Belgium Centre for the Evaluation of Vaccination, WHO collaborating Centre for the Prevention and Control of Viral Hepatitis, Epidemiology and Community Medicine, University of Antwerp, Antwerp, Belgium c GlaxoSmithKline Biologicals, Rixensart, Belgium

Received 7 May 2001; received in revised form 21 February 2002; accepted 21 February 2002

Abstract About 5–10% of the general adult population respond inadequately to hepatitis B vaccination. The histocompatibility leucocyte antigen (HLA) DQ2, DR3 and DR7 phenotypes have been linked with non-responsiveness to hepatitis B vaccination. A first part of our study determined the prevalence of the HLA DQ2 allele in a healthy population, aged 15–50 years. We found 35% of our study population (n = 1008) positive for the HLA DQ2 allele. Positive subjects for HLA DQ2 were subsequently invited to participate in a trial and were to be given either the HBsAg/AS04 hepatitis B vaccine or a licensed hepatitis B vaccine (EngerixTM -B).1 Both contained 20 ␮g of recombinant HBsAg. The HBsAg/AS04 vaccine was administered on a 0 and 6 months schedule whilst the comparator vaccine was given according to the standard 0, 1 and 6 months schedule. The experimental vaccine was formulated on a novel adjuvant containing 3 deacylated monophosphoryl lipid A (3D-MPL) and alum. A total of 230 subjects were enrolled into the vaccination study. At month 7, 99% of the subjects had a protective titre (≥10 mIU/ml) with a geometric mean titre (GMT) of 6613 mIU/ml in the group receiving HBsAg/AS04 versus 97% seroprotected with a GMT of 2315 mIU/ml in the other group. Both vaccines, with their respective schedule, give very high seroprotection rates (>96%). Our data suggest that HLA DQ2 positivity is not a good marker for non- or poor-responsiveness. The HBsAg/AS04 vaccine was more reactogenic mainly because of an increased local reactogenicity. Both vaccines, especially HBsAg/AS04, are highly immunogenic and well tolerated by the study subjects. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: HLA DQ2; EngerixTM -B; Hepatitis B vaccination

1. Introduction Hepatitis B vaccination remains the best tool available to prevent and to control hepatitis B infection and disease, which still threatens millions of people world-wide. Current recombinant hepatitis B vaccines achieve seroprotection in >95% of the vaccinated healthy adult population [1,2]. The 5–10% who respond less well or even not at all to standard vaccination schedules are often referred to as non- or poor-responders. Poor- or non-responsiveness is considered in those people who, after at least three doses (primary schedule) of hepatitis B vaccine, have an anti-HBs titre 夽 This study was in part presented at the 10th International Symposium on Viral Hepatitis and Liver Diseases, Atlanta, GA, USA, 9–13 April 2000. ∗ Corresponding author. Tel.: +32-9-240-3422; fax: +32-9-240-4985. E-mail address: [email protected] (G. Leroux-Roels). 1 Note: EngerixTM -B is a trademark of GlaxoSmithKline Biologicals, Rixensart, Belgium.

that never exceeded 10 mIU/ml. Various constitutional and behavioural factors have been described which could have a negative influence on the immune response and play a role in non-responsiveness: age, male gender, obesity and smoking habits [3]. In addition, several genetic factors, and more specifically, polymorphisms of the major histocompatibility complex have also been linked to variations in immune responses ([4] and references therein). One study could link the presence or absence of several human histocompatibility leucocyte antigens (HLAs) to poor-responsiveness [4]. Certain HLA alleles, some of which are genetically linked with each other, are associated with non-responsiveness in a statistically significant manner. In particular, a positive status for HLA DR3 and DR7, which are in linkage disequilibrium with HLA DQ2, had a strong association with non-responsiveness [4], an observation made also by other investigators [5]. This linkage disequilibrium is a consequence of the proximity of HLA DQ2 with either HLA DR3 or DR7 which

