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Endocrinology Vol. 145, No. 1 228-233
Copyright © 2004 by The Endocrine Society

Low-Dose Immunization with Adenovirus Expressing the Thyroid-Stimulating Hormone Receptor A-Subunit Deviates the Antibody Response toward That of Autoantibodies in Human Graves’ Disease

Chun-Rong Chen, Pavel Pichurin, Gregorio D. Chazenbalk, Holly Aliesky, Yuji Nagayama, Sandra M. McLachlan and Basil Rapoport

Autoimmune Disease Unit (C.-R.C., P.P., G.D.C., H.A., S.M.M., B.R.), Cedars-Sinai Research Institute and School of Medicine, University of California, Los Angeles, California 90048; and Department of Pharmacology 1 (Y.N.), Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8523 Japan

Address all correspondence and requests for reprints to: Basil Rapoport, M.D., Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California. E-mail: rapoportb{at}cshs.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Immunization with adenovirus expressing the TSH receptor (TSHR) induces hyperthyroidism in 25–50% of mice. Even more effective is immunization with a TSHR A-subunit adenovirus (65–84% hyperthyroidism). Nevertheless, TSHR antibody characteristics in these mice do not mimic accurately those of autoantibodies in typical Graves’ patients, with a marked TSH-blocking antibody response. We hypothesized that this suboptimal antibody response was consequent to the standard dose of TSHR-adenovirus providing too great an immune stimulus. To test this hypothesis, we compared BALB/c mice immunized with the usual number (1011) and with far fewer viral particles (109 and 107). Regardless of viral dose, hyperthyroidism developed in a similar proportion (68–80%) of mice. We then examined the qualitative nature of TSHR antibodies in each group. Sera from all mice had TSH binding-inhibitory (TBI) activity after the second immunization, with TBI values in proportion to the viral dose. After the third injection, all groups had near-maximal TBI values. Remarkably, in confirmation of our hypothesis, immunization with progressively lower viral doses generated TSHR antibodies approaching the characteristics of autoantibodies in human Graves’ disease as follows: 1) lower TSHR antibody titers on ELISA and 2) lower TSH-blocking antibody activity without decrease in thyroid-stimulating antibody activity. In summary, low-dose immunization with adenovirus expressing the free TSHR A-subunit provides an induced animal model with a high prevalence of hyperthyroidism as well as TSHR antibodies more closely resembling autoantibodies in Graves’ disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANIMAL MODELS ARE vital tools for investigating the pathogenesis of autoimmune diseases. Spontaneous development of disease, such as diabetes mellitus (type I) in NOD mice, provides an excellent model. Only humans, however, spontaneously acquire Graves’ hyperthyroidism, a prototypic organ-specific autoimmune disease even more common than diabetes mellitus type I (1). Moreover, only after 40 yr of effort was the first induced animal model of Graves’ disease developed. Shimojo et al. (2) succeeded in inducing thyroid-stimulating antibodies (TSAbs) by injecting mice with fibroblasts coexpressing the TSH receptor (TSHR) and major histocompatibility complex class II molecules However, limitations to this model included the relatively low frequency (~25%) of hyperthyroidism. Other immunization approaches involved injecting TSHR-expressing B cells (3) and naked TSHR-plasmid DNA vaccination (4, 5). Unfortunately, neither of these models proved generally applicable, either because of their complexity (3) or because of uncertain factors that resulted in very low incidences of hyperthyroidism in some laboratories (6, 7, 8, 9). Further progress toward a more efficient induced murine model of Graves’ disease involved the im injection of an adenovirus vector expressing the human TSHR DNA (8). This procedure, more practical than the plasmid DNA vaccination protocol, resulted in approximately 50% of immunized animals developing hyperthyroidism. Finally, by adapting the adenovirus model to express the free A subunit of the TSHR rather than the wild-type TSHR, we raised the incidence of hyperthyroidism in BALB/c injected mice to 65–80% (10).

