1. Introduction
Type 1 diabetes mellitus (T1DM) is an autoimmune disease in which antibodies and T cells target a range of host autoantigens associated with beta cell production of insulin (INS), resulting in loss of INS production and consequent hyperglycemia. While much of the focus of T1DM research is on the autoimmune targeting of INS itself [
1,
2,
3], antibodies and T cells also target glutamic acid decarboxylases (GAD) [
1,
2,
3], protein tyrosine phosphatase non-receptor types (related to islet-associated protein or PTPN-IA-2) [
1,
2,
3], the INS receptor (INSR) [
4,
5,
6,
7,
8], and glucagon [
9,
10]. This combination of autoantigenic targets helps to explain why pancreatic beta cells are particular targets of T1DM pathogenesis. However, the major mystery concerning T1DM pathogenesis is the disease’s etiology: what triggers the autoimmunity directed at these pancreatic targets?
Determining the causes of autoimmune diseases such as T1DM has turned out to be a recalcitrant problem. Despite over a century of epidemiological and experimental studies of autoimmunity, the natural cause of no human autoimmune disease has yet to be discovered. It is generally believed that predisposition to autoimmune diseases is determined by genetic factors but that infectious (or other environmental) factors are required to trigger the disease process (e.g., [
11,
12,
13,
14,
15,
16,
17]). Epidemiological methods in conjunction with individual patient case reports are generally used to try to identify what these infectious triggers may be. The general assumption is that causative microbes present antigens to the immune system that mimic the host autoantigens that the disease subsequently targets.
The onset of T1DM has been associated epidemiologically with both viral and bacterial infections, and the best clinical correlations for the onset of T1DM are probably the coxsackieviruses (COX), both A and B strains [
18,
19,
20,
21,
22,
23]. However, other enteroviruses [
18,
23,
24,
25], such as rubella, mumps, rotaviruses, cytomegalovirus (CMV), Epstein–Barr virus (EBV), and hepatitis C virus (HCV), have also been associated with T1DM initiation [
13,
26,
27,
28,
29]. Bacterial infections associated with onset of T1DM include
Bordatella pertussis and
Mycobacterium species [
30,
31] and
Helicobacter pylori [
32]. Studies using T cells specific for the INS B chain, which is often considered to be the main target of T1DM autoimmunity, have identified
Streptococci,
Clostridia,
Escherichia coli, and
Pseudomonas [
33] as potential mimics. T cells specific for GAD65 identified
Streptococci,
Staphylococci,
Haemophilus,
Legionella, and
Chlamydia as the most likely triggers [
34]. Most recently, significant differences in gut microbiota between children who have just been diagnosed with T1DM and those who have not suggest that intestinal bacteria may also play a critical role in triggering or regulating the development of diabetes [
35,
36,
37,
38,
39,
40]. The focus on the gut microbiome has led to the identification of
Parabacteroides distasonis as a possible trigger of T1DM because its bacterial antigens activated both human T cell clones from T1DM patients and T cell hybridomas from nonobese diabetic (NOD) mice specific to the INS B chain residues 9–23 [
40]. However,
P. distasonis was not identified by previous studies of T cells reactive to INS [
41,
42]. Another study identified peptides from
Bacteroides fragilis and
Clostridium asparigiforme as potent activators of human T1DM T cells responsive to pre-pro-INS [
41], of which only
Clostridia were identified in previous studies [
41,
42].
Unfortunately, the numerous agents associated with T1DM leave significant questions regarding the sufficiency and necessity of any one microbe as a trigger for diabetes, a problem that has persisted for decades [
28,
43]. Attempts to model the onset of T1DM using individual infectious agents from the list above have thus far failed. No one has been able to produce T1DM in any animal using any of the single infectious agent listed above. COX and CMV exacerbate or accelerate disease in rodents already producing autoantibodies (e.g., NOD mice) or pretreated with the pancreatic toxin streptozotocin [
21,
44] but produce only transient pancreatic pathology in select strains of non-diabetic mice [
45]. Monkeys infected with coxsackie B virus types 3 and 4 also develop transient pancreatitis but fail to develop chronic diabetes [
46]. Cross-reactivity between COX antigens (strains B1-B6) and human T1DM antibodies reactive to either pro-INS or GAD could not be identified [
47,
48,
49,
50,
51,
52,
53]. COX antibodies do not appear to be cross-reactive with INS, nor are INS antibodies cross-reactive with COX [
54]. Moreover, although evidence of COX infections appears in temporal relationships with subsequent T1DM diagnosis [
50,
55,
56], at least one live enterovirus vaccine—oral polio—is not associated with any increased risk of T1DM, even among genetically high-risk individuals [
57,
58]. However, COX infections have been linked to the development of INS receptor antibodies [
59].
