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Celiac-like Disorder in Developmentally-delayed Children

from Bacteria Surface ProteinsJune 16, 2011

jeffc3497 – at – gmail – dot – com

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Bottom Line Up Front• B. pertussis pertactin and S. pneumoniae surface proteins contain regions with

a strong homology to the deamidated wheat gluten fragments that cause the immunogenic response in celiac disease.

• These regions appear to have a strong binding affinity for APC HLA-DQ2 and would also appear to trigger T-cell recognition/stimulation. This is caused by the repeating proline/glutamate patterns found within. This is the same mechanism that leads to autoimmunity toward small bowel tissue in celiac.

• These surface proteins are found as a component of the acellular pertussis vaccine (DTaP) and as contaminants in the pneumococcal vaccine (PCV7, PCV13, and PPSV). DTaP and PCV7/PCV13 are routinely and repeatedly administered as part of the pediatric vaccine schedule.

• The aluminum salt adjuvants used in these vaccines forms an antigen depot within the muscle after injection. These depots cause a prolonged, continuous antigen persistence that has been shown to last months.

• In those genetically predisposed, it is hypothesized that homology toward gluten peptides and antigen persistence produce a continuous autoimmune response toward small bowel tissue. Repeated vaccine boosters result in a constant inflamed bowel state present for years.

• Malnutrition materializes as the inflamed small bowel impedes nutrient absorption. Destruction of the intestine mucosa reduces peptidase secretion required for protein digestion. It may also reduce secretin and cholecystokinin mucosa secretion needed to stimulate bile and pancreatic enzyme release. Malnutrition leads to neurological manifestations and developmental delay.

• It is conceivable that the bowel inflammation seen in celiac disease is actually intended to be a transitory state that purges the digestive tract of these pathogens. Gluten reactivity inadvertently results from its homology toward these pathogens after the fortuitous deamidation by tissue transglutaminase. As gluten is continually present, the inflammatory state becomes chronic.

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Outline

• Celiac-like symptoms in developmentally-delayed children• Review of celiac disease• Alpha-gliadin, 33-mer, and immunogenic epitopes• Homologs in pertussis pertactin and pneumococcal surface

proteins• The bacteria surface protein hypothesis• Review of published literature regarding bacteria surface

proteins and celiac disease• Bacteria surface proteins and vaccines• Conclusions• References

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Introduction• This presentation puts forth a hypothesis that some developmentally-

delayed children children suffer from an autoimmune disorder closely related to celiac disease. It is suspected that triggers for this disorder are surface membrane proteins found in respiratory tract bacteria and used in vaccines. Regions of these proteins have a strong homology and potential stimulatory equivalence to immunogenic peptides found in wheat gliadin. Findings supporting this hypothesis are presented.

• This conclusion was reached after reviewing celiac-disease related studies, researching case reports, reading numerous parental descriptions of their children’s symptoms, personal interviews with parents, and the author’s own experience with his son’s regression into autism spectrum disorder (ASD) and subsequent dramatic recovery. The author has a sibling with celiac disease.

• It is thought that in this distinct ASD phenotype, this celiac-like disorder sets off a catastrophic nutritional downward spiral. The neurological impact is amplified by the immature state of the child’s neurological system– Inflammation – continuous adaptive and innate immune response– Maldigestion – leading to protein energy malnutrition– Malabsorption – particularly essential fatty acids and fat soluble vitamins– Bacterial overgrowth – due to the constant presence of maldigested food– Food protein allergies – from increased intestinal permeability

• The author strongly believes many affected children can improve significantly and some can recover completely using diet restriction, targeted nutritional assistance, and supplemental digestive support

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Celiac-like Symptoms in ASD• As early as the 1960s, there have been observations in medical literature

noting gastro-intestinal (GI) and stool similarities between those with autism and untreated celiac disease [1,2]

• Parents and clinicians have long reported that many ASD children exhibit GI symptoms and manifestations that appear to mimic those seen in celiac disease [3-5].– Diarrhea often with alternating constipation– Extremely malodorous stools– Maldigestion – undigested food in stool– Evidence of malnutrition

• Protein malnutrition – mild kwashiorkor-like appearance (muscle wasting, distended abdomen, skin de-pigmentation, etc.)

• Essential Fatty Acid and fat soluble vitamin malnutrition – elevated markers for lipid peroxidation and oxidative stress

• Parents also report autistic symptoms often improve with a gluten-free diet. Unlike celiac disease, symptoms rarely resolve completely.– The gluten-free, casein-free diet is consistently one of the highest-rated

treatment by parents in the Autism Research Institute’s comprehensive survey [6]

• Improvement in bowel function• Neurological improvement (eye contact, alertness, tantrum frequency)• Sleep improvement

• However, these children typically don’t have strongly elevated antibody markers seen in celiac (anti-gliadin, anti-tissue transglutaminase), as such they are not considered to have celiac disease. [7]

• Intestinal biopsies are rarely performed. The incidence rate of villous atrophy and brush border damage is unknown.

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The Distinct Phenotype• Although not applicable to all with ASD, there does appear to be a

distinct phenotype that shares common symptoms and responds remarkably well to treatment [8-10]– Gastrointestinal - celiac-like symptoms as described on the previous page– Neurological

• High functioning – able to talk, although speech may be delayed or difficult• Minimal stereotypy – unlike many children with autism, obsessive behavior and

repetitive actions appears to be limited in this group• Apathetic, not alert – needs name called or questions repeated several times• Attention difficulties – can’t focus on specific tasks as required, has trouble

quickly shifting focus from one task to another when needed• Irritable and irrational – petulant, tantrums at minor annoyances, punishment

makes tantrums worse, acts in the moment without considering consequences– Physical

• Muscle wasting – low amounts of muscle tissue, condition may be hidden from view by body fat at young ages

• Hypotonia – low muscle tone, poor motor skills, lacks physical coordination• Pallor – extremely pale or ashen tone face despite a more typical skin tone on

other parts of the body. Allergic shiners may be pronounced.• Many of the symptoms associated with this group are shared with

advanced untreated celiac disease and also kwashiorkor, a relatively common third-world disease caused by protein deficiency [11] . This is not surprising as malnutrition appears to be the common element in all three.

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Celiac Process

BIOMARKERS USED CURRENTLYpresence of antibodies to gliadin/tTg

1Gluten peptide

2Passage to

serosa(intestinal

permeability)

3Deamidation

by tTG

4Consumption

by APC

5Presentation by DQ2 or DQ8

(epitope dependant)

6Recognition by T cell (CD4)

7aTh1 response initiated,

inflammation begins

7bTh2 response initiated,

Gliadin specific antibodies producedrole in inflammation uncertain

8Gluten-specific,

DQ2 or DQ8-restrictedT cells proliferate

9Innate immune response

triggering peptides

10Innate immune response

adds to inflammation

11Tissue destruction,

increased permeability, malabsorption

12Food proteins enter

bloodstream, immune response, chronic

inflammation, allergies

Illustration from Bethune and Khosla

2008 [12]Annotations added.

7cTh2 response initiated,

tTG specific antibodies producedrole in inflammation uncertain

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Celiac Review (page 1 of 3)• To better understand the mechanism at work, a review of celiac

disease is presented. The understanding of this process is evolving; the consensus view in described [13-15]. The chart on page 7 accompanies this discussion.

• In celiac disease, wheat gluten triggers a destructive response toward small bowel tissue resulting in tissue damage, malabsorption, and eventual malnutrition.

