Journal of Hepatology
Volume 37, Issue 3 , Pages 396-399, September 2002

Influence of C4A deficiency on nonresponse to HBsAg vaccination: a new immune response gene

The Vaccine Research Institute of San Diego, P.O. Box 17439, San Diego, CA 92177, USA

Accepted 2 July 2002.

See Article, pages 387–392

Article Outline

 

The hepatitis B surface antigen (HBsAg) is a rather unique antigen in terms of the extensive immunogenetic analysis that has been performed in both mice and humans. Early studies in mice indicated a strong influence of major histocompatibility complex (MHC)-linked class II genes on the immune response to HBsAg and identified high (H-2d,q), intermediate to low (H-2 a>b>k), and nonresponder (H-2s,f) haplotypes [1]. The suggestion that MHC-linked genes may also control the human immune response to HBsAg was first made by Walker et al. who observed a significant excess of HLA-DR7 and a total absence of HLA-DR1 in HBsAg-vaccinated low or nonresponders [2]. Subsequent studies demonstrated that the HLA class II alleles DRB1* 3, 7, and 14 and DQB1* 2 are associated with low or nonresponsiveness and DRB1* 1, 13, and 15 and DPB1* 4 are associated with efficient humoral responses to HBsAg vaccination [3], [4], [5], [6], [7], [8]. Several groups have demonstrated that vaccine antibody low or nonresponders also fail to demonstrate efficient HBsAg-specific Th cell activation in vitro [9], [10]. MHC class II molecules mediate Th cell activation by binding T-cell epitopes and these MHC-peptide complexes are presented on the surface of antigen presenting cells (APC) to Th cells. T-Cell nonresponse can result from the failure of a given MHC class II molecule to bind a T-cell epitope from the antigen (i.e. presentation defect) or from the lack of a T cell that can recognize a particular MHC–peptide complex (i.e. a hole in the T-cell repertoire). Both defects have been proposed to explain nonresponse to HBsAg vaccination in humans [6], [11], [12], [13]. In the murine system it appears that nonresponse is due to a presentation defect because the H-2s and H-2f haplotypes are nonresponsive to both the d and y subtypes of HBsAg, which are non-crossreactive at the T-cell level [14], [15]. Human vaccine studies to determine if nonresponsiveness to both the d and y subtypes of HBsAg correlates with the same MHC class II alleles are warranted and may resolve the mechanism of MHC class II-mediated nonresponse in humans.

The study by Höhler et al. [16] in this issue demonstrates that MHC class III genes can also influence responsiveness to HBsAg vaccination and do so at the B-cell level. Although associations of C4A alleles with the response to HBsAg vaccination have been previously suggested [17], [18], [19], Höhler et al. convincingly demonstrated in a study involving 4269 vaccinees that nonresponders to HBsAg have a significantly increased prevalence of C4A gene deletions and of non-expressed C4AQO alleles compared to responders. Höhler et al. demonstrate that while the C4A*Q and the DRB1*0701 alleles show strong independent associations with nonresponse (as well as an additive effect), no independent association for DRB1*0301 with nonresponse was observed. Because the HLA A1,B8 DRB1*0301 haplotype is in strong linkage disequilibrium with C4A gene deletions, the authors suggest that the often reported association of DRB1*0301 with nonresponse to HBsAg vaccination may be primarily due to a C4A deficiency.

How might C4A deficiency impair anti-HBs production? As the authors explain, C4 is crucial to the cleavage of C3 and the attachment of C3d or C4d fragments to antigen which targets antigen to follicular dendritic cells (FDC) and B cells via CD21 and CD35 receptors. Further, coligation of the CD21/CD19 receptor complex with the B-cell receptor (BCR) enhances signal transduction and reduces the amount of antigen required by 10–100-fold [20]. This effect has been directly demonstrated by coupling three copies of the C3d molecule to the N-terminus of lysozyme, which dramatically enhanced immunogenicity in vivo [21], These results should not be surprising since mice genetically deficient in C4 demonstrate normal T-cell responses and impaired B-cell responses [22]. This raises an important question. If C4A deficiency primarily effects the B-cell response why do DRB1*3+ nonresponders fail to demonstrate T-cell proliferative responses to HBsAg [10], [12]? Either DRB1*3+ nonresponders have an accompanying T-cell APC defect which only prevents anti-HBs production in the absence of adequate levels of C4A or insufficient HBsAg-specific B-cell activation negatively effects HBsAg-specific T-cell activation via lack of B-cell APC function, etc.

It was also interesting that the majority of HBsAg nonresponders in this study were heterozygous for C4AQO alleles. This suggests that a mere partial deficiency in C4A can have severe consequences on the humoral anti-HBs response. This raises another question. Does the immune response to HBsAg require especially high levels of C4A? If not, it is likely that nonresponders to HBsAg will be nonresponsive to other antigens as well since C4A deficiency represents an antigen-nonspecific defect.

This study by Höhler et al., the advances in understanding of MHC gene products, and the role of complement components in regulating the immune response have prompted me to revisit some murine immunogenetic data published almost 20 years ago. The murine humoral anti-HBs response is regulated by at least two H-2-linked genes, one mapped to the I-A subregion (Ir-HBs-1) and the other (Ir-HBs-2) mapped to the I-C subregion [15]. Subsequently, the I-C subregion was abandoned along with the I-B and I-J subregions because Ia-like protein products could not be demonstrated. The H-2 class II region was simplified to include only the A and E regions [23]. Therefore, the Ir-HBs-2 gene actually maps to a region to the right of the E region and to the left of the class I D region designated the S region, which encodes the MHC class III molecules including the C4 and C2 complement components. The mapping of a gene affecting the anti-HBs response to the S region is demonstrated in Table 1.