Despite this progress, the search for the optimal induced animal model of human Graves’ disease continues. Although a high proportion of mice injected with TSHR A-subunit adenovirus develop TSAbs, goiter, and thyrotoxicosis (10), some dissimilarities with human disease remain, particularly in the qualitative nature of the TSHR antibodies. TSHR autoantibody concentrations in human disease are very low (11, 12, 13), and only a minority of patients have concomitant thyroid-blocking antibody (TBAb) activity in their serum [for example 18.5% of 200 untreated Graves’ patients (14)]. In contrast, in the adenovirus A subunit-injected mice, TSHR antibodies levels are extremely high, and the great majority have TBAb activity.

Because of the importance in optimizing the induced animal model of Graves’ disease for future studies on pathogenesis and therapeutic approaches, we hypothesized that the number of adenovirus particles injected in different studies (1011) (8, 10, 15) was excessive and could be contributing to very high TSHR antibody titers with TBAb activity. Therefore, in the present study, we immunized mice with up to 10,000-fold lower doses of adenovirus. Indeed, we find that injecting fewer viral particles does reduce TSHR antibody titers and TBAb activity, importantly without altering TSAb activity and incidence of hyperthyroidism. These findings optimize the induced animal model of Graves’ disease and also provide new insight into the balance between stimulating and blocking TSHR autoantibodies in human disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Immunization of mice with adenovirus expressing the TSHR A-subunit
Construction and purification of adenovirus containing TSHR amino acid residues 1–289 (Ad-TSHR-289) has been described previously (10). Because this segment contains nearly the entire TSHR A-subunit, for simplicity we now refer to this virus as A-subunit-Ad. In brief, adenoviruses A-subunit-Ad and control adenovirus (Con-Ad) expressing ß-galactosidase were propagated in HEK293 cells (American Type Culture Collection, Manassas, VA) and purified by CsCl density gradient centrifugation. Viral particle concentration was determined by measuring the absorbance at 260 nm (16). All viruses used in this study were from the same preparation, stored in aliquots at -80 C.

Female BALB/c mice (age, 6–7 wk; Jackson Laboratories, Bar Harbor, ME) were injected im with the following doses of A-subunit-Ad: 1011 (25 mice), 109 (10 mice), and 107 (10 mice) particles per injection (in 50 µl PBS). Mice were injected three times at three-weekly intervals and blood was drawn 1 wk after the second injection. The mice injected with 1011 viral particles have been reported previously (10), although some sera were reassayed in parallel with the new groups immunized with fewer viral particles. Animals were euthanized 4 wk after the third injection to obtain blood, spleen cells, and thyroid glands. All animal studies were approved by the Institutional Animal Care and Use Committee and performed with the highest standards of care in a pathogen-free facility.

Serum thyroxine levels
Total T4 in mouse sera was measured in undiluted serum (25 µl) by RIA using a kit (Diagnostic Products Corp., Los Angeles, CA).

TSHR antibodies measured by ELISA
TSHR antibodies were measured as previously described (6) using ELISA wells coated with purified TSHR A-subunit protein [1 µg/ml in 10 mM Tris (pH 7.4), 50 mM NaCl]. TSHR A-subunits secreted into the culture medium by transfected Chinese hamster ovary (CHO) cells with an amplified transgenome (13) were purified by affinity chromatography (17). Duplicate aliquots of sera diluted 1:102 and 1:103 were analyzed, and antibody binding was detected with horseradish peroxidase-conjugated mouse anti-IgG (Sigma Chemical Co., St. Louis, MO). The signal was developed with o-phenylenediamine and H2O2 and optical density (OD) read at 490 nm.

TSAb, TBAb, and TSH-binding inhibition (TBI) assays
TSAb and TBAb were measured and calculated as previously described (10). In brief, monolayers of CHO cells expressing the wild-type TSHR in 96-well plates were incubated with 3% test serum in 100 µl Hank’s buffer without NaCl and supplemented with 20 mM HEPES (pH 7.4), 1 mM isobutylmethylxanthine, 220 mM sucrose, and 0.3% BSA. After 3 h at 37 C, total cAMP content (medium and cells) was measured by RIA. TSAb was expressed as a percentage of basal cAMP generated in the presence of serum from normal, untreated mice. TBAbs were calculated as follows:

TBI was measured using a commercial kit according to the protocol of the manufacturer (Kronus, Boise, ID). In brief, duplicate serum aliquots (35 µl unless indicated otherwise) were incubated with detergent-solubilized TSHR; 125I-labeled TSH was added and the TSHR-antibody complexes were precipitated with polyethylene glycol. TBI values were calculated from the following formula:

Values greater than the normal range (mean ± 2 SD of TBI activity in Con-Ad mouse serum) were considered positive.