• Research suggests the tissue damage results from both adaptive and innate immune responses. To date, the adaptive immune response is better understood.

• The following sequence triggers the adaptive immune response– Gliadin and glutenin (wheat proteins from the diet) are partially

digested leaving protein fragments (peptides) that are resistant to proteolytic digestion due to their high proline content

– Via intestinal permeability, these peptides reach the intestinalserosa (the lamina propria) and ultimately the bloodstream

– Some of these peptides are ideal substrates for the tissue transglutaminase enzyme (tTG). They are selectively deamidated;glutamine (Q) is converted to glutamate (E) at specific locations

– The deamidated peptides are consumed by antigen presenting cells(APCs).

1

2

3

4

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Celiac Review (page 2 of 3)• The adaptive immune response to gluten (continued)

– The peptides are presented on the APC surface bound to the HLA-DQ2 or HLA-DQ8 haplotype molecules

• The epitopes (immunogenic fragments) of many gluten peptides have a high binding affinity for HLA-DQ2 or HLA-DQ8

• More than 90% of celiac cases carry the DQ2 haplotype, most of the rest are DQ8 [19]

• Roughly a third of the population has the DQ2 and/or DQ8 haplotype [15], yet the incidence of celiac disease is far lower. Other genetic or environmental factors must be involved.

– APCs with HLA-DQ2/DQ8 presenting gluten epitopes are recognized by T-cells. The presence of proline and glutamate in specific positions bound to the DQ2/DQ8 molecule appears to playa key role in T-cell recognition.

– T-cells initiate the adaptive immune response• Cell-mediated Th1 response – cross reactive with small bowel

tissue, causes inflammation and ultimately small bowel tissue destruction

• Humoral Th2 response – production of antibodies– It is unclear if these play a role in tissue destruction. If so, the

role is secondary to the Th1 response– Antibodies are produced against gliadin and tTG. These

antibodies can be used as markers for celiac disease as they are easily measured without invasive procedures.

– If gluten consumption persists, gluten-specific, HLA-DQ2 or HLA-DQ8-restricted T-cells proliferate

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6

7a

7b 7c

8

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Celiac Review (page 3 of 3)• The inflammatory response from the innate immune response

appears to be involved but the mechanism is not as clear– Undigested gluten fragments (different from those involved in the

adaptive immune response) trigger activation of the innate immune response

• Relationship between the adaptive and innate response is uncertain• The p31-43/49 fragment of alpha-gliadin appears to be involved as it

induces interleukin 15 production in celiac mucosa. This sets off a chain of events that increases stress on small bowel tissue

– The innate immune response adds to tissue inflammation leading to further small bowel tissue destruction

• Inflammation and tissue destruction lead to malabsorption, maldigestion, and increased intestinal permeability. A vicious downward cycle results as the body’s malnourished state leads to a further decline in overall health.

• Removal of gluten breaks the cycle halting activation of the adaptive and innate immune response. Unless the case is very advanced, full recovery usually results if a gluten-free diet is maintained.

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10

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33-mer in Alpha-gliadin (Shan 2002)

• Shan et al [16] discovered that the 33-mer portion (marked in red above) of gliadin wheat protein was highly resistant to digestion by proteolytic enzymes

– Note high frequency of Proline (P) and Glutamine (Q)– Proline bonds are difficult to hydrolyze due to its unusual cyclic structure of the side

chain. Repeating proline residues also gives the peptide a unique helical shape.• They also discovered 33-mer contains six overlapping sequences of immunogenic

epitopes that invariably produce a response in adults with celiac disease [17]• Once deamidated by tTG (glutamine → glutamate) at specific positions, these

epitopes will tightly bind to the HLA-DQ2 presenting molecule of the APC and trigger strong T-cell recognition [18]

Red (1 place) PFPQPQLPY Alpha-IGreen (3 places) PQPQLPYPQ Alpha-IIBlue (2 places) PYPQPQLPY Alpha-III

MVRVPVPQLQPQNPSQQQPQEQVPLVQQQQFPGQQQPFPPQQPYPQPQPFPSQQPYLQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPFRPQQPYPQSQPQYSQPQQPISQQQQQQQQQQQQKQQQQQQQQILQQILQQQLIPCRDVVLQQHSIAYGSSQVLQQSTYQLVQQLCCQQLWQIPEQSRCQAIHNVVHAIILHQQQQQQQQQQQQPLSQVSFQQPQQQYPSGQGSFQPSQQNPQAQGSVQPQQLPQFEEIRNLALETLPAMCNVYIPPYCTIAPVGIFGTNYR

Alpha2-gliadin amino acid sequence (290 amino acid residues)

Key: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn;P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Note Pattern

Similarity

33-mer (Shan 2002)overlapping epitopes

shownIllustration from Sollid 2002 [13]

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HLA-DQ2 and DQ8 haplotypes

• Although genetic factors are not fully understood, There is a strong association with the HLA-DQ2 and HLA-DQ8 haplotypes in those with celiac disease

• HLA-DQ2 and HLA-DQ8 are molecules used by the Antigen Presenting Cell (APC) to present the gliadin epitope on its surface to T-cells. Upon recognition, the T-cell initiates the adaptive immune response.

• The amino acid sequence of the gliadin epitopes are well matched to the DQ2 and DQ8 register structure by virtue of the proline (P) locations and glutamate presence. There is a high binding affinity of the epitopes for DQ2 and DQ8.

• The glutamate (E) residue (deamidated from glutamine by tTG) presence and location are also critical for T-cell recognition of the APC presenting the epitope. Evidence also suggests some proline locations are crucial for T-cell recognition.

• The 33-mer epitopes bind tightly to DQ2 as do epitopes from other gliadin peptides• Epitopes from other gliadin and glutenin fragments bind tightly to DQ8

Epitope from 33-mer

Registers P1-P9 of HLA-DQ2

Glutamate (E) deamidated from glutamine by tTG prior to APC consumption of peptide

Examples of epitope binding to HLA registers. Note pattern similarities and differences between DQ2 & DQ8

Illustration from Sollid 2002 [13]

Table from Tollefson et al

2006 [19]

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33-mer Epitope Example (Tollefson 2006)

Binding for epitopes with HLA-DQ2 and HLA-DQ8 (Tollefson et al 2006) [19]:• Proline - DQ2 binds gluten peptides with the proline residues localized in P1, P3, P5, P6, and P8 but not in P2, P4, P7, or P9. This pattern is similar for DQ8, which bind peptides with proline residues in P3, P6, and P8, but not in P1, P2, P4, P7 or P9.• Glutamate - For DQ2, glutamate in P4 or P6 is crucial for T cell recognition. For DQ8, glutamate is needed in P1 or P9 for T cell recognition. Due to the negative charge of its side chain, glutamate also increases binding strength.

