Calibración, Configuración y Puesta en Marcha del Sensor CLF10sc

In this chapter, the HLA analysis of 86 ‘S’ vaccine non-responders that were recruited into a preSl/preS2/S (Hepagene™) vaccine trial is described. This group represented the largest study of antibody non-responders to hepatitis B vaccine and HLA. The objective was to analyse the frequency and distribution of HLA alleles within the non­ responder population and to correlate these findings with HBs-antibody response

following vaccination with Hepagene™ vaccine, which includes additional

immunogenic components of the surface coat of HBV, not present in the ‘S’ containing vaccines.

We found significantly increased frequencies of the HLA genes encoding DRB 1*0701 and DQB 1 *02 both individually and when found in linkage disequilibrium on the HLA haplotype DRB 1*0701; DQB 1*02 compared to HLA and ‘S’ vaccine control populations. This is in agreement with previous reports (Walker et al., 1981; Craven et al., 1986; Alper et a i, 1989) that identified an excess of HLA-DR7 in a population of

‘S’ vaccinated antibody non-responders. The extended HLA haplotype B8, DR3, SCOl which has been strongly implicated in hepatitis B vaccine nonresponse (Craven et al.,

1986; Alper et al., 1989; Kruskall et al., 1992) was found to be carried by 16.3% of our ‘S’ vaccinated antibody non-responders, compared to 11.9% in the vaccine control group. However, this did not reach significance by our stringent analysis. The HLA- DQ2 antigen was found at a frequency of 71.6% in the S’ vaccinated antibody non­

responder population. The genes encoding the HLA-DQ2 are subtyped into

DQB 1*0201 (in linkage disequilibrium with DR3) and DQB 1*0202 (in linkage disequilibrium with DR7) (Hall et al., 1993). The lack of antibody response to ‘S’ vaccination is significantly associated with the HLA haplotype DRB 1*0701; DQB 1*0202. However, a second HLA haplotype DRB 1*0701; DQB 1*03032 was not

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associated with the lack o f antibody response to ‘S ’ vaccination indicating a crucial role for the DQB 1*0202 allele.

Milich first described the effects of vaccination with the products o f the pre-S 1 and pre- 82 regions of the HBV genome in 1985 from mouse studies (Milich and Chisari, 1985). Previous vaccination in humans with the pre-S regions has yielded variable results

(Suzuki et ai, 1994; Leroux-Roels et ai, 1997). H ow ever, no previous com parative

study, equivalent to the num ber o f individuals in the current study has addressed the influence of H L A upon response to a preS l/preS 2/S containing vaccine (Hepagene™)

exists. The revaccination o f the trial subjects with Hepagene™ vaccine elicited an

antibody response in 56/86 o f the previously non-responder individuals. The H L A

haplotypes in the Hepagene"' vaccinated responder population were analysed and found that all those individuals expressing the HLA haplotypes D R l; DQ5 and DR 15; DQ6

demonstrated an antibody response >10 IU/1. Indeed those with the DRB 1*1501;

DQB 1*0602 haplotype all responded with a titre >100 IU/1. In this study we have also observed a low frequency of individuals expressing the DQB 1*0602 allele within the antibody non-responder population, suggesting that the presence o f this allele strongly predisposes to antibody response following ‘S ’ vaccination.

T he most striking observation, however, indicates that the H L A haplotype DRB I * 0 7 0 1; DQ B 1*0202 is significantly associated with antibody nonresponse to the p re S l/p re S 2 /S containing vaccine. The association suggests a role for the H L A -D Q B 1*0202 allele, only found in the context o f this haplotype, in m odulating the im m une response to the

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Hepagene™ vaccine. The H L A -D Q molecules are less well characterised^HLA-DR and have shown to have the ability to present peptides, although expressed at com paratively

lower levels than H L A -D R (Sontheimer et ai, 1986; Brooks and M oore, 1988). The

structure o f the H L A -D Q molecule differs from that o f H L A -D R displaying polym orphism in both o f its a and (3 chains thereby potentially creating restricted

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peptide binding motifs (Kwok et a l, 1995; Raddrizzani et a l, 1997). This may increase the number of possible peptides binding to the HLA-DQ molecules. The principles governing peptide-binding interactions have been gradually defined in the context of disease models (Dorman et al., 1990; Lundin et al., 1993; Jackson and Capra, 1995; Johansen et al., 1996; Kwok et al., 1996; Spurkland et al., 1997; van der Wal gr al., 1997). In order to address the peptide binding motifs involved in response to hepatitis B vaccine peptides, the motifs from a HLA-DQ(a1*0201, p*0202) model

were considered (figures 3.2). From the HLA-DQ(a1*0201, p*0202) model the

characteristic binding pockets located at positions 1, 4 and 9 were identified. However, subsequent experimental data has indicated that anchor residues at positions 6 and 7 also participate in peptide binding specificities (Johansen et al., 1996; van der Wal et al., 1997). Despite polymorphism in both chains of the heterodimer, the p chain polymorphism appears to play the most important role in peptide binding (Johansen et al., 1996; Raddrizzani et al., 1997). However, unlike HLA class I, the principles for peptide binding for the HLA class II molecules and particularly HLA-DQ, remain to be defined.

It has been proposed that in HLA-DQ heterozygote individuals the D Q aand DQP chains may potentially cross pair resulting in functional DQ-heterodimers that may alter peptide binding and T-cell interactions (Kwok et a l, 1993). An association with HLA- DQ (ap) trans-encodQd dimers have been investigated in relation to disease however, in- vivo expression has yet to be resolved (SoUid et al., 1989; Lundin et al., 1990). An analysis for the distribution the potential rran^-encoded dimers could not be investigated in our study population given the low numbers involved.

In a Japanese population, HLA-DQ has previously been linked to a nonresponder phenotypes to ‘S’ containing vaccination, streptococcal cell wall, mycobacterium and pollen antigens (Matsushita et al., 1987; Sasazuki, 1989; Ottenhoff et al., 1990;

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Watanabe et a l, 1990). Although the mechanisms involved still remain unclear, it has been suggested that HLA-DQ linked immune suppression is mediated by CD8^ lymphocytes but the evidence for this remains inconclusive (Sasazuki, 1989). Indeed molecular mimicry of peptides, derived from the regions of HBV in the vaccine, with self peptides could lead to anergy of T cells leading to deletion in the peripheral tissues (Nossal, 1989; Salgame et a l, 1991; Simpson et a l, 1994). Moreover this may also influence the T-cell repertoire during thymic maturation resulting in lack of response to hepatitis surface antigen epitopes.

It is interesting to speculate on why nonresponse is found to be associated more strongly with a DQB 1*0202 allele on a DRB 1*0701 haplotype, when the only difference between the DQB 1*0202 allele and the closely related DQB 1*0201 lies within the second domain, residue 135. The position of this residue does not contribute directly in the formation of the peptide binding groove yet remains influential. This might indicate a distal role for this polymorphism in modifying the conformation of the peptide binding groove or affect the binding of accessory molecules to HLA-DQ as demonstrated for HLA-DR (Fleury et a l, 1995) or, as above, may lead to the education of a specific T-cell repertoire.

Moreover, the observed polymorphism may simply be in linkage disequilibrium with a nonresponse or Ir gene elsewhere within the haplotype. Resolution of this question should facilitate a better understanding of the contribution of HLA-DQ in mediating

vaccine responses. Functional analyses considering the HLA background of the

individual may also be important in elucidating the mechanism underlying hepatitis B vaccine nonresponse.

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CHAPTER 4

Human leukocyte antigens influence the immune response to a Pre-S/S