Even more confusingly, some putative triggers of T1DM have actually prevented the development of T1DM in animal models (reviewed in [
60]). For example, pertussis vaccine protected streptozotocin-treated CD-1 mice against developing diabetes [
61]; immunization with
Mycobacterium. leprae [
62], Bacillus Calmette-Guerin (BCG) [
63], or
Clostridium butyricum [
64] also prevented diabetes in NOD mice [
65].
Taken as a whole, the results summarized above demonstrate that mono-infectious approaches to modeling T1DM have universally failed or yielded results that seem to contradict the role of any particular microbe as a cause of the disease. These failures led Horwitz et al. [
20,
66,
67] to suggest that the role of COX may not be as direct triggers of T1DM via molecular mimicry but rather as bystander infections supporting some other infectious agent. As Filippi and von Herrath [
68] suggested, “This could be explained by the fact that viral association with T1D will likely be multifactorial”.
So, perhaps the difficulty identifying “the cause” of T1DM stems from the assumption that “the cause” is mono-factorial and resides in the unique identification of one of the microbes listed above as “the” T1DM trigger. However, perhaps no single microbe is both necessary and sufficient. Perhaps a new paradigm is required, based on multiple, concurrent infections as autoimmune disease initiators stimulating the production of complementary or synergistic sets of autoantibodies and TCR directed at multiple targets simultaneously.
The major aim of our research is to explore the possibility that sets of microbes may cooperate to induce T1DM. This aim requires a shift in the types of experiments and logic employed to test possible T1DM etiologies. The standard approach to elucidating autoimmune disease etiology is based on Koch’s postulates, which assume a single etiological agent. A concurrent-infection model requires that two or more etiological agents be involved. The use of multiple agents is consistent with the observation that there are multiple autoantigen targets in T1DM. Therefore, one aim of our research is to use proteonomic methods to identify microbes that mimic the key autoantigens in T1DM, INS, and INSR. A second aim is to use the mimicry data as the basis for experimentally testing whether antibodies against the identified microbes cross-react with INS and INSR. Because INS and INSR are molecularly complementary, logically it follows that the microbial antigens inducing cross-reactive immunity to INS and INSR will also be complementary to each other. Thus, a third aim is to test for possible antigen complementarity. Three such tests are presented. The first test is based on the proposition that some microbial antigens mimicking INS and INSR will bind to each other just as INS and INSR themselves bind to each other. A second test is predicated on the assumption that antibodies against these INS and INSR mimics will act like idiotype–anti-idiotype pairs. A third test is whether or not T cell receptor sequences from T1DM patients that have previously been demonstrated to bind to INS and INSR peptides also recognize these microbial antigens. The final aim is to test whether sera from T1DM patients recognize the microbial antigens identified in testing the previous aims.
The results consistently reveal that COX mimic INSR antigens; Clostridia mimic INS; and that the resulting immune responses whether based on animal-derived antibodies, human TCR sequences, or T1DM sera, involve idiotype–anti-idiotype relationships to sets of complementary antigens. No set of control antibodies displayed a similar range of interactions. These results suggest a new role for COX and other enteroviruses (inducing INSR, rather than INS, cross-reactivity) in T1DM etiology; identify Clostridia as the triggers of INS cross-reactivity for the first time; and provide the first evidence suggesting a multifactorial, synergistic mechanism for T1DM etiology.