Alpha-gliadin peptide

Selective deamidation from tTG (glutamine → glutamate)

Peptide with glutamate residue in position 4 of epitope

Consumption by APC

Presentation on APC surface bound with DQ2 (see below)

… P F P Q P Q L P Y P Q P Q …

… P F P Q P E L P Y P Q P Q …

1 2 3 4 5 6 7 8 9

… P F P Q P E L P Y P Q P Q …

HLA-DQ2 P Registers

Epitope in Blue

Recognition by T cellAdaptive immune response starts

tTG

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• Tollefson, Vader, Shan, Kim, Milea, Stepniak and others [16,17,19-23] have characterized the alpha I, alpha II, and alpha III gliadin epitopes seen in 33-mer that frequently cause a response in adult celiac cases

• For these reactive peptides, a distinct pattern emerges for the nine residue binding cores– An iterative proline sequence causing a type II polyproline helical character

that results in strong HLA-DQ2 binding• Proline in the 1st, 3rd, {5th or 6th}, and 8th position

– Residues in key positions also appear critical for T-cell recognition• Proline in the 1st (sometimes), 3rd (often), and 8th (regularly) positions• Glutamate in the 4th or 6th position – note that in gliadin, glutamate is

deamidated from glutamine by tTG prior to binding with HLA-DQ2– A review of the literature found preferences, but not apparent criticality to

the 2nd, 7th or 9th position (also {4th/5th} depending on proline sequence) with a few caveats

• They can not be proline as this distorts the helical character• Glutamine (Q), leucine (L), phenylalanine (F), and tyrosine (Y) are seen in these

epitopes. However, this seems to be due to the nature of these gluten proteins. It does not appear that these residues are exclusively required or even optimum for HLA-DQ2 binding.

• Thus with these reactive deamidated gliadin peptides, there is seen an overlapping of two nine-residue patterns

HLA-DQ2 Binding and T-cell Recognition

Where x is L, F, Y, Q or E (tTG-deamidated Q)PxPxPExPx

PxPExPxPx

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Binding and Recognition (Stepniak et al 2008) [17]

The peptides found in alpha-gliadin are uniquely suited for both DQ2 binding and T-cell recognition. PxPxPExPx and PxPExPxPx tend to fill both criteria.

HLA-DQ2 Binding

T-cell Recognition

PQPELPYPQ PFPQPELPY

Helical shape from “P” sequence matched to DQ2

pocket structure

Glutamate negative charge strengthens bond, position 4 and 6 matched to DQ2 pocket

DQ2 is the only known DQ allele to accept “P”

in the first position

Bulky side chains fit in

large P9 pocket

35% of gluten stimulatory peptides

have “P” in position 1

> 50% of gluten stimulatory peptides

have “P” in position 3

85% of gluten stimulatory peptides

have “P” in position 8

> 80% of gluten stimulatory peptides

have “E” in position 4/6

33-mer Alpha-II and Alpha-I examples

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33-mer Homologs (Shan 2002)• Shan et al also discovered that the 33-mer peptide has two strong

homologs among non-gluten proteins [16]– Pertactin - a highly immunogenic surface protein that Shan et al identified

as being from Bordetella pertussis – Tyrosine phosphatase – a mammalian protein of unknown function

• They characterized these homologs and noted both share unique characteristics with 33-mer– High frequency of occurrence of proline and glutamine along with a

similar pattern structure– Comparable to 33-mer, both are excellent substrates for tTG (high levels

of specificity)• Pertactin kcat/KM = 121/min/mM• Tyrosine Phosphatase kcat/KM = 37/min/mM

– Both exhibit a strong type II polyproline helical character• typical of peptides bound to class II MHC proteins (i.e. APCs)• likely to enhance their binding affinity to these proteins

• Shan 2002 did not comment on the possible involvement of either homolog in the pathogenesis of celiac disease. They did brieflycomment on their conceptual use in oral vaccines.

LQLQPFPQPQLPYP-QPQLPYPQPQLPYPQPQPF (SHAN 33-mer)---QPGPQPPQPPQ-PPQPPQPQPQPEAPAPQP- (SHAN PERTACTIN)-QLQPQPQPQPQPQP-PPQPQPQPQ-PQPQPQP- (SHAN TYROSINE PHOSPHATASE)

(sequence alignment from Shan 2002 Supplemental Information [24])

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B. Bronchiseptica vs. B. Pertussis Pertactin• Shan 2002 appears to have some inconsistencies between the text of

the published paper and the footnotes regarding pertactin. These inconsistencies extend to the supplemental information (SI) [24] .

• The homolog identified in the footnotes as “pertactin” is actually from B. bronchiseptica (P.68), not pertactin from B. pertussis (P.69) as identified in the text. This was confirmed via the BLASTP [25] database.

• B. bronchiseptica is closely related to B. pertussis, with some distinctions– B. bronchiseptica is rare in humans, but is found in small mammals such as

cats, dogs, and rabbits– B. bronchiseptica does not produce the toxin as seen in B. pertussis,

despite having the genes required. This feature, along with other similarities, suggests common ancestry for both strains.

• Despite the discrepancy, the BLASTP database also confirms pertactin from B. pertussis (P.69) is structurally similar to P.68 and is also a strong 33-mer homolog

APQPGPQPGPQPPQPPQPPQPPQRQPEAPAPQPPAGRELS P.68 (Shan footnote)GAKAPPAPKPAPQPGPQPPQPPQPQPEAPAPQPPAGRELS P.69 (from BLASTP)

Comparison of pertactin from B. bronchiseptica (P.68) and B. pertussis (P.69), both are strong homologs of 33-mer

Pertactin fragment shown is from an area referred to as “region 2” in pertussis-related literature and is known to have highly immunogenic qualities [26]

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Pertactin and HLA-DQ2 / T-cell Recognition• Upon closer inspection, pertussis pertactin is not only a homolog of

33-mer, but it also contains two overlapping sequences that appear will bind tightly with HLA-DQ2 and trigger T-cell recognition.

• These sequences obey all the HLA-DQ2 binding and T-cell recognition guidelines (see page 14 and 15), they do not match HLA-DQ8– They share all proline and some glutamine locations with 33-mer epitopes– They already include a glutamate (E) residue at the correct position. They

do not need deamidation by tTG prior to binding with DQ2

PFPQPELPY PYPQPELPY PQPELPYPQPQPQPEAPA PQPQPEAPA PQPEAPAPQ

33-merPertactin

Epitope 1 Epitope 3, 5 Epitope 2, 4, 6

Epitope A Epitope A Epitope B

Red indicates glutamine (Q) deamidated to glutamate (E) by tTG. Pertactin epitopes A an B do not require deamidation. They are already a good match for HLA-DQ2 and have proline and glutamate at the correct positions for T-cell recognition. Note sequence similarities between 33-mer and pertactin sequences.

LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (33-mer)

KPAPQPGPQPPQPPQPQPEAPAPQPPAGRELSA (P.69 pertactin)

12

3

45

6

AB [27]

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Pertactin Epitope B HLA-DQ2 Binding Example

Pertussis pertactin sequences A & B share a close similarity with those that are found in 33-mer • Prolines are located in the exact same positions as 33-mer epitopes• Glutamate resides in the position where glutamine → glutamate via tTG in 33-mer• The residues that are different between the two have very similar properties

• Ala (A), leu (L), phe (F) are all non-polar, and neutrally charged. Tyr (Y) and Gln (Q) are polar and neutrally charged. However, polar Y and Q appear to exchange freely with non-polar F and L in 33-mer.

Pertussis pertactin sequence B

No Selective deamidation from tTG is needed

glutamate residue in position 4 of epitope without needing tTG

Consumption by APC

Presentation on APC surface bound with DQ2

… P Q P Q P E A P A P Q P P …

… P Q P Q P E A P A P Q P P …

1 2 3 4 5 6 7 8 9

… P Q P Q P E A P A P Q P P …

HLA-DQ2 P Registers

Epitope in Blue

Recognition by T cellAdaptive immune response starts

tTG

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Pneumococcal Surface Protein A (PspA)

• Similar to pertussis pertactin, Streptococcus pneumoniae also contains proline-rich surface proteins. These are found in Pneumococcal surface protein A (PspA) and surface protein C (PspC).

