anti-polysialic acid antibodies and autism

PRELIMINARY DRAFT – FOR REVIEW

PRELIMINARY DRAFT – FOR REVIEW 1

Jeffrey J. Calvagna 4/20/2012 Rancho Palos Verdes, California [email protected] Hypothesis: A Link between Anti-Polysialic Acid Antibodies and Autism (abridged) Abstract: The Neisseria meningitidis type B (MenB) capsular antigen contains long-chain polysialic acid (PSA). This substance is also present in embryonic and infant mammal brains acting as a temporal and spatial regulator of nervous system development. In neonatal cultured neurons, the presence of anti-PSA antibodies causes antibody-PSA cross-linking and internalization by the neuron. The resulting growth dysregulation led to increased cortical axon outgrowth, reduced thalamic axon outgrowth, and greatly accelerated myelination. These alterations closely resemble the characteristic physical brain anomalies documented in those with autism: rapid infant head circumference growth, frontal cortex overgrowth, thalamic undergrowth, unusual myelination maturity patterns, and Purkinje cell abnormalities. The last is notable as myelination terminates Purkinje cell regenerative capability. For two decades, the Haemophilus Influenza B (HiB) conjugate vaccine has been routinely administered to infants in the United States and other developed countries. One HiB vaccine variant uses the Neisseria meningitidis type B Outer Membrane Protein Complex (OMPC) as the protein conjugate. MenB is a surprising choice for a pediatric vaccine due to PSA autoimmunity concerns. Published patent information indicates the HIB vaccine OMPC product contains residual PSA. Experimental vaccine research demonstrates PSA readily complexes with OMPC, the ensuing product is immunogenic and sharply increases anti-PSA antibodies when administered to humans. Furthermore, the HIB-OMPC vaccine introduction corresponds to the initial increases in the autism diagnosis birth year cohorts. Hypothesis: The physiological CNS changes brought on by anti-PSA antibodies may play a role in the motor skill, cognitive, and communication developmental disorders found in those with autism. Abbreviations used

MenB - Neisseria meningitidis B PSA – Polysialic Acid HiB – Haemophilus Influenza B OMPC – Outer membrane protein complex NCAM – Neural Cell Adhesion Molecule MRI - magnetic resonance imaging HiB-OMPC – the HiB conjugate vaccine variant using the MenB OMPC as the

protein carrier CNS - Central nervous system ENS – enteric nervous system FDA – United States Food and Drug Administration ACIP - Advisory Commission on Immunization Practices BBB – Blood brain barrier



1. Introduction Despite long being recognized as contributing significantly to human morbidity, there is presently no widespread immunization effort directed toward Neisseria meningitidis B (MenB). The reasons are multi-faceted, but the primary basis stems from mammalian immunogenic tolerance toward the MenB capsular antigen. Unlike many other bacterial polysaccharide capsules, isolated MenB capsules do not induce an immune response even in mature mammals [1]. In the 1980s Finne and others discovered that long chain polysialic acid (PSA) is present in both MenB capsules and human brain tissue, specifically the embryonic form of neural cell adhesion molecule (NCAM) [1][2][3]. Finne et al’s findings have been widely cited; the presence of PSA in host tissues is accepted as the cause of mammalian immunogenic tolerance toward MenB capsules. Further research has shown PSA is crucial regulator of newborn nervous system development, with premature removal resulting in physiological aberrations in brain growth [4][5]. Experiments with cultured neurons show that the presence of anti-PSA antibodies leads to accelerated cortical neuron maturity, stunted thalamic neuron axon outgrowth, and accelerated myelination; alterations that may also cause premature loss of Purkinje cell regenerative capacity [6][7][8][9]. As will be presented in this publication, these changes strikingly resemble those found in the brains of autistic individuals via post-mortem examinations and magnetic resonance imaging (MRI) studies. Early PSA removal is a biologically-plausible explanation for the anomalies found in the autistic brain. Haemophilus Influenza B (HiB) conjugate vaccines were first introduced in the late 1980’s. There are several conjugate variants using diverse protein carriers; one of these uses a MenB outer membrane protein complex (OMPC) as the conjugate [10]. In light of the embryonic NCAM cross-reactivity findings, the choice of MenB OMPC in a pediatric vaccine appears questionable. Presumably, autoimmunity concerns would dictate such a vaccine be purified of PSA content. There is no public evidence such concerns were considered when choosing MenB as the conjugate carrier despite the early warnings regarding autoimmune potential. Supporting this contention are published patents documenting the vaccine’s manufacturing process indicating the final OMPC product contains substantial residual PSA [11][12]. MenB vaccine research from the same era also demonstrates that unlike the pure capsules alone, PSA complexed with MenB OMPC forms a highly immunogenic compound that provokes a relatively long-lasting IgM anti-PSA antibody response in humans [13][14][15]. This complexing occurs spontaneously without requiring specialized catalysts [15]. When considered in total, these findings suggest the HiB-OMPC vaccine variant may inadvertently produce anti-PSA antibodies when administered to humans, antibodies that are cross-reactive with PSA found within the developing infant brain. The introduction of the HiB conjugate vaccine corresponds with initial increases in autism spectrum disorder. Analysis of autism caseload data indicates a “change point” occurred between the birth year cohorts of 1987 and 1988 [16]. From the 1988 through 2003 birth year cohorts the autism rate increased ten-fold faster than seen in the previous seventeen years. Richmand 2011 provided extensive documentation suggesting that the autism rate change point coincided directly with the introduction of the HiB conjugate vaccine not only in the United States, but also in other countries [17]. Richmand further discussed the evolutionary



rational for newborn immunogenic tolerance toward carbohydrate antigens, hypothesizing that antibodies toward these capsules could be cross-reactive with substances related to brain development. This hypothesis ties together the findings of Finne with the Richmand hypothesis, supported by analysis of publications related to the HiB-OMPC vaccine. Richmand’s general hypothesis is extended to specifics; the HiB-OMPC conjugate vaccine may provoke antibody cross-linking of PSA thus causing its internalization and destruction. Since PSA is a crucial regulator of newborn nervous system development, artifacts of the now dysregulated process could result in physiological aberrations in brain growth. Thus the pediatric HiB-OMPC conjugate vaccine is put forth as a potential contributor to the dramatic increase in autism incidence.

2. MenB Capsules, Polysialic Acid, and Autoimmunity In 1983, Finne, Leinonen, and Makela published an important paper regarding MenB capsular polysaccharides notable for both its findings and conclusions. They determined that MenB IgM antibodies were cross-reactive with polysialic acid (PSA) glycoproteins isolated from both human and rat brains [1]. Prior research had determined that MenB capsules were poorly immunogenic in humans. Finne et al 1983 provided an explanation for the MenB capsule’s poor immunogicity - self tolerance. In strong terms rare for journal vaccine discussions, they urged caution in the development of