0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 2 ) 0 0 1 5 0 - 0

2598

I. Desombere et al. / Vaccine 20 (2002) 2597–2602

means both alleles do not behave independently of each other. Certain diseases have also been linked to the HLA DQ2 positive status like type 1 diabetes and celiac disease [6,7]. A clinical study in established non-responders to a fourdose hepatitis B vaccination schedule, compared a hepatitis B vaccine formulated with a novel adjuvant (HBsAg/AS04) with a commercially available recombinant hepatitis B vaccine (EngerixTM -B) and showed promising results [8]. Three doses of the novel study vaccine induced a seroprotection rate of 98% in the non-responders compared to 68% for the conventional hepatitis B vaccine. Previously, this HBsAg/ AS04 vaccine was shown to be well tolerated and to have a superior immunogenity over current recombinant hepatitis B vaccines in healthy adults [9–11]. We report here on two studies. The first study determined the prevalence of the HLA DQ2 allele in a healthy population, aged between 15 and 50 years, residing in Belgium. Subjects who were positive for HLA DQ2 and thus were expected to be positive for HLA DR3 and DR7 [4], and as a consequence had an increased likelihood to be nonor slow-responders to hepatitis B vaccination were subsequently invited to participate in a clinical trial were they were to be given either the HBsAg/AS04 hepatitis B vaccine or an established recombinant hepatitis B vaccine for comparison.

2. Materials and methods

by PCR. The amplified DNA is subsequently hybridised to membrane-bound sequence specific oligonucleotides and visualised using an alkaline phosphatase based reaction. Subtypes of DQ2 were not determined since the test used at the time of the study, did not discriminate between the main subtypes (DQB1*0201 and DQB1*0202). 2.3. The hepatitis B/AS04 vaccination study This open, randomised study compared the immunogenicity and reactogenicity of HBsAg/AS04 with that of EngerixTM -B in HLA DQ2 positive subjects. 2.3.1. Design The group receiving the HBsAg/AS04 vaccine was immunised according to a 0 and 6 months schedule while the other group received an established hepatitis B vaccine (EngerixTM -B) on a 0, 1 and 6 months schedule. Blood samples for antibody evaluation were drawn at study entry and at months 1, 6 and 7. Subjects had to be HLA DQ2 positive and negative for HBsAg, and anti-HBs and -HBc antibodies. Volunteers were excluded if they had received any previous hepatitis B vaccination or any previous administration of a vaccine adjuvanted with AS04, if they had a history of significant and persisting hematologic, hepatic, renal, cardiac or respiratory disease. Simultaneous administration of any other vaccine or participation in another clinical trial or administration of immunoglobulins was prohibited during the study period.

2.1. Study ethics and approval Both studies were conducted according to the European Commission Directive on Good Clinical Practice (91/507/EEC) and the Declaration of Helsinki as amended in Somerset West, South Africa, October 1996. The study was approved by the Ethical Review Committees of both study centres and written informed consent was obtained from the subjects and/or their parents/guardian. 2.2. The HLA DQ2 prevalence study This study was conducted with a 1000 subjects planned, aged 15–50 years, at two clinical trial centres in Belgium. Subjects were healthy males and females. There were no specific exclusion criteria but a medical history was taken at study entry to determine if certain diseases were more prevalent in HLA DQ2 positive subjects. For all subjects, a 10 ml blood sample was taken to test for anti-HBs antibodies (AUSAB, Abbott, IL, USA), anti-HBc antibodies (CORAB, Abbott, IL, USA) and for HBsAg (AUSRIA, Abbott, IL, USA). In addition 10 ml heparinised blood was collected for HLA typing. The HLA class II DQ type of all subjects was determined with INNO-LiPA strips (Innogenetics, Ghent, Belgium). This typing method is based on a technique developed by Buyse et al. [12] and involves an amplification of the polymorphic second exon of the different HLA DQ genes

2.3.2. Vaccines Each 0.5 ml dose of HBsAg/AS04 contained 20 ␮g of recombinant HBsAg, 50 ␮g of 3 deacylated monophosphoryl lipid A (3D-MPL) and 0.5 mg alum as adjuvant (AS04 = adjuvant system 04). Each 1 ml dose of EngerixTM -B contained 20 ␮g HBsAg and 0.5 mg alum as adjuvant. 2.3.3. Assessment of immunogenicity Blood samples were tested for anti-HBs antibodies with a RIA (AUSAB, Abbott, IL, USA). The lowest sensitivity level of the test was 1 mIU/ml. Subjects who had a titre ≥10 mIU/ml were considered to be seroprotected against hepatitis B. 2.3.4. Assessment of reactogenicity Each participant was closely observed for 15 min following each vaccine administration. Local and general signs and symptoms were recorded on individual diary cards on the day of vaccination and the three subsequent days. Volunteers were asked to record any local (soreness, redness and swelling) and general (fever, headache, malaise, fatigue and gastrointestinal symptoms) reactions on the day of vaccination and the three subsequent days. Systemic and local reactions were scored as absent, grade 1 (easily tolerated), grade 2 (interfered with daily activities) and grade 3 (prevented normal daily activities). The size of redness and swelling