• PspA contains proline-rich blocks (“P” above) separated by non-proline blocks (“N”). These proline-rich blocks are highly-reactive and have been shown to produce an antibody response [28, 29].

• The structure of these proline-rich blocks are notable in their similarity to the epitopes of alpha-gliadin 33-mer. They contain multiple overlapping sequences of the patterns PxPxPExPx and PxPExPxPx

Illustration from Moreno et al

2010 [28]

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PspA and HLA-DQ2 / T-cell recognition (1 of 2) • Beall et al 2000 [30] did extensive work sequencing the clade defining

region and the proline-rich region of PspA for pneumococcal serotypes 6B, 9V, 14, 19A, 19F, and 23F. These serotypes cause the majority of pneumonia cases and are the basis of the PCV and PPSV vaccines [31-33].

• Beall 2000 listed 33 distinct accession number variants spread across the sequenced serotypes listed above. All were retrieved from BLAST and checked for pattern matches to PxPxPExPx and PxPExPxPx; the patterns seen in 33-mer.

• Matches were found in every serotype/strain. The number of matching sequences ranged from a low of three to an astonishingly high of fourteen overlapping sequences in two variants of serotype 9V.

PneumococcalSerotype variant 1 variant 2 variant 3 variant 4 variant 5 variant 6 variant 7

6B 10(AF255547.1)

8(AF255548.1)

3(AF255549.1)

5(AF255550.1)

5(AF255551.1)

3(AF254257.1)

7(AF254258.1)

9V 14(AF252286.1)

14(AF253404.1)

6(AF253405.1)

14 10(AF255908.1)

4(AF253406.1)

4(AF255546.1)

19A 3(AF254255.1)

3(AF254254.1)

6(AF253408.1)

19F 6(AF254256.1)

9(AF255544.1)

9(AF255545.1)

23F (1 to 7) 6(AF254259.1)

4(AF255542.1)

6(AF255543.1)

3(AF288751.1)

4(AF255552.1)

3(AF253407.1)

3(AF255900.1)

23F (8 to 14) 3(AF255901.1)

3(AF255902.1)

3(AF255903.1)

3(AF255904.1)

3(AF255905.1)

3(AF255906.1)

3(AF255907.1)

Number of PxPxPExPx or PxPExPxPx matches(accession number from Beall et al 2000 in parenthenses)

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PspA and HLA-DQ2 / T-cell recognition (2 of 2) • A Sampling of PspA proline-rich sequences from various serotypes

– Red lettering – match for PxPxPExPx or PxPExPxPx– Blue lettering – non-proline blocks– 33-mer included for comparison

DEPETPAPAPAPKPAPAPAPTPEAPAPAPKPAPAPKPAPAPKPAPAPKPAPAPKPAPAPKPETPKTGWKQENGMSerotype 23F PspAAF255906.1

ETPAPAPQPEQPAPAPAPKPEKSADQQAEEDYARRSEEEYNRLTQQQPPKAEKPAPAPAPKPEQPAPAPKTGWKQENGMSerotype 23F PspAAF255542.1

ETPAPAPQPEQPAPAPKPEQPAPAPKPEQPAPAPKPEQPAPAPKPEQPAKPEKPAEEPTQPEKPATPKTGWKQENGMSerotype 19F PspAAF255544.1

ETPAPAPQPEKPAPAPAPKPEQPAPAPKPEKSADQQAEEDYARRSEEEYNRLTQQQPPKAEKPAPAPAPKPEQPAPAPKTGWKQENGMSerotype 19A PspAAF253408.1

ETPAPAPQPEQPAPAPAPKPEQPAPAPAPKPEQPAPAPAPKPEQPAPAPAPKPEQPAPAPKTGWKQENGMSerotype 14 PspAAF255908.1

ETPAPAPQPEQPAPAPKPEQPTPAPKPEQPTPAPKPEQPAPAPKPEQPAPAPKPEQPAPAPKPEQPTPAPKTGWKQENGMSerotype 9V PspAAF253404.1

EPEKPVPAPETPAPAPQPAPAPKPEQPAPKPEKPAEQPKAEKPADQQAEEDYARRSEEEYNRLTQQQPAPAPKPEQPAPAPAPAPKTGWKQENGMSerotype 6B PspAAF254258.1

ETPAPAPQPEKPAPAPKPEQPAPAPKPEQPTPAPKPEQPTPAPKPGWKQENGMSerotype 6B PspAAF255548.1

LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF33-mer

SequenceIDAccession

6 sequences

8 sequences

14 sequences

10 sequences

7 sequences

6 sequences

9 sequences

4 sequences

3 sequences

Note the variety of arrangements and overlapping patterns seen. It is similar to the overlapping structure seen in 33-mer and other gliadin sequences.

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PspA Epitopes

Pattern PxPxPExPx PxPExPxPxPFPQPELPYPYPQPELPYPAPAPEAPA PAPEAPAPKPAPAPETPA PAPETPAPAPAPKPEKPA PAPETPAPEPAPKPEQPA PKPEQPAPAPAPKPEQPT PKPEQPAPKPAPKPEQPV PKPEQPTPAPAPKPETPK PKPEQPVPAPAPQPEKPA PQPEKPAPAPAPQPEKPE PTPEAPAPAPAPQPEQPAPAPTPEAPAPVPAPETPAPVPKPEQPA

PQPELPYPQ

Pattern Sequences

PspA

33-mer

• All sequence matches found in PspA from the Beall sequenced serotypes [30] are shown

• Most appear multiple times, PKPEQPAPA appears 21 times

• All have exact proline matches to the 33-mer epitopes. All have glutamate (E) correctly located in position 4 or 6 and would not required deamidation from tTG

• Other positions are from a small group of amino acids– Alanine (A)– Threonine (T)– Glutamine (Q)– Valine (V)– Lysine (K) (positive charge)

• All except lysine have similar properties to the residues seen in the same positions in 33-mer

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Pneumococcal Surface Protein C and HLA-DQ2• Although fewer strains have been sequenced than PspA, a check of

the BLASTP database revealed that Streptococcus pneumoniae surface protein C (PspC) also exhibited pattern matches to 33-mer.

• Sequences A, B, and C all have exact proline matches and glutamate (E) at the same position where glutamine is deamidated by tTG in 33-mer. They overlap in the same manner as 33-mer epitopes 1, 2, and 3.

• Also as with pertactin, the amino acid substitutes from 33-mer have similar properties to those they replace (uncharged). Again, lysine (K) appears to be the exception with it’s positively-charged side chain.

• Many more matches almost certainly exist as the pattern is very similar to that seen in PspA. To date, PspC has been sequenced in fewer strains.

PFPQPELPY PYPQPELPY PQPELPYPQ PYPQPELPYPAPQPEKPA PAPQPEKPA PQPEKPAPK PAPKPEKPA

33-merPspC

Epitope 1 Epitope 3, 5 Epitope 2, 4, 6 Epitope 3,5

Epitope A Epitope A Epitope B Epitope C

LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (33-mer)

KVKEKPAEQPQPAPAPQPEKPAPKPEKPAEQPK (Pneumococcal surf C)A

BC

12

3

45

6

[34]

25

Other Interesting Sequences• Pattern searches using BLAST reveal that PxPxPExPx and PxPExPxPx

appear in other pathogenic microorganisms, usually in surface membrane proteins. Many more probably exist.– Bacillus cereus BDRD-ST26 Collagen adhesion protein (PQPEQPKPQPEKP)

• A soil-dwelling, Gram-positive beta hemolytic bacterium. Some strains are harmful to humans and cause food borne illness

– Campylobacter rectus RM3267 Surface-layer-RTX protein (PQPQPEQPKPNPE)

• A species of Campylobacter implicated as a pathogen in chronic periodontitis– Corynebacterium amycolatum SK46 hypoth. protein (PAPAPEAPAPAPEAP)

• Causes of endocarditis in patients who have underlying structural heart disease or are immunocompromised, as well as of prosthetic-valve endocarditis.