3. Discussion
3.1. Detailed Summary and Interpretation of Results
Section 2.1 demonstrated that pathogenic
Clostridia species mimic INS, PTPN(IA2), and GAD65 better than any other human pathogen or commensal microbe, while COX was the most likely T1DM-associated mimic of INSR, a result consistent with a previous similarity study that found very significant similarities between INS and PTPN(IA-2) [
74].
Lactobacilli and
Bifidobacteria might play a similar role, but their lack of pathogenicity may mediate their autoimmunogenic potential.
Section 2.2 demonstrated that, as predicted, COX antibodies bound to INSR and some of its constituent peptides, while
Clostridia antibodies (and most of the other microbial antibodies tested) did not. Conversely,
Clostridia antibodies bound to INS, but COX antibodies (and most of the other microbial antibodies tested) did not. Very few other microbial antibodies tested had any affinity for either INS or INSR. The notable exceptions were
Streptococcal antibodies binding to INS and Epstein–Barr virus (EBV) antibodies recognizing INSR peptides. Notably, COX antibodies were demonstrated to be complementary to
Clostridium antibodies and EBV antibodies to
Staphylococcal antibodies, mimicking INS–INSR complementarity (
Section 2.3).
Section 2.4 then demonstrated that because COX antibodies mimic INS by binding to INSR, COX antibodies also bind to INS antibodies. Similarly, because
Clostridia antibodies mimic INSR by binding to INS,
Clostridia antibodies bind to INSR antibodies. It follows that at least some COX antibodies are not only complementary to
Clostridia antibodies, as demonstrated in the previous Section, but that this complementarity involves INS- and INSR-specific idiotypes. It was further demonstrated that, as predicted from the similarity data in
Section 2.1, PTPN(IA-2) and GAD antibodies could be substituted for INS and
Clostridia antibodies, yielding the same binding to COX antibodies. The basic logic of these experiments is summarized in
Figure 17.
The complementarity of INSR-like COX sequences and INS-like
Clostridia sequences were demonstrated using U.V. spectroscopy in
Section 2.5 and
Section 2.6, and it was then shown that TCR derived from T1DM patients recognized these COX and
Clostridia peptides as antigens, providing direct evidence of autoimmunity against both microbes in three sets of patient TCR. These results indicated that each patient had TCR against both INS–
Clostridia and against INSR–COX. Similar results were obtained using sera from T1DM patients, which recognized inactivated
Clostridium sporogenes and COX antibodies (
Section 2.7 and
Section 2.8), thus demonstrating the existence within the sera of anti-idiotype antibodies against both
Clostridia antibodies and COX antibodies. Not surprisingly, given the prevalence of the microbes, some of the healthy and T2D control sera also recognized
Clostridium antigen,
Clostridium antibodies, and COX antibodies, though generally with lower affinities than the T1DM sera (
Figure 15).
In short,
Clostridia antigens mimic INS; both are in turn mimicked by some INSR antibodies as well as some COX antibodies. COX antigens mimic INSR so that COX antibodies mimic INS as well as INS-like
Clostridia antigens. Thus, COX antigens are complementary to
Clostridia antigens; COX antibodies are complementary to (idiotype–anti-idiotype)
Clostridia antibodies; which means that COX antigens mimic antibodies against
Clostridia as well as INSR, while
Clostridia antigens mimic antibodies against COX as well as INS (
Figure 17). The result is that the immune system loses the ability to differentiate between “self” and “non-self” because any simultaneous response to both COX and
Clostridia necessitates an active response against its own antibodies (and TCR) as well INS and INSR. Moreover, each antibody mimics one of the microbial antigens, creating further confusion. A combination of EBV with Staphylococci may induce similar self–non-self confusion, leading to T1DM.