Splenocyte response to TSHR antigen
The response of mice spleen cells to TSHR antigen was performed as described previously (6). In brief, splenocytes (quadruplicate 200-µl aliquots of ~5 x 105 cells) were seeded in round-bottomed 96-well plates in the presence or absence of purified TSHR A-subunits (10 µg/ml) or concanavalin A (Con A; 5 µg/ml; Sigma). Culture medium was RPMI 1640, 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 50 µg/ml gentamycin, 50 µM ß-mercaptoethanol, and 100 U/ml penicillin. Supernatant collected on d 6 was centrifuged to remove cell debris and stored at -80 C. Interferon (IFN)-{gamma} was measured by ELISA using capture and biotinylated detection antibodies from BD PharMingen (San Diego, CA) and following the manufacturer’s protocol. Cytokine production was expressed as picograms per milliliter using standard curves of recombinant murine IFN-{gamma} (PharMingen).

Statistical analyses
Fisher’s exact test was used to determine the significance of differences between the number of mice in a group positive or negative for a particular parameter. Significant differences between the magnitudes of responses of mice in different groups were determined by ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hyperthyroidism in relation to viral particle dose used for immunization
We measured serum T4 levels to diagnose hyperthyroidism in the A-subunit-Ad-injected mice. One week after the second injection, 21 of 25 mice immunized with the standard, high dose of A-subunit-Ad (1011 particles per injection) had elevated serum T4 levels relative to Con-Ad-immunized mice (Fig. 1AGo). Seven of 10 mice immunized with an intermediate dose (109 particles per injection) and six of 10 mice challenged with low dose (107 particles per injection) had elevated serum T4 levels. The proportions of hyperthyroid mice were not statistically different between the group receiving the standard, high dose vs. either of the two groups injected with fewer particles (Fisher’s exact test).



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  		FIG. 1. Similar proportions of BALB/c mice develop hyperthyroxinemia despite immunization with up to 10,000-fold fewer A-subunit-Ad viral particles than used previously (10 ). Mice were immunized with 1011, 109, or 107 particles per injection on three occasions at three-weekly intervals. Serum T4 levels were analyzed 1 wk after the second injection and at euthanasia 4 wk after the third injection. Values for mice immunized with the high dose (1011 particles) were reported previously (10 ). Data are shown for individual mice, and the number of hyperthyroid vs. the total number for each group is indicated. One mouse injected with 109 viral particles and two mice injected with 107 viral particles died 3 wk after the third injection, shortly before scheduled euthanasia. These animals were classified as thyrotoxic because of previously documented hyperthyroxinemia, progressive weight loss, and large goiters at autopsy. None of the mice receiving the highest viral load died. The shaded arearepresents the mean ± 2 SD for serum T4 levels in mice immunized with Con-Ad.

 
Four weeks after the third injection (at the time of euthanasia) there were no premature deaths in the 25 mice receiving the high viral dose, and the prevalence of hyperthyroidism decreased from 21 to 17 animals (Fig. 1BGo). In contrast, the prevalence of hyperthyroidism was maintained in mice injected with fewer A-subunit-Ad particles. Indeed, a number of these animals lost weight and died prematurely, consistent with a previous report of fatal thyrotoxicosis in TSHR-immunized mice (18). At autopsy, these mice had extremely large goiters and were already thyrotoxic after the second injection. Categorizing these mice as thyrotoxic, hyperthyroidism remained high in the intermediate-dose and in the low-dose groups (eight of 10 and seven of 10 animals, respectively). Remarkably, despite up to a 10,000-fold decrease in the viral dose, the prevalence of hyperthyroidism was similar in all groups of animals.