– Trichomonas vaginalis surface antigen BspA-like (PNPTPETPNPAPETP)• An anaerobic, flagellated protozoan, the causative agent of trichomoniasis, and is

the most common pathogenic protozoan infection of humans in industrialized countries.

• Corn (Zea mays) has an interesting pattern match that appears multiple times in a cell-wall protein [35]– Vegetative cell wall protein gp1 precursor (PKPEVPHPVPELPKPE)– Further research is needed as the documentation is unclear if this protein is

found in the consumable portion of corn– This is an interesting finding as some with celiac disease report sensitivity

to corn and at least one study has shown heightened antibody responses to corn protein in celiac patients [36]

26

Bacteria Surface Protein-mediated ProcessGluten peptide

Deamidation by tTG

not required

Intestinal permeability not required

No tTG specific antibodies produced

CELIAC BIOMARKERS NEGATIVEno antibodies to gliadin or tTg

E

Pertactin

Pertactin

Pertactin

Pertactin

1Pertactin from bloodstream

2Consumption

by APC 3Presentation by

DQ2 only(not DQ8)

4Recognition

by T cell (CD4)

5aTh1 response initiated,

inflammation begins

5bTh2 response initiated,

Pertactin specific antibodies producedNo gluten-specific antibodies

6Pertactin-specific,

DQ2-restrictedT cells proliferate

7Innate immune response

gluten triggering peptides

8Innate immune response

adds to inflammation

9Tissue destruction,

increased permeability, malabsorption

10Food proteins enter

bloodstream, immune response, chronic

inflammation, allergies

Illustration from Bethune and Khosla

2008 [12]Annotations added.

27

Bacteria Surface Proteins Hypothesis (1 of 2)• The chart on page 26 accompanies this hypothesis• It is hypothesized that early exposure to bacteria surface proteins can

trigger a Th1 autoimmune response toward small bowel tissue in those genetically predisposed. Exposure can be from vaccines or illness.

• Damage is inflicted via comparable mechanism to that seen in celiac disease. This results from the remarkable structurally similarity of certain gluten (i.e. 33-mer) and bacteria surface protein sequences.

• This genetic disposition overlaps those criteria that predispose an individual for celiac disease– HLA-DQ2 haplotype, HLA-DQ8 does not appear susceptible– Other unknown genetic factors

• Repeated vaccine administration (i.e. primary series and boosters) causes surface protein-specific DQ2-restricted T cells to proliferate– Use of aluminum compound adjuvants causes a muscular antigen

deposition and long-term persistence that can last several months [37]• This specificity to surface proteins may act analogously to a

“vaccination” for 33-mer. The HLA-DQ2 positive individual is less likely to develop an adaptive immune response to 33-mer as the offending APCs and T-cells are now more specific to surface proteins.– Few anti-gliadin antibodies present– Would expect a negative correlation between anti-33-mer and anti-pertactin

or pneumococcal surface protein antibodies– Consistent with observation that children with celiac (more likely to be

recently vaccinated) are less reactive to 33-mer epitopes than adults [17,20]

28

Bacteria Surface Proteins Hypothesis (2 of 2)• Vaccine-mediated autoimmunity has a faster onset and higher rate of

incidence than classic celiac disease in those genetically predisposed– The immunogenic epitope has direct access to the bloodstream– Time and additional triggers are not required to develop intestinal

permeability and possibly required protein maldigestion• As the individual does not need a gluten reaction, and deamidation

with tTG is not required for surface protein epitopes, neither anti-gliadin or anti-tTG antibodies are present in large quantities– Negative result from standard celiac disease biomarker tests

• Protein maldigestion may result as tissue destruction reduces brush border peptidases. Partially digested gluten fragments lead to further tissue destruction as the innate immune response is activated.

• Removing gluten from the diet stops the innate immune response, but not necessarily the adaptive immune response

• The manufacturers could potentially make a safer and more effective pertussis vaccine if they were motivated to do so.– A study by Hinjen et al [38] intriguingly showed that immunization using an

engineered pertactin without region 2 (includes the homologs to the 33-mer epitopes) had the highest protection level of multiple vaccine variations. This could indicate that some pertactin is being “sidetracked” from it’s intended immune response target in genetically predisposed individuals (i.e. HLA-DQ2).

– If this hypothesis is born out, Hinjen’s observation indicates a safer and more effective pertussis vaccine without PRN region 2 is possible

29

Pertactin – Celiac Link Studies• An exhaustive search of the scientific literature turned up only one

paper pursuing the 33-mer/pertactin homology noted in Shan 2002• Citing Shan 2002, He et al (Vaccine [23] 2005) [39] conducted a study to

determine if there was evidence of pertussis or pertussis vaccination contributing to celiac disease

• He 2005 looked for cross-reactivity between anti-pertactin and anti-33-mer antibodies in two populations– Those exposed to pertussis

• Infants and toddlers vaccinated with the aP vaccine• Individuals with pertussis

– Those with or suspected of having celiac disease• Those with the clinical diagnoses of celiac disease as determined by lab tests• Those with celiac symptoms but negative lab tests• Healthy controls

• He et all concluded, “We found no cross-reactivity between human antibodies to the two different components, suggesting that neither pertussis immunization nor disease contributes to the pathogenesis of CD (celiac disease)”

• In light of the novel findings presented in this summary, it is reasonable to conclude that He et al may have misinterpreted their findings.

30

33-mer / Pertactin Cross-reactivity (He 2005)

• 33-mer/pertactin (Prn) cross reactivity data from He et al 2005. Charts are X-Y scatter plots of 33-mer IgA or IgG antibodies vs. pertactin IgG antibodies in various populations. The population He et al calls “non-CD patients” actually were suspected of having celiac disease but were not confirmed serologically.

• Note large scale changes from plot to plot making visual comparison difficult• Note weak negative correlation in some plots. Further examination of the data

raises interesting questions regarding the conclusions of He et al

(33-

mer

)(3

3-m

er)

γ = -0.127

γ = -0.019

γ = -0.001

γ = -0.142

γ = -0.088

γ = 0.016

Plots from He et al 2005 [39]

31

PRN IgG vs. 33-mer IgA/IgG - Healty Controls

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PRN IgG

33-m

er Ig

A/Ig

G

33 mer IgA 33 mer IgG

Another Look at He et al 2005

• He 2005 scatter plots (previous page) did not appear to show a correlation between Pertactin IgG (x axis) and 33-mer IgA or IgG (y-axis)

• The scatter plots were digitized and reproduced here using identical scaling between plots and also placing the 33-mer IgA and IgG data on the same graphs [40].