I. Desombere et al. / Vaccine 20 (2002) 2597–2602

was obtained by measuring their largest diameter and was scored by the sponsor as grade 1 (1 to ≤10 mm), grade 2 (>10 to ≤50 mm) or grade 3 (≥50 mm). Fever was defined as axillary body temperature above 37.4 ◦ C and was scored as grade 1 (37.5–38 ◦ C), grade 2 (>38–39 ◦ C) or grade 3 (>39 ◦ C). Unsolicited symptoms were recorded for 30 days after each vaccination and serious adverse events were recorded throughout the whole study period. All solicited local symptoms were considered as related to vaccination.

2599

Of the 1008 subjects which could be tested, 657 (65.2%) were negative and 351 (34.8%) were positive for the HLA DQ2 genotype. Of the positives, 308 (88%) were heterozygous. All subjects were negative for HBsAg, 41 (4.1%) were anti-HBs positive and 7 (0.7%) were anti-HBc positive. Similar percentages (0, 3.7 and 0.6%) were obtained for the HLA DQ2 positive subjects. There were no significant differences between hetero- and homozygous subjects. We could not establish a correlation between HLA DQ2 positive status and past or current medical conditions (based on medical history) in this particular study population.

2.3.5. Statistical methods Seroprotection rates, geometric mean titres (GMTs) were calculated and tabulated. The GMTs were calculated using the log transformation for anti-HBs titres above the cut-off value of the assay and taking the anti-log of the mean of these transformed values. The primary objective of this study was to demonstrate that the HBsAg/AS04 vaccine was better than EngerixTM -B in terms of seroprotection rates 1 month after the last vaccine dose in a population expected to be poor- or non-responders. Assuming that the seroprotection rate was equal to 85% in the EngerixTM -B group and 95% in the HBsAg/AS04 group, 130 evaluable subjects in each group were needed to have at least 80% power to reject the null hypothesis that HBsAg/AS04 was not better than EngerixTM -B, using a one-sided (α = 5%) Fisher’s exact test. Allowing for 13% of subjects not evaluable for analysis 150 subjects per treatment group (a total of 300 subjects) had to be enrolled. Since only 230 subjects could be enrolled in the study, the power of the statistical test for the primary endpoint drops to 76.2%. This means that the probability to declare a statistically significant difference when the null hypothesis is actually not true is only 76.2%.

3.2. The hepatitis B vaccination study Of the 337 subjects identified as being HLA DQ2 positive and being negative for hepatitis B markers, 230 accepted to participate in the vaccination study and were thus enrolled after control of the inclusion and exclusion criteria. Only one subject did not complete the study because the subject of “lost to follow-up” after the second dose of the licensed hepatitis B vaccine. Of the 230 enrolled, five were excluded for the according-to-protocol (ATP) reactogenicity analysis because they received other vaccines during the study and two more subjects were excluded because of a randomisation violation. From the 223 remaining subjects five more were excluded from the ATP immunogenicity analysis (one because of the use of concomitant medication which was not allowed in the protocol, one because of missing essential data and three for non-compliance with intervals specified within the protocol). The immune response (ATP, according-to-protocol analysis) of both vaccine groups is detailed in Table 1. Nearly all subjects in both study groups achieved seroprotection levels ≥10 mIU/ml at month 7. The largest difference between both groups is seen at month 1 were about one-third of those receiving HBsAg/AS04 are protected versus about 1 in 10 for the comparator vaccine. Differences in GMTs are most pronounced at month 7 (P < 0.0001, Wilcoxon rank sum two-sided test). The results from the intention-to-treat (ITT) analysis were similar to that of the ATP analysis (data not shown). We could not demonstrate superiority in seroprotection rates of the HBsAg/AS04 vaccine over the comparator hepatitis B vaccine in HLA DQ2 positive subjects, which was the primary objective of the study.