3.2. Relationship of the Experimental Findings to Previous Results
The experimental findings reported here are consistent with much of the previously published literature concerning T1DM etiology and pathogenesis. While a variety of viruses and bacteria have been associated with T1DM onset (reviewed in [
76]), enteroviruses, and in particular COX, have been the ones most consistently identified through methods ranging from direct virus isolation to antibody cross-reactivity to microbiome studies [
12,
13,
14,
15,
16,
17,
18,
19,
19,
20,
21,
22,
23,
48,
49,
50,
51,
52,
53,
54,
55,
56,
76,
77,
78,
79,
80,
81]. Our results also make a strong case of a role for enteroviruses in T1DM etiology but suggest that the main target of enterovirus antibodies is not INS (as most previous research has attempted to demonstrate) but rather INSR, a T1DM target other investigators have previously reported [
4,
5,
6,
7,
8,
9,
54,
82,
83,
84,
85,
86]. Additionally, previous studies have found that COX does not elicit anti-INS antibodies, nor antibodies against GAD or PTPN(IA-2) [
38,
51,
53,
54,
87,
88,
89,
90,
91]. These results are consistent with our previous report that GAD, PTPN(IA-2), and INS share many homologies [
73,
74] and the results shown in
Figure 11 demonstrating that COX antibodies are complementary to, rather than mimics of, GAD and PTPN(IA-2) antibodies.
What is missing from the enterovirus story is how INS becomes a primary target of autoimmunity in T1DM. Given the importance of INS autoantibodies [
40,
41,
42,
66,
92,
93,
94,
95,
96,
97,
98], as well as anti-GAD and anti-PTPN(IA-2) autoantibodies and TCR [
99,
100,
101,
102,
103], in T1DM, and the failure of COX antibodies to recognize these antigens, a critical question is whether there needs to be a second microbe that triggers concomitant anti-INS autoimmunity. Our results strongly implicate
Clostridia for this role.
While few studies have directly linked the presence of a
Clostridium infection to initiation of T1DM [
104,
105], a very large number of studies have implicated dysregulation in the number and types of gut
Clostridia. In particular,
Bifidobacteria, Bacteroides, and
Lactobacilli all decrease significantly, while the number of
Clostridia increases and is directly correlated with the degree of glucose dysregulation observed in the patient [
26,
37,
106,
107,
108,
109,
110,
111]. Thus, it is plausible that
Clostridia plays a role in T1DM etiology, either to produce bystander activation of the immune system or, as is more likely in view of the complementarity to COX demonstrated here, by synergizing with COX via the production of complementary immune responses. Microbiome constituents such as
Lactobacilli,
Bifidobacteria, and
Bacteroides may become accidental targets of the resulting autoimmunity because they, too, express antigens that mimic INS (
Figure 1 and
Figure 2) [
112]. Additionally, our results are consistent with the observation that pro-INS and GAD65 are both recognized as antigens by the same T cells [
1,
38,
39,
113] so that
Clostridia could initiate autoimmunity against both antigens via shared mimicry and/or epitope drift.
3.3. Animal Models
Two animal models support the theory that T1DM has a multifactorial etiology involving a combination of viral and bacterial infections and, in particular, that Clostridia has a role in triggering the disease.
T1DM can be triggered in Lewis rats by infecting them with Kilham rat virus (KRV). Studies found that KRV infection significantly increased the abundance of intestinal
Bifidobacterium and
Clostridium species, indicating a possible synergism between the KRV and these bacteria. Furthermore, treating KRV-infected rats with a combination of trimethoprim and sulfamethoxazole (Sulfatrim) beginning on the day of infection prevented the increase in
Bifidobacterium and
Clostridium abundance, and also T1DM development [
114].
T1DM can also arise “spontaneously” in genetically-predisposed nonobese diabetic (NOD) mice. However, several studies suggest that bacteria, and in particular
Clostridia, are involved in triggering diabetes. Tanca et al. [
115], for example, found that NOD mice, compared with genetically modified NOD mice protected from T1D (Eα16/NOD), differed in the significant depletion of commensal
Clostridial butyrate biosynthesis species. Consistent with this finding, Jia et al. [
64,
116] demonstrated that supplementing the gut microbiome of NOD mice with the probiotic
Clostridium butyricum protected them against diabetes onset. Fecal transplants from non-NOD mice into NOD mice also prevented onset of T1DM, specifically increasing colonization by commensal
Clostridia species [
117]. Conversely, vancomycin-treated NOD mice were much more prone to develop T1DM than non-treated NOD mice, and the accelerated risk was again associated with a significant decrease in commensal
Clostridia species in the gut providing a niche for pathogenic forms [
118].