• Using identical scaling, it becomes clearer that the healthy control group has a pronounced clustering of data points in the lower left corner of the graph

• See next page for expanded view

PRN IgG vs. 33-mer IgA/IgG - Celiac Confirmed

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33mer IgA 33mer IgG PRN IgG vs. 33-mer IgA/IgG - Celiac Not Confirmed

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PRN IgG

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33 mer IgA 33 mer IgG

Celiac Confirmed Celiac Suspected, But Not Confirmed

Healthy Controls

Data Cluster

Data from [39] replotted using [40]

32

He 2005 Healthy Controls (40 subjects)

• 33-mer and pertactin (PRN) are both specific to the HLA-DQ2 haplotype only. It is suspected that those inside the rectangle (84% of the healthy controls) do not carry the HLA-DQ2 haplotype or if they do, have not have had APCs exposed to the immunogenic portions of gluten or pertactin. Presumably, this is the “normal” response in an unaffected individual.

PRN IgG vs. 33-mer IgA/IgG - Healty Controls

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0 10 20 30 40 50 60 70 80PRN IgG

33-m

er Ig

A/Ig

G

33 mer IgA 33 mer IgG

Data ClusterThose that do not exhibit an exaggerated responseto either 33-mer or pertactin

Healthy Controls

Data from [39] replotted

using [40]

33

He 2005 Celiac Cases (19 Subjects)

• He 2005’s confirmed celiac cases plotted on the same scale as controls. Note that 5 of 19cases (26%) have 33-mer IgA/IgG and pertactin (PRN) IgG entirely within the “normal” range of controls (red box). Since 33-mer and PRN are both HLA -DQ2 specific, it is suspected these 5 cases are HLA-DQ8 or may be susceptible to epitopes other than those in 33-mer.

• Also note that 8 of 19 cases (42%) have PRN IgG above the “normal” range. Controls were 4 of 40 cases (10%)

PRN IgG vs. 33-mer IgA/IgG - Celiac Confirmed

0

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0 10 20 30 40 50 60 70 80PRN IgG

33-m

er Ig

A/Ig

G

33mer IgA 33mer IgG

PRN IgG above “normal” range of controls

Celiac Confirmed

Data from [39] replotted

using [40]

34

He 2005 “Non-Celiac” Cases (16 Subjects)

• He 2005’s “non celiac” cases plotted on the same scale as controls. Note that 9 of 16 cases (56%) have 33-mer IgA/IgG and pertactin (PRN) IgG entirely within the “normal” range of controls (red box). From He 2005 - non-celiac cases include “those who were suspected to have CD but not serologically confirmed at the department”. Suspected celiac is more appropriate.

• Also note that 5 of 16 cases (31%) have PRN IgG above the “normal” range. Controls were 4 of 40 cases (10%)

PRN IgG vs. 33-mer IgA/IgG - Celiac Not Confirmed

0

40

80

120

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200

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280

320

0 10 20 30 40 50 60 70 80PRN IgG

33-m

er Ig

A/Ig

G

33 mer IgA 33 mer IgG

PRN IgG above “normal” range of controls

Celiac Suspected, But Not Confirmed

Data from [39] replotted

using [40]

35

He 2005 All Cases Combined (75 Subjects)

• He 2005’s all subject case categories plotted together. Of those that fall outside the “normal” range (red box) there is a distribution that appears non-random.

– 15 (IgA) and 7(IgG) cases have a 33-mer exaggerated immune response– 17 cases have a pertactin (PRN) IgG exaggerated immune response– 6 (IgA) and 1 (IgG) case(s) have exaggerated immune responses to both 33-mer and PRN IgG. This

group appears to be strongly under represented if the two variables were truly independent.

PRN IgG vs. 33-mer IgA/IgG - All Cases

0

40

80

120

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200

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320

0 10 20 30 40 50 60 70 80PRN IgG

33-m

er Ig

A/Ig

G

33 mer IgA 33 mer IgG

PRN IgG above “normal” range of controls

33-mer IgA or IgG above “normal” range of controls

Both 33-mer IgA and PRN IgG high

6 cases

Both 33-mer IgG and PRN IgG high

1 case

Data from [39] replotted

using [40]

36

He 2005 Celiac Cases Reanalyzed (14 Subjects)

• He 2005’s confirmed cases after removing 5 cases with “normal” 33-mer IgA, 33-mer IgG, and pertactin (PRN) IgG (the suspected HLA-DQ8 haplotype or 33-mer non-responders). Linear trend lines calculated using remaining data from 14 cases.

• Note strong negative 33-mer IgA to PRN IgG correlation. 33-mer IgG to PRN IgG exhibits weaker negative correlation.

PRN IgG vs. 33-mer IgA/IgG - Confirmed CeliacThose with "normal" PRN and 33-mer removed

0

40

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320

0 10 20 30 40 50 60 70 80PRN IgG

33-m

er Ig

A/Ig

G

33 mer IgA33 mer IgGLinear (33 mer IgA)Linear (33 mer IgG)

Correlation with PRN IgG33-mer IgA γ = -0.67233-mer IgG γ = -0.345

Data from [39] replotted

using [40]

37

He et al 2005 Review Summary (1 of 2)• He et al concluded that they found no evidence of cross reactivity

between 33-mer and pertactin antibodies. What they missed is that in affected individuals the pattern is not random, there is a negative correlation between 33-mer and pertactin antibodies. This is consistent with the hypothesis that APCs and T-cells will be selective toward either 33-mer or pertactin depending upon exposure.– The under representation of cross-reactors suggests a dependence between

variables• Pertactin exaggerated response 17 IgG cases• 33-mer exaggerated response 7 (IgG) 15 (IgA) cases• Cross exaggerated response 1 (IgG) 6 (IgA) cases

– There was a strong negative correlation in “confirmed” celiac cases with exaggerated immune responses to either 33-mer or pertactin

• Correlation between pertactin IgG and 33-mer IgA γ = - 0.672• Correlation between pertactin IgG and 33-mer IgG γ = - 0.345

• They also failed to note that the “confirmed celiac” and “suspected but not confirmed celiac” groups exhibit an exaggerated pertactin antibody response at a much higher rate than the healthy control group.– Confirmed Celiac 8 of 19 cases 42%– Suspected Celiac but not confirmed 5 of 16 cases 31%– Healthy Controls 4 of 40 cases 10%

38

He et al 2005 Review Summary (2 of 2)• He 2005’s conclusions appear to have blunted further investigations

into pertactin-celiac links as there is nothing in the literature after it [41]• Their methods of data presentation and analysis may have led

subsequent reviewers to overlook important trends– Failure to note the lack of cross-reactors to 33-mer and pertactin despite it

being statistically unlikely– Plot auto-scaling used when presenting side-by-side study population

comparisons gives the impression of data randomness that is not born out under more detailed scrutiny

– Misleading labeling of “non-celiac cases” on plots when the paper’s text states “those who were suspected to have CD but not serologically confirmed at the department in 2002”. “Suspected celiac but not confirmed” is a far more appropriate description and could alter conclusions in light of the findings presented here.

• Far from closing the book on pertactin, the raw data from He 2005 supports the hypothesis that APCs and T-cells can be selective toward either pertactin or 33-mer peptides. It also indicates pertactin antibodies are higher in confirmed and suspected celiac cases than in healthy controls. Although there could be many explanations for this, the conclusion that some “celiac cases” are actually pertactin-mediated can’t be easily dismissed.

• To facilitate further analysis, the published paper, raw data, and processing algorithms should placed online with full open-access to independent researchers.

39

Pneumococcal – Celiac Link Studies• A review of the scientific literature turned up no publications studying

the relationship between immunogenic gluten peptides and Streptococcus pneumoniae.– There are many publications regarding the high incidence and serious

nature of pneumococcal sepsis among those with celiac disease. This phenomena is attributed to hyposplenia (splenic atrophy) and is well-documented in studies and case reports [42-44].