3. Results 3.1. The HLA DQ2 prevalence study A total of 1013 subjects were recruited (472 in one centre and 541 in the other centre) of which 692 were female and 321 were male. The mean age of the subjects was 24.7 years (S.D. 9.38). A total of 7 (0.7%) subjects were of non-Caucasian origin.

Table 1 Anti-HBs seroprotection levels and antibodies titres (ATP analysis) in both study groups Group

Time

N

SP

CI (95%)

GMT

CI (95%)

HBsAg/AS04

PI (m 1) PI (m 6) PII (m 7)

104 104 104

29.8 63.5 99.0

21.1–39.6 53.4–72.7 94.8–100

4 12 6613

3–5 9–17 4268–10247

EngerixTM -B

PI (m 1) PII (m 6) PIII (m 7)

114 114 114

10.5 79.8 96.5

5.6–17.7 71.3–86.8 91.3–99.0

1 49 2315

1–2 34–73 1494–3587

N, number of subjects; SP, seroprotection rate (%); CI, confidence interval; P, post-dose; m, month.

2600

I. Desombere et al. / Vaccine 20 (2002) 2597–2602

Table 2 Immunogenicity in homozygous and heterozygous HLA DQ2 positive subjects (ATP analysis) Group Heterozygous HBsAg/AS04

EngerixTM -B

Homozygous HBsAg/AS04

EngerixTM -B

Time

N

PI (m 1) PI (m 6) PII (m 7)

90 90 90

PI (m 1) PII (m 6) PIII (m 7)

SP

CI (95%)

GMT

CI (95%)

31.1 65.6 98.9

21.8–41.7 54.8–75.3 94.0–100

11 18 7696

7–17 14–24 5025–11785

97 97 97

11.3 83.5 96.9

5.8–19.4 74.6–90.3 91.2–99.4

6 76 3068

4–9 54–108 2006–4693

PI (m 1) PI (m 6) PII (m 7)

14 14 14

21.4 50.0 100

4.7–50.8 23.0–77.0 76.8–100

7 10 4969

3–19 4–24 1315–18778

PI (m 1) PII (m 6) PIII (m 7)

17 17 17

5.9 58.8 94.1

0.1–28.7 32.9–81.6 71.3–99.9

65 39 776

0–0 12–130 199–3026

N, number of subjects; SP, seroprotection rate (%), CI, confidence interval; P, post-dose; m, month.

We further investigated whether we could detect differences in the immune response to both vaccines, between homozygous and heterozygous HLA DQ2 positive subjects. We found no statistically significant differences in seroprotection rates at month 7 (Table 2). However, when we looked at the GMTs at month 7 we observed some trends towards a difference. For those receiving EngerixTM -B the GMT for the heterozygous subjects was approximately four times higher than that of the homozygous subjects 3068 versus 776); for the group receiving HBsAg/AS04, the corresponding figure was 1.5 times (7696 versus 4969). Within the homozygous group, HBsAg/AS04 induced 6.4 times higher GMTs than EngerixTM -B (4969 versus 776) and within the heterozygous group this was 2.5 times (7696 versus 3068).

Those numbers indicate that despite the fact that the GMTs were higher in the heterozygous subject, the HBsAg/AS04 vaccine was more immunogenic in homozygous HLA DQ2 positive subjects when compared to the licensed hepatitis B vaccine. Data on the reactogenicity after 218 doses of HBsAg/ AS04 and 361 doses of the comparator vaccine was available for analysis. A total of 62% of the established hepatitis B vaccine doses were followed by at least one symptom versus 91% for the other vaccine group. This increase in reactogenicity is for a large part due to an increased local reactogenicity in the group receiving HBsAg/AS04 (Table 3). For both study groups pain at the injection site is the most frequently reported local symptom whilst fatigue is the most

Table 3 Local and general symptoms after vaccination (per dose analysis) on the ITT population EngerixTM -B (N = 361)

HBsAg/AS04 (N = 218)

Total Grade 3

33.0 1.1

87.6 10.5

Redness

Total Grade 3

13.5 0.0

26.1 0.5

Swelling

Total Grade 3

4.8 0.0

22.0 0.5

Total Grade 3

24.1 0.6

30.7 0.9

Gastrointestinal symptom

Total Grade 3

9.1 0.3

13.8 0.9

Headache

Total Grade 3

18.6 0.8

22.0 2.8

Temperature

Total Grade 3

1.7 0.0

3.2 0.0

Local Pain

General Fatigue

N, number of completed symptom sheets (one per subject after each dose); grade 3 is defined in Section 2.