Quantitation of TSHR antibodies
We determined TSHR antibody levels in A-subunit-Ad-immunized mice using two assays. The TBI assay is based on the ability of TSHR antibodies to inhibit radiolabeled TSH binding to its cognate receptor. After the second immunization, all immunized mice had high TBI antibody levels, even when injected with only 107 viral particles. TBI levels were slightly, but significantly, lower in the intermediate- and low-dose groups than in the high-dose group (Fig. 2AGo). Four weeks after the third injection, at the time of euthanasia, TBI values were very high, without statistically significant differences between groups (Fig. 2BGo). Because values were close to maximum, we reassayed TBI activity after the final injection using less serum (5 µl instead of 35 µl). Now within the midrange of the assay, there was clearly no significant difference among groups. TBI values in the mice injected with 1011 viral particles were 58.3 ± 14.1 (n = 25). After injection of 109 and 107 viral particles, TBI values were 49.9 ± 19 (n = 9) and 54.1 ± 17.1 (n = 8), respectively.



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  		FIG. 2. TSHR receptor antibodies in TSHR A-subunit adenovirus injected determined by the TBI assay. BALB/c mice were immunized with the indicated number of A-subunit-Ad viral particles. TBI activities were measured in 35-µl serum aliquots 1 wk after the second injection (A) and 4 wk after the third injection (B). After the second injection, TBI levels in the mice receiving the high dose (1011 particles) were significantly higher than in mice injected with intermediate (109) or low (107) numbers of viral particles (*, P < 0.05, ANOVA on ranks, Dunn’s method). There were no significant differences between the groups of mice injected with 109 and 107 particles or between any of the groups after the third injection. The shaded area represents the mean + 2 SD for TBI levels in mice immunized with Con-Ad.

 
In the second assay, we measured TSHR antibodies by ELISA using purified TSHR A-subunit-coated wells. Sera of mice injected with different doses of adenovirus expressing the TSHR A-subunit, as well as control mice injected with adenovirus expressing ß-galactosidase, were studied at euthanasia, 4 wk after the final immunization. Sera from the control mice, diluted 1:100, provided very low OD values, similar to background (<0.1). At the same dilution, sera from all animals immunized with A-subunit-Ad, regardless of dose, had detectable TSHR antibodies (Fig. 3Go). Compared with animals receiving the standard, high adenovirus dose (1011 particles), mice injected with the lowest adenovirus dose (107 particles), but not an intermediate dose (109 particles), had lower TSHR antibody levels (P < 0.05, ANOVA on ranks, Dunn’s method). At a 1:1000 dilution, ODs were in a more accurate range of the ELISA, and values for both intermediate- and low-dose injected mice were significantly lower than in mice receiving the standard high viral dose (P < 0.05). The difference between the intermediate- and low-dose groups was not statistically significant.



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  		FIG. 3. TSHR antibody levels measured by ELISA. BALB/c mice were immunized with the indicated number of A-subunit-Ad particles as well as with Con-Ad expressing ß-galactosidase. Sera obtained 4 wk after the final (third) immunization, at the time of euthanasia, were tested at the indicated dilutions on TSHR protein-coated ELISA wells. Data shown are the OD values for individual mice (mean of duplicate wells). The shaded area indicates the mean ± 2 SD of values for mice immunized with Con-Ad. *, P < 0.05 (ANOVA on ranks, Dunn’s method) for differences between groups injected with the intermediate or low doses of A-subunit-Ad particles compared with the high-dose group (1011 particles).

 
Functional TSHR antibody activity
Although the TSHR A-subunit ELISA provides quantitative information on TSHR antibody titers, it does not indicate the qualitative, functional properties of TSHR antibodies, an important question in the present study. We, therefore, performed bioassays for both TSAb and TBAb activities using CHO cells stably expressing the wild-type TSHR. Assays could not be performed on the three hyperthyroid mice that died prematurely, shortly before euthanasia. In contrast to the ELISA, neither the level of TSAb activity nor the proportion of mice in each group with detectable TSAb activity was significantly influenced by the viral dose injected. This was the case after the second immunization (Fig. 4AGo), as well as at euthanasia 4 wk after the third injection (Fig. 4BGo).