3.4. Epidemiology of Clostridium and Enterovirus Infections
The epidemiology of Clostridium and enterovirus infections is also consistent with the possibility that T1DM is a result of their co-infection. Such a multifactorial mechanism helps to explain one of the great mysteries of autoimmune disease epidemiology, and that of T1DM in particular, which is why many of the putative triggers are so common and the incidence of disease so rare.
Overt
Clostridium difficile infections are relatively common, particularly in children, with an incidence of 4 in 1000 American children or 6 per 10,000 patient days [
119]. The rate is approximately half that in most European nations, and adults contract
Clostridia infections at somewhat lower rates worldwide [
120]. However, studies of asymptomatic
C. difficile carriage demonstrate that approximately 12% of children less than 18 years of age are infected with non-toxigenic variants, while 6% carry toxigenic variants (reviewed in [
121]). In most countries, there is no seasonal variation in incidence [
122].
There are also approximately 10 million (1 in 35) COX infections each year in the U.S., the majority among infants and children, with similar rates in most other nations [
123,
124]; however, asymptomatic carriage of enteroviruses as a whole is approximately 5% among children under 18 worldwide [
125]. Cases follow cyclical patterns that vary seasonally across the globe. In North America, cases tend to increase sharply during summer and again in late fall, with a peak in August [
126], while, for instance in China, the peak is in January–February [
127]. Like Clostridia, COX infections also occur in adults at slightly lower rates than in children [
123,
124,
125,
126,
127].
The epidemiology of COX and
Clostridium infections broadly correspond to T1DM epidemiology. The incidence of new T1DM diagnoses is approximately equal among individuals less than 19 years of age and in those above 19 years of age [
128,
129], making children approximately four or five times more likely to develop T1DM than adults in any given year of life. This epidemiology is consistent with the majority of COX and
Clostridia infections occurring in children. Additionally, the seasonal incidence of new T1DM diagnoses correlates reasonably well with COX incidence. The peak of new T1DM diagnoses in China occurs between December and February [
130], which corresponds to the January peak in COX infections. In the U.S., there are two peaks, one of them in August and the other in early winter [
130], also corresponding again to the variations in COX incidence. The incidence of new T1DM diagnoses in the southern hemisphere tends to be inverted from that in the northern hemisphere [
130], which again corresponds with peak incidences of COX infections [
126]. Thus, although a genetic component to T1DM susceptibility is well-recognized among children with relatives with T1DM [
131,
132], and their risk can be documented by the development of an increasing number and diversity of T1DM-related autoantibodies over many months or years preceding T1DM diagnosis [
133,
134], the seasonality of new T1DM diagnoses suggest that triggering full-blown autoimmune disease, even against this genetic background, may require an appropriate combination of infectious triggers.
Note, however, that the putative triggers of T1DM identified here—COX with Clostridia—are very common infections, while T1DM is very rare. The reported rates of diagnosed infections and asymptomatic carriage of COX (or enteroviruses more generally) and
Clostridia raise serious problems for any T1DM mechanism that is based on a mono-infectious trigger model. The estimated number of new, annual T1DM diagnoses worldwide is many orders of magnitude less than the number of new enterovirus and
Clostridia infections. Estimates of the annual incidence of new T1DM diagnoses in the United States range from approximately 40,000 cases, or 1.2/10,000 individuals [
128] to approximately 60,000 cases, or 2.0/10,000 individuals [
129], the latter figure being typical of most of the rest of the world [
129]. While genetic predisposition certainly accounts for some of T1DM risk [
131,
132,
135], it is important to stress that 90% of new T1DM cases have no known relative with T1DM or any defined genetic risk [
136], and known genetic risk factors appear to be involved in fewer new cases each year [
137].