Table 1. Influence of the H-2 class III S region on immune response to HBsAga
StrainH-2Anti-HBs titer (1/dilution)
KAESD
A/Jkkkdd65536
A.ALkkkkd4096
B10.A(2R)kkkdb16384
B10.BRkkkkk2048
B10.S(9R)sskdd2048256 (ay)
B10.HTTsskkd2560 (ay)

a Pairs of intra-H2 recombinant strains were immunized with two doses of 4.0 μg of HBsAg/ad, except the B10.S(9R)/B10.HTT strains, which were immunized with the ad and ay HBsAg subtypes. Sera were analyzed for anti-HBs antibody by RIA. Titers are expressed as the highest serum dilution to yield 2.5 times the counts of preimmunization sera. Modified from [15].

Comparisons of the anti-HBs response in intra-H2 recombinant murine strains, which are virtually identical except in the S region, reveal dramatically different responses. In all cases mice possessing Sk region are inferior responders to mice possessing a Sd region or a Sb, Ss regions (not shown). Although the S region contains a number of genes, only one S region gene is defective only in H-2k-bearing mice. Mouse strains carrying the C4k gene derived from the H-2k haplotype express a serum C4 level that is 1/10 to 1/20 of that in non-H-2k strains due to a retroposon-like insertion of the B2 sequence into intron 13 of the C4k gene [24]. Therefore, it seems likely that the inferior anti-HBs antibody responses observed in Sk-bearing mice is due to a C4 deficiency, which supports the observations of Höhler et al. for the human response. Note that the effect of the Sk gene in the context of the Ak responder gene is quantitative and results in an 8–16-fold reduction in anti-HBs titer. This represents a dramatic effect mediated by only a partial deficiency in C4 serum levels. In the context of a nonresponder gene in the A region (As) the presence of the Sk gene correlates with nonresponse as seen when the B10.S(9R) and B10.HTT strains are immunized with the HBsAg/ay subtype (Table 1). Although the B10.S(9R) and the B10.HTT strains both respond to the HBsAg/ad subtype, the B10.S(9R) response is 10-fold higher. The Ek molecule most likely serves as a restricting element in these low responder strains. Therefore, a reinterpretation of the original data suggests that at least two MHC class II genes in the A and E regions and one MHC class III gene in the S region regulate the anti-HBs antibody response in mice. Interestingly, the Sk region also negatively affects the HBsAg-specific T-cell proliferative response as compared to Sd,b,s regions in the context of the Ak responder gene [25]. Therefore, it appears that reduced C4 levels can adversely effect both anti-HBs antibody production and HBsAg-specific T-cell activation in mice.

Unlike C4 knockout mice, the H-2k haplotype is not a low antibody responder to all antigens. For example, H-2k mice are low responders to HBsAg, but high responders to a similar particulate HBV antigen, the hepatitis core antigen [26]. This reflects the variability of MHC-linked regulation of immune responses and most likely differences in antigen structure and complexity. The combined influences of the MHC class II and class III genes are apparent in the murine anti-HBs response. This level of complexity in inbred mice predicts far greater complexity in an outbred human population. In this regard, even the responders in the Höhler et al. study demonstrate a large range of anti-HBs levels (200–20 000 IU/ml). Of course, human MHC class II genes are very polymorphic; however, the C4 protein is the most polymorphic of the complement components and other than the immunoglobulins the most polymorphic serum protein [27], [28]. It would be interesting to determine if C4A deficiency also correlates with low response status among the HBsAg responder population. Considering that partial C4 deficiency is the most common inherited immune deficiency in humans and the combined prevalence of C4A and C4B deficiency is over 31% among Caucasians [27], the Höhler et al. study reveals a very important variable affecting the regulation of the human anti-HBs immune response and most likely responses to a host of other antigens as well.

In hindsight, the murine literature may also address the question of how unique the HBsAg may be in terms of ‘sensitivity’ to C4 serum levels. Immune response genes have been mapped to the I-C subregion in the responses to both lysozyme [29] and sperm whale myoglobin [30], and in both cases a I-Ck/Sk region gene conferred low responsiveness as compared to a I-Cd/Sd region gene. In fact, the old literature suggested that the I-C subregion influenced the generation of suppressor T cells [23]. These observations should be re-evaluated in light of the C4k gene defect, our increasing appreciation for the role of C4 in immune regulation and the results of Höhler et al.

In summary the demonstration by Höhler et al. that a MHC class III gene (C4A) can independently and in association with MHC class II genes regulate the immune response to HBsAg vaccination most likely at both the B- and T-cell levels is important in terms of future HBsAg vaccine development, implications for natural HBV infection and most likely is relevant to other antigens as well. Publication of this paper may send a number of investigators ‘scurrying’ back to reanalyze and possibly reinterpret their old data, as I did.

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Acknowledgements 

The author thanks Isabelle Desombere, Geert Leroux-Roels and Jean-Noel Billaud for helpful comments, and Rene Lang for editorial assistance.

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References 

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PII: S0168-8278(02)00237-4

Journal of Hepatology
Volume 37, Issue 3 , Pages 396-399, September 2002

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