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  		FIG. 4. TSAb and TBAb activities in the sera of mice immunized with different doses of A-subunit-Ad. BALB/c mice were immunized with the indicated number of A-subunit-Ad particles. TSAb activity (A and B) and TBAb activity (C and D) were measured in sera obtained 1 wk after the second injection and 4 wk after the third injection. CHO cells expressing the wild-type TSHR were used to measure TSAb and TBAb activities as described in Materials and Methods. Data shown are the values of individual mice in each group. The shaded areas indicate the mean ± 2 SD of values for mice immunized with Con-Ad. The proportions of TSAb-positive mice were not statistically different among the different groups. However, the proportions of TBAb-positive mice were significantly lower in the animals injected with 107 A-subunit-Ad particles than in animals receiving the standard, high-dose number of particles (1011) (C and D); *, P = 0.022; **, P = 0.036 (Fisher’s exact test).

 
In contrast to the TSAb values, there was a clear relationship between viral particle dose and TBAb activity. Thus, after the second immunization, a high proportion of mice receiving the standard, high viral dose (17 of 25; 68%) had TBAb activity greater than that in control animals (Fig. 4CGo). After the third high-dose injection, the prevalence of TBAb activity increased to 80% (20 of 25) of animals (Fig. 4DGo). In contrast, with the lowest A-subunit-Ad dose (107 viral particles), TBAb activity was present in only 2 of 10 (20%) and three of eight (38%) mice after the second and third injections, respectively, both significantly fewer than in the high-dose group (P = 0.022 and P = 0.036, respectively; Fisher’s exact test). Intermediate TBAb prevalences were observed in the intermediate viral dose groups: five of 10 (50%) and five of nine (56%) mice after the second and third injections, respectively (not significantly lower than in the high dose group).

T cell responses to TSHR antigen
Splenocytes from BALB/c mice immunized with the standard number of TSHR adenovirus particles (1011) produce IFN-{gamma} when challenged in vitro with TSHR antigen (19). However, in the present study we wished to determine whether this IFN-{gamma} response to antigen was quantitatively proportional to the viral dose administered. We cultured splenocytes harvested from mice 4 wk after the third immunization with 1011, 109, and 107 viral particles, as well as with 1011 Con-Ad. Splenocytes from Con-Ad-immunized mice generated no IFN-{gamma} when cultured with TSHR protein (Fig. 5AGo). In contrast, all groups of A-subunit-Ad-immunized mice responded strongly to challenge with TSHR antigen. There was no significant difference in the amplitude of the IFN-{gamma} response between the groups of mice receiving high, intermediate, and low doses of A-subunit-expressing adenovirus. Splenocytes from all animals, including controls, generated IFN-{gamma} (Fig. 5BGo) in response to nonspecific stimulation with Con A.



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  		FIG. 5. Splenocyte response to challenge with TSHR antigen in vitro. Splenocytes were obtained from BALB/c mice immunized with Con-Ad or with high (1011), medium (109), or low (107) numbers of A-subunit-Ad. After culture for 6 d in medium alone or in medium supplemented with TSHR A-subunit protein (10 µg/ml; A) or in Con A (5 µg/ml; B), IFN-{gamma} was measured in the culture supernatant by ELISA (see Materials and Methods). Each bar represents the mean + SD of values for net IFN-{gamma} production by splenocytes from four animals after subtracting the value obtained in medium alone. IFN-{gamma} production in response to antigen or to Con A was not significantly different among groups of mice immunized with different numbers of A-subunit-Ad particles.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Adenovirus vector expressing the TSHR injected im (8) or by transfecting dendritic cells (20) has overcome many of the limitations of previously described animal models of Graves’ disease. Moreover, immunization with an adenovirus expressing the TSHR A-subunit, as opposed to the holoreceptor, refined the induced model further with hyperthyroidism resulting in the great majority of mice (10). Yet differences with human Graves’ disease remain. TSHR antibody levels are far higher than observed in the typical Graves’ patient. Moreover, we noted that the prevalence of hyperthyroidism tended to be higher after the second than after the third immunization with the suggestion that TBAbs were concomitantly increased (10). These observations suggested similarity with some unusual Graves’ patients in whom TBAb activity is high, sometimes even to the point of producing hypothyroidism. Such patients (as in our mice) typically have extremely high titers of TSHR autoantibodies (for example Ref. 21).