The incidence of new T1DM diagnoses is much more in line with a dual, concurrent-infection model of etiology. Assuming 12% of children have C. difficile carriage and 5% contract COX each year, then 60/10,000 would be exposed to both in a single year. However, the dual, concurrent-infection model requires that both infections be present simultaneously. Therefore, it seems reasonable to divide the 60/10,000 figure by 52 to yield the probability that an individual would contract both infections during the same week. This yields a probability of 1.2/10,000, which approximates the actual incidence of new T1DM diagnoses. Multiple Clostridia species might be involved as triggers of T1DM, so this number might increase, but on the other hand, a requirement for active (or activated) infection with at least one microbe (see the KRV rat model above) might decrease the probability.
Our data also hint at a combination of EBV with Streptococci as possible co-triggers of T1DM, which would have a probability of occurring concurrently at approximately the same order of magnitude of occurrence as COX + Clostridia. The essential point is that a dual-infection model gets the probability of a new T1DM diagnoses within the right order-of-magnitude estimation, whereas any single-agent model is several orders of magnitude off. Variations in the geographic incidence of T1DM would then be a function of the seasonal variations in the infections and their specific co-incidences in that location.
3.5. Prevention Implications of Complementary Antigen Theory
If both COX and
Clostridia are necessary to trigger T1DM, then several implications concerning the prevention of T1DM follow. One is that anyone diagnosed with either an enterovirus infection or a
Clostridium infection should be tested for the complementary infection. In light of the fact that T1DM could be prevented in KRV-infected rats with a combination of trimethoprim and sulfamethoxazole (Sulfatrim), beginning on the day of KRV infection [
114], if both COX and
Clostridia infections are present, appropriate antibiotic therapy might be an effective T1DM preventative. The assumption that the clinically obvious infection is the only infection present may be putting patients at risk for post-vaccinal autoimmune complications such as T1DM.
Next, vaccination against COX should be effective in preventing most cases of T1DM. Each individual vaccine should be completely safe in light of
Figure 17 because any host cross-reactive epitopes will either be non-antigenic or tolerized. Only in the rare instance that an individual contracts a Clostridium infection concurrently with their COX vaccination would there be a risk of triggering post-vaccinal T1DM (and thus individuals should be screened before vaccination). COX vaccines for this purpose are already under development [
138], but these are being developed under the assumption of a mono-infectious etiology for T1DM acting by means of one of three mechanisms: “Beta-cell death may be primarily induced by CVB [COX type B] itself, possibly in the context of poor immune protection, or secondarily provoked by T-cell responses against CVB-infected beta cells. The possible involvement of epitope mimicry mechanisms skewing the physiological anti-viral response toward autoimmunity has also been suggested… Understanding which [mechanisms] are at play is critical to maximize the odds of success of CVB vaccination, and to develop suitable tools to monitor the efficacy of immunization and its intermingling with autoimmune onset or prevention” [
138]. If, in fact, T1DM etiology involves complementary antigens, then the safety of COX vaccines may require antigen deletion of regions mimicking INSR and other TIDM autoantigen sequences identified here.
A third approach would be to develop
Clostridium vaccines, which should be safe for the same reasons provided above for COX vaccines but carry the same caveat regarding COX co-infection. Such
Clostridia vaccines are also in development, although at present for different purposes [
75,
139,
140,
141,
142]. The use of such vaccines again depends upon understanding the mechanism by which
Clostridium is involved in T1DM etiology and including (or excluding) appropriate antigenic regions. For example, our data suggest (but certainly do not prove) that
Clostridium toxin A is not involved in T1DM pathogenesis so that a toxin-based vaccine might be particularly safe from the perspective of preventing
Clostridium infections without risk of T1DM as a post-vaccinal side effect. However, if non-toxin-producing strains of
Clostridia can trigger T1DM, then a toxin-based vaccine may not be optimal for preventing T1DM.
Whether COX or Clostridia-based, optimization of T1DM prevention strategies rely, in the end, on having an appropriate animal model with which to test such strategies.