I. Desombere et al. / Vaccine 20 (2002) 2597–2602

frequently reported general symptom. In the group receiving HBsAg/AS04, all symptoms are reported at a higher frequency than in the other group but this is especially the case for pain and to a lesser extent for swelling at the injection site. Except for pain at the injection site, there are few symptoms reported which are scored grade 3 (interfering with normal daily activities) (Table 3). A total of 201 doses (of which 82 in the comparator group), administered to 143 subjects were followed by 252 unsolicited symptoms (ITT analysis). Two subjects, one from each study group, reported a serious adverse event during the study period, none of which was deemed to be related to vaccination. One case was diverticulitis and one case was ligament ruptures caused by a sport injury. Both resolved and the both vaccinees received the subsequent dose(s) of vaccine.

4. Discussion The first study we report on, was conducted to determine the prevalence of the HLA DQ2 allele in a general healthy population residing in Belgium. The HLA DQ2 phenotype, which is genetically linked to the HLA DR3 and DR7 phenotype, has been associated with a poor- or non-response to recombinant hepatitis B vaccination ([4,13] and references therein). HLA DQ2 positive subjects were subsequently recruited into a comparative clinical trial to investigate the immunogenicity and reactogenicity of an experimental adjuvanted hepatitis B vaccine, which was previously shown to induce higher titres and higher seroprotection levels than an established recombinant hepatitis B vaccine both in healthy subjects [9] and in non-responders to hepatitis B vaccination [8]. We found that about 35% of our test population was positive for the HLA DQ2 allele. This compares to the 33% described as typical for a general Caucasian population (sample size of 2016) [14] and a frequency of 40% found in 247 British Caucasians [15] but is somewhat lower than the 47% found in a population of 1789 Welsh blood donors [16]. The presumption of our study was that, in this group of potential poor- or non-responders to hepatitis B vaccination, as identified by their HLA DQ2 status, the immune response would be as observed before [8]: higher titres and seroprotection rates with HBsAg/AS04 compared to a moderate response to the comparator hepatitis B vaccine. In that particular study [8], non-responders were defined as subjects who after having received four doses of hepatitis B vaccine still had titres <10 mIU/ml whilst in the study presented here, the subjects were never vaccinated with a hepatitis B vaccine before. Contrary to the expectation, both study groups achieved a high level of seroprotection (>95%). A seroprotection level >95% is similar to the level normally observed in the general population with current recombinant hepatitis B vaccines [1,2]. However, even if the superiority in terms of seroprotection rates of HBsAg/AS04 over