In the present study, we confirmed our hypothesis that a weaker antigenic stimulus would alter the balance between stimulating and blocking TSHR antibody activity toward that found in the typical Graves’ patient. As would be expected, immunizing mice with fewer TSHR-expressing adenovirus particles reduced the TSHR antibody titer as detected by ELISA. TBAb activity in serum declined in parallel. Remarkably, despite immunization with a far lower viral dose, there was no reduction in serum TSAb activity, nor did the prevalence of hyperthyroidism decrease. Indeed, it is likely that the most severe hyperthyroidism occurred in some animals immunized with fewer A-subunit-Ad particles. The only premature deaths occurred in these groups (three of 20 mice), unlike survival until euthanasia in all 25 animals injected with 100–10,000 times more viral particles. Moreover, these deaths occurred long (3 wk) after the final immunization, in animals already hyperthyroid after the second injection, and with large goiters and progressive weight loss, consistent with fatal thyrotoxicosis in mice (18).

Although the major focus in this study was on TSHR antibodies and thyroid cell function, we also examined T cell responses to TSHR antigen in vitro. We found comparable T cell responses (at least for the Th1 cytokine IFN-{gamma}) regardless of A-subunit-Ad viral dose administered. These data are reminiscent of the previous finding with naked TSHR-DNA vaccination of the same mouse strain that the T cell response to TSHR antigen was independent of the development of TSHR antibodies (6). It should be appreciated that many studies of immune responses examine antibodies induced by one protocol and T cell responses induced by different, typically short-term, immunization. The present findings are of value because they demonstrate that this optimized induced model of Graves’ disease is amenable to the simultaneous study of both antibody and T cell responses.

Besides optimizing the animal model, the present observations on TSHR antibodies provide insight into the pathophysiology of human Graves’ disease. Our data indicate that with a greater immune stimulus to TSHR antibody generation, the balance shifts away from TSAbs toward TBAbs. Furthermore, TBAbs have more diverse epitopes than TSAbs (discussed in Ref. 22), consistent with our previous finding in which progressively greater antigenic stimulation led to B cell epitope spreading from the critical TSHR N-terminal region to additional epitopic determinants further downstream in the TSHR ectodomain (23). Indeed, the latter study is likely to underestimate epitope spreading because linear peptide scanning is blind to discontinuous or highly conformational epitopes. B cell epitope spreading is a well recognized phenomenon in autoimmunity (reviewed in Ref. 24). Finally, the present data suggest a possible reason why radioiodine therapy may occasionally lead to transient hypothyroidism in association with TBAb activity (25, 26). Boosting the TSHR antigen stimulus after thyrocyte damage may, in some patients, enhance TBAb generation. TSHR antigen denaturation may also contribute to the preferential induction of TBAb.

In summary, in the present study, by immunizing BALB/c mice with up to 10,000-fold fewer TSHR A-subunit adenovirus particles than the standard dose, we attained lower TSHR antibody titers without any decrease in the high incidence of hyperthyroidism. In contrast to TBAb activity, which declined, TSAb activity was unchanged. Therefore, with a lower immunization dose, the induced animal model more closely resembles Graves’ disease in humans. These data explain, in part, the clinical observation in human disease that significant TSH-blocking activity is typically associated with high TSHR autoantibody titers.


   Acknowledgments
 
We are grateful for contributions by Dr. Boris Catz, Los Angeles.


   Footnotes
 
This work was supported by NIH Grants DK 19289 and DK 54684.

Abbreviations: A-subunit-Ad, Adenovirus expressing TSHR A-subunit; bTSH, bovine TSH; CHO, Chinese hamster ovary; Con A, concanavalin A; Con-Ad, control adenovirus; OD, optical density; TBAb, thyroid-blocking antibody; TSAb, thyroid-stimulating antibodies; TSHR, TSH receptor.

Received August 29, 2003.

Accepted for publication October 6, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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