3.6. The Need for New Animal Models of T1DM
If T1DM is, in fact, triggered by a combination of COX and Clostridium infections (or EBV + Streptococci), then new animal models of the disease need to be developed, not least in order to test potential preventative approaches such as those described in the previous section. Such models might be implemented in several ways. One would be to infect susceptible animals with combinations of COX and Clostridia. An alternative would be to inoculate animals with combinations of inactivated COX and inactivated Clostridia. Success of the second type of experiment would also demonstrate that the pathogenesis is immunologically mediated (and therefore purely autoimmune) rather than requiring damage to the pancreas due to active infection. This second type of experiment might also be used to screen COX and Clostridia vaccines for their potential synergy and thus be used as a screen to increase their safety or to warn against their co-administration. Finally, another way to develop a novel T1DM animal model would be to inoculate animals with combinations of COX polyclonal antibodies and Clostridia polyclonal antibodies induced in the same species as that inoculated. According to the antigenic complementarity demonstrated in this paper, the resulting immune complexes should mimic the key complementary antigens triggering T1DM, and their idiotypes should therefore function equally as antigenic epitopes to initiate an autoimmune process.
Animal models should also explore the possibility suggested by the data we have provided here that an EBV–Streptococci combination may trigger T1DM.
3.7. Limitations of the Study
This study has limitations. One obvious one that has just been addressed is the lack of an animal model to test whether a combination of COX and Clostridia induces T1DM. The previous section lays out three ways to test this prediction.
A second limitation is that we tested our human sera only for binding to C. sporogenes rather than pathogenic species of Clostridia such as C. perfringens, C. difficile, etc., and may therefore have missed important cross-reactivity to additional Clostridia antigens or found cross-reactivities that do not extrapolate to other Clostridia species. Similarly, we were unable to obtain even relatively pure whole-COX antigens and therefore could not directly demonstrate whether our human T1DM sera were positive for COX antibody. COX antibody presence was inferred from a demonstration of anti-idiotypic responses to Clostridia antibodies combined with a demonstration of COX–Clostridia antibody anti-idiotype.
A third limitation is that the actual molecular complementarity between COX and Clostridium antigens has only been tested in the most cursory way in the present study using two pairs of peptides. Clearly much additional work needs to be performed to characterize this antigenic complementarity.
Similarly, the role of TCR in mediating T1DM through both antigenic complementarity and TCR idiotype–anti-idiotype interactions has only been explored cursorily here. While much more comprehensive studies have previously been published [
72,
73], the role of TCR complementarity needs much further investigation. The possibility that the TCR sequences expanded in T1DM may identify not just the particular microbes triggering the disease but also the specific antigenic sequences that are involved also needs further research. Both of these possibilities therefore stand as important predictions that can be used to test the validity and utility of the complementary antigen theory presented here.
Additionally, the COX–
Clostridia combination and its corresponding anti-idiotype may not be the sole triggers of T1DM. Our data also suggest that an EBV–
Streptococci combination be investigated more thoroughly. Additionally,
Bifidobacteria and
Lactobacilli both appeared as possible INS mimics in our similarity studies (
Figure 1 and
Figure 2) and have also been implicated in many T1DM microbiome studies (
Section 3.2 and
Section 3.3). Unfortunately, no antibodies against these bacteria could be located and therefore their possible synergy with COX or other viruses could not be explored.
Correspondingly, various viruses other than COX and EBV have also been associated with T1DM initiation, including CMV [
89,
143,
144,
145] and rotaviruses [
145,
146]. Thus, it is possible, if not likely, that other microbial combinations (probably bacterial–viral but possibly bacterial–bacterial or viral–viral) can also express complementary antigens that mimic human glucose-regulatory proteins and peptides and thus induce some form of T1DM. This possibility is supported by the fact that not all of the T1DM sera tested in this study reacted strongly to
Clostridia antigens (
Section 2.7) and some cross-reactivity was observed between non-COX virus antibodies or bacteria to INSR peptides (
Section 2.2). Thus, it is important not to over-interpret the data presented here as meaning that a COX–
Clostridium combination is the only possible cause of T1DM or predict that COX and/or
Clostridium vaccines will prevent all T1DM cases in the future.
Finally, there is thus far no direct evidence from human studies (either clinical or epidemiological) demonstrating that individuals newly diagnosed with T1DM have recently been exposed to both enteroviruses and Clostridia infections. Such studies will be needed to test the hypothesis proposed here.