– While hyposplenia is undoubtedly a major factor, it is an interesting coincidence that the surface protein sequence of a pathogen that plagues celiac patients so closely match that of 33-mer.

• The negative correlation seen between pertussis pertactin and 33-mer antibodies in the re-analyzed He 2005 data raises an interesting question. Could the proliferation of APCs and T-cells selective toward 33-mer reduce the quantity of pneumococcal antibodies? If so, thiscould be a contributing factor beyond hyposplenia in explaining the high rate of pneumococcal infection in those with celiac disease.

• A single small study by McKinley et al 1995 [45] appears to contradict this theory. They found 10 celiac patients all demonstrated appropriate acute antibody responses to a polyvalent pneumococcal vaccine. More investigation is warranted as their raw data has not been reviewed in detail.

40

Selectivity Toward 33-mer or Surface Proteins• Stepniak [17] and Vader [20] have noted the differences in epitope

reactivity patterns between adults and children with celiac disease– Adults routinely produce a response to alpha I, alpha II, and alpha III; the

three epitopes found in 33-mer. Although many other epitopes have been identified, response to these is less certain (see list next page)

– Children tend to exhibit a much more varied response, with reactivity to the three epitopes in 33-mer being far less frequent than seen in adults

• They have hypothesized that at the start, celiac disease produces a response to a wide range of epitopes. As the disease progresses, response becomes more focused on the immunodominant epitopes found in 33-mer.

• The findings presented here regarding the homology between 33-mer epitopes and bacteria surface proteins suggest a different explanation– Children are far more likely to have received a recent vaccination that

includes pertactin or pneumococcal surface proteins as these are included in the routine pediatric vaccine schedule

– APC and T-cell selectivity toward bacterial surface proteins would diminish reactivity toward the epitopes in 33-mer. This is consistent with the negative 33-mer/pertactin reactivity correlation found in the reanalyzed He 2005 data.

• The observed reactivity patterns support the hypothesis that recent vaccination will drive APC/T-cell selectivity away from 33-mer and toward the bacteria surface proteins

41

Gluten Stimulatory Epitopes

• Gluten stimulatory epitopes, 33-mer sequences boxed in red at the top of the list. In adults with celiac disease, responses to these three sequences are invariably present, less so for other sequences. Interestingly, responses to the others are seen more often in children with celiac disease, responses to the 33-mer sequences are often not found [17,20].

Found in

33-mer

Table from Stepniak et al

2008 [17]

42

Pertussis Pertactin and Vaccines• Pertactin is a highly immunogenic virulence factor of Bordetella

pertussis, the bacteria that causes whooping cough. It is a surface membrane protein that promotes adhesion to the trachea. – Pertactin is purified from Bordetella pertussis and is used in production of the

acellular pertussis vaccine [46]– The acellular pertussis (aP) vaccine replaced the whole-cell pertussis (wP)

vaccine used previously due to adverse reaction concerns with wP.• The pertussis vaccine is administered simultaneously with diphtheria and

tetanus toxins in a tri-valent combination known as the “DTaP” vaccine (acellular version) or the “DTP” vaccine (whole-cell version) [47]

• Timeline of the pertussis vaccine introduction in the United States– In the late 1940’s, the whole cell pertussis vaccine (DTP) was introduced to

widespread use in the U. S. It has since been almost entirely replaced by the acellular vaccine (DTaP) in the developed world.

– The acellular pertussis vaccine (DTaP) was recommended for use for only the 4th and 5th doses in the pediatric series in February 1992 [48]

– DTaP was recommended for all doses in the pediatric series in March 1997 [49]

• The pertactin motif that contains the alpha-gliadin 33-mer pattern matches is known to be highly immunogenic and exhibits virtually no variation among pertussis strains– Known as “region 2” in pertussis literature, it has been extensively studied

and is well characterized [26,38].– Pertactin-based vaccines would contain the 33-mer pattern match region

43

Pneumococcal Surface Proteins and Vaccines• Pneumococcal surface membrane polysaccharides are used in the

production of the pneumococcal conjugate vaccine (PCV) [31,32]– Saccharides of the capsular antigens from multiple serotypes are individually

conjugated to diphtheria CRM197 protein (used as a carrier)• Timeline of the pneumococcal vaccine introduction in the United States

– PPSV (23 serotypes, non-conjugate) was recommended for high-risk groups over two years of age in March 1997 [50]

– PCV7 (7 serotypes) was recommended for routine pediatric use in June 2000, and added to the CDC schedule in 2001 [51]

– PCV13 (13 serotypes) was recommended for pediatric use in February 2010 [52]• Yu et al 2003 [53] studied surface protein PspA contamination of the PCV7

and PPSV pneumococcal vaccines. They concluded these vaccines are contaminated with PspA though lot variability appears to be high.– Adults given a single dose of PPSV or PCV7 produced a significant increase in

anti-PspA antibodies. PPSV response appeared manufacturer/lot dependant.– They noted that the pediatric schedule frequency and dosage may produce

higher anti-PspA responses than seen in this study.• The serotypes sequenced by Beall et al 2000 are found in these vaccines.

Vaccine recipients would receive PspA contaminants that contain the sequence pattern matches to alpha-gliadin 33-mer.

Sequenced by Beall et al 2000

6A 6B 9V 14 19A 19F 23F

PCV7 [31] 4 6B 9V 14 18C 19F 23F

PCV13 [32] 1 3 4 5 6A 6B 7F 9V 14 18C 19A 19F 23F

PPSV [33] 1 2 3 4 5 6B 7F 8 9V 10A 11A 12F 14 15B 17F 18C 19A 19F 20 22F 23F 33F

Pnuemococcal Serotypes

44

Haemophilus influenzae B and Vaccines• The Haemophilus influenzae B (Hib) conjugate vaccine components were

checked for pattern matches to the gliadin 33-mer sequence. A review of the relevant sequences in the BLAST database found no matches.– No matches among the Hib capsular antigens– No matches for the tetanus, diphtheria, or Neisseria meningitidis proteins used

as carriers for the conjugate• However, Hib has a unique interaction with pneumococcal strains in

eliciting an immune system response against Streptococcus pneumoniae. The mechanism is not well understood [54].– The presence of both Hib and pneumococcal strains together provokes a

vigorous immune system response against the pneumococcal strains. Hib is not affected.

– The immune response is not elicited if Hib is absent• Due to this interaction, widespread use of the Hib vaccine in infants may

have led to a proliferation of the pneumococcal strains– The Hib vaccine was recommended for routine pediatric use in 1991 in the U.S.,

a decade prior to the pneumococcal conjugate vaccine [55]– Although findings in studies have been inconsistent, Baer et al 1995 [56] did

find that pneumococcal infections in children increased after widespread introduction of the Hib vaccine in Finland

• The potential change in the mucosal flora brought on by the Hib vaccine could be a contributing factor in this hypothesis. A surge in pneumococcal proliferation after Hib vaccine administration may trigger the autoimmune response toward small bowel tissue in those genetically susceptible.

45

Use of Aluminum Compound Adjuvants• The vaccines discussed use aluminum compound adjuvants intended

to amplify and prolong the adaptive immune response– DTaP - aluminum hydroxide [46]– PCV7/PCV13 - aluminum phosphate [31,32]– Hib - aluminum hydroxide [57] (manufacturer dependant)

• The aluminum adjuvant serves to form a depot within the muscle providing a long-duration persistence of the antigen [37]. This is key to the surface protein hypothesis as it explains the chronic nature of the small bowel tissue destruction. – Unlike a pathogenic acute infection that leads to either rapid antigen

clearance (via the immune system/antibiotics) or death, there is low-level, continuous persistence of the antigen

– Analogous to the continued presence of gluten in the diet, the antigen persistence causes a chronic inflammatory state of the small bowel

• Although data is sparse, studies have shown this antigen persistence from aluminum depots continues for several months– Using an aluminum compound adjuvant, Harrison 1935 [58] demonstrated

diphtheria antigen persistence in guinea pigs lasted at least seven weeks, the duration of this study.

– Verdier et al 2005 [59] demonstrated persistence at the injection site of up to 3 months with aluminum phosphate and at least 6 months with aluminum hydroxide using a tetanus/diphtheria vaccine in monkeys

• Despite use for over 80 years, the mechanisms at work with aluminum compound adjuvants are not well understood

46

CDC Pediatric Vaccine Schedule

As can be seen in the CDC schedule [47], genetically-susceptible infants and toddlers would be repeatedly injected with vaccines that contain pertactin (DTaP) or pneumococcal surface protein A (PCV) contaminants. The repeated administration of the Hib vaccine may also play a role. The long duration persistence from the aluminum adjuvant deposition may appear to the immature pediatric immune system as a nearly continuous exposure.

Table from CDC 2011 [47]

47

Bacteria Surface Proteins and Vaccines• In developed countries, DTaP is routinely administered to infants,

toddlers, and children • PCV and Hib are also routinely administered to infants and toddlers under

2 years of age in the United States and other countries • All are often administered together and with other vaccinations as the

CDC schedule combines vaccinations at single age milestones [47]– 2 months – DTaP, PCV plus vaccinations for Hib, polio, rotavirus, and hepatitis

B– 4 months – DTaP, PCV plus Hib, polio, rotavirus, and hepatitis B– 6 months – DTaP, PCV plus Hib, polio, rotavirus, hepatitis B, and influenza– 12-15 months – PCV plus Hib, polio, measles, mumps, rubella, varicella,

hepatitis A, and hepatitis B– 18 months - DTaP plus hepatitis A and influenza– ~ 5 years (prior to kindergarten admission) – DTaP, plus polio, measles,

mumps, rubella, varicella, and influenza• Although anecdotal, many parents report autistic regression shortly after

vaccination. The temporal relationship between the introduction of the DTaP and Hib vaccines and the increase in autism rates deserves further review. – DTaP was introduced for the 4th dose (at 18 months) and 5th dose (at 5 years) in

the series in 1992. These correspond to 1990-1991 and 1987 birth year cohorts, respectively. Hib was introduced in 1991 for ages 2 to 15 months.

– Although it is not presumed to be the sole trigger of autism and developmental delays, increases in autism rates do appear to be correlated with the DTaP and Hib vaccine introduction when plotted by birth year cohort (see next page).

48

Bacteria Surface Proteins and Vaccines

• California data shown. Due to the Lanterman Act of 1969, California has the most comprehensive autism data prior to 1990.

California Autism Rate by Birth Year CohortSource: ideadata.org and

California Health and Human Services Agency

0

20

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1975 1980 1985 1990 1995 2000 2005Birth Year

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hs

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Birth Year Cohort Introduction

DTaP 5th dose(5 years)

Birth Year Cohort Introduction

DTaP 1st to 3rd dose(2 to 6 mos)

Birth Year Cohort Introduction

PCV7Birth Year Cohort

Introduction

Latest Data Shown (2009) Drop in ASD rates after 2003

is due to age of birth year cohort (< 6 years old)

HibBirth Year Cohort

Introduction

49

Conclusion - Tying It All Together• Celiac disease may have its roots in an entirely appropriate evolutionary

response to surface proteins in pathogenic microorganisms. During exposure to a pathogen, the immune system may induce a temporary state of inflammation and malabsorption to quickly purge infectious bacteria from the digestive tract. Once the acute stage passes, normal digestive function returns.

• In those genetically predisposed, the immune system is fooled by partially digested gluten peptides selectively deamidated by tTG. As wheat is continually present, malabsorption continues to the point of malnutrition.

• Celiac disease may take decades to manifest itself, probably due to the need for an additional environmental trigger or prior health deterioration. Intestinal permeability and possibly protein maldigestion need to develop first to allow undigested gluten fragments access to the APCs.

• In the case of pediatric vaccinations, bacterial surface membrane proteins have direct and immediate access to the bloodstream and APCs without any need for intestinal permeability, protein maldigestion, or deamidation by tTG.

• The vaccine regimen’s high concentration of surface membrane proteins, the simultaneous introduction of multiple strains, the presence of powerful aluminum adjuvants that produce antigen persistence, and the reintroduction over the course of multiple boosters may trigger the celiac-like disorder in those genetically disposed. Analogous to gluten, the immature immune system interprets the unorthodox, subclinical, and persistent re-exposure to the epitopes as a continuous infection.

• The use of a one-size-fits-all pediatric vaccination schedule should be reexamined. Results from genetic screening and a review of family history may warrant a more cautious approach to vaccination. An individual risk-benefit analysis seems reasonable, rather than a presumption of low risk/high benefit.

50

References (1 of 4) 1. Goodwin MS, Goodwin TC. In a dark mirror. Ment Hyg. 1969 Oct;53(4):550-63.2. Goodwin MS, Cowen MA, Goodwin TC. Malabsorption and cerebral dysfunction: a multivariate and

comparative study of autistic children. J Autism Child Schizophr. 1971 Jan-Mar;1(1):48-62.3. Horvath K, Papadimitriou JC, Rabsztyn A, Drachenberg C, Tildon JT. Gastrointestinal abnormalities in children

with autistic disorder. J Pediatr. 1999 Nov;135(5):559-63.4. Valicenti-McDermott MD, McVicar K, Cohen HJ, Wershil BK, Shinnar S. Gastrointestinal symptoms in children

with an autism spectrum disorder and language regression. Pediatr Neurol. 2008 Dec;39(6):392-8. 5. Genuis SJ, Bouchard TP. Celiac disease presenting as autism. J Child Neurol. 2010 Jan;25(1):114-9.6. Parent Ratings of Behavorial Effects of Biomedical Interventions. Autism Research Institute San Diego, CA

92116. ARI Publ. 34/March2009.7. Pavone L, Fiumara A, Bottaro G, Mazzone D, Coleman M. Autism and celiac disease: failure to validate the

hypothesis that a link might exist. J Child Neurol. 2010 Jan;25(1):114-9. Epub 2009 Jun 29.8. Stories of recovered children, Autism Research Institute. http://legacy.autism.com/treatable/recovered/

recovered.htm.9. Generation Rescue Recovery Stories. http://generationrescue.com/recoverystories.10. Author’s interviews with parents of ASD children and own personal experience with his son’s diagnosis of

autism (high functioning) at age four and full recovery by age six. Tests for anti-gliadin and anti-tTG antibodies were negative. The recovery methodology was steered by biomedical testing along with trial and observation. The far most effective treatments were the gluten-free, dairy-free diet, supplemental digestive enzymes with meals, an elemental amino acid formula with meals, and high dose essential fatty acids and fat soluble vitamins. He continues on this regimen with periodic plasma amino acid and vitamin status testing. Neurological symptoms have resolved completely with the exception of very mild verbal apraxia that continues to improve. Physical symptoms have improved substantially including weight gain, dramatic increases in skeletal muscle, and improved pallor. Bowel habits have normalized, all celiac-like symptoms have disappeared.

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