2601

EngerixTM -B could not be proven in this study, HBsAg/ AS04 appears to elicit a better immune response. The GMT after two doses of HBsAg/AS04 vaccine was significantly higher than after three doses of EngerixTM -B. In the present study, both vaccines contained 20 ␮g of HBs antigen, thus the difference in GMTs is most likely to be a consequence of the different adjuvants in both vaccines. The immune responses to both study vaccines in the HLA DQ2 positive subjects, suggests that a positive HLA DQ2 phenotype is not the only or best marker for pooror non-responsiveness. However, the HLA DQ2 haplotype could be a marker for sub-optimal response to hepatitis B vaccination, especially in homozygous subjects. After the full vaccination course with EngerixTM -B, the GMT observed in the homozygous population of this study were markedly lower (776 mIU/ml) than in the heterozygous population (3068 mIU/ml). Further studies, in larger numbers of subjects could clarify this. Noteworthy, is the fact that at the time we assessed the HLA DQ2 phenotype, we could not yet discriminate between the 0201 and 0202 subtypes. It could thus be possible that being of either subtype, is important in the determination of the responsiveness towards hepatitis B vaccination. The HBsAg/AS04 vaccine was shown to be more reactogenic than the comparator hepatitis B vaccine, which is almost exclusively due to local injection site events. However, when considering the severe events all except for local pain at the injection site were reported below 3%. This increased local reactogenicity has been described before with the HBsAg/AS04 vaccine [11]. Although higher in incidence and somewhat more severe the events are transient and have all resolved during the study period. In conclusion, both study vaccines are well tolerated by the study subjects and are highly immunogenic in this population of HLA DQ2 positives. The reactogenicity profile confirms that which was observed during other trials with the HBsAg/AS04 vaccine [8–11]: an increased local reactogenicity but the vaccine is well tolerated by the vaccinees. Our data suggest that the use of the HLA DQ2 typing as a genetic marker for poor- or non-responsiveness to hepatitis B vaccination may not be a good option. References [1] Coates T, Wilson R, Patrick G, André F, Watson V. Hepatitis B vaccines: an assessment of the seroprotective efficacy of two recombinant DNA vaccines. Clin Ther 2001;23:392–403. [2] Safary A, André F. Over a decade of experience with a yeast recombinant hepatitis B vaccine. Vaccine 1999;18:57–67. [3] Hollinger BF. Factors influencing the immune response to hepatitis B vaccine, booster dose guidelines and vaccine protocol recommendations. Am J Med 1989;87(Suppl 3A):36–40. [4] Desombere I, Willems A, Leroux-Roels G. Response to hepatitis B vaccine: multiple HLA genes are involved. Tissue Antigens 1997;51:593–604. [5] McDermott AB, Zuckerman JN, Sabin CA, Marsh SG, Madrigal JA. Contribution of human leukocyte antigens to the antibody response to hepatitis B vaccination. Tissue Antigens 1997;50:8–14.

2602

I. Desombere et al. / Vaccine 20 (2002) 2597–2602

[6] Thorsby E, Ronninger K. Particular HLA DQ molecules play a dominant role in determining susceptibly resistance to type 1 diabetes. Diabetologica 1993;36:371–7. [7] Farré R, Humbert P, Vilar P, et al. Serological markers and HLA DQ2 haplotype among first-degree relatives of celiac patients: Catalonian Coeliac Disease Study Group. Dig Sci 1999;44: 2344–9. [8] Jacques P, Moens G, Desombere I, Dewijgaert J, Leroux-Roels G, Thiriart C, et al. A hepatitis B vaccine with a novel adjuvant: immunogenicity and reactogenicity profile in adult vaccine nonresponders. In: Proceedings of the 39th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), 26–29 September 1999. San Francisco, [Abstract 373]. [9] Thoelen S, Van Damme P, Mathei C, et al. Safety and immunogenicity of a hepatitis B vaccine formulated with a novel adjuvant system. Vaccine 1998;16:708–14. [10] Ambrosh F, Wiedermann G, Kundi M, et al. A hepatitis B vaccine formulated with a novel adjuvant system. Vaccine 2000;18:2095– 101.

[11] Thoelen S, De Clercq N, Tornieporth N. A prophylactic hepatitis B vaccine with a novel adjuvant system. Vaccine 2001;19:2400–3. [12] Buyse I, Decorte R, Baens M, et al. Rapid DNA typing of class II HLA antigens using the polymerase chain reaction and reverse dot blot hybridisation. Tissue Antigens 1993;41:1–14. [13] Desombere I, Gijbels A, Verwulgen A, Leroux-Roels G. Characterization of the T cell recognition of hepatitis B surface antigen (HBsAg) by good responders to hepatitis B vaccines. Clin Exp Immunol 2000;122:390–9. [14] Baur MP et al. Population analysis on the basis of deduced haplotypes from random families. In: Albert ED, Baur MP, Mayr WR., editors. Histocompatibility testing. Berlin: Springer, 1984. [15] Awad J, Navarrete C, Festenstein H. Frequency and associations of HLA-DR and HLA-DQ antigens in a British Caucasoid panel. Tissue Antigens 1987;29:55–9. [16] Darke C, Guttridge MG, Thompson J, McNamara S, Street J, Thomas M. HLA class I (A, B) and II (DR, DQ) gene and haplotype frequencies in blood donors form Wales. Exp Clin Immuonogenet 1998;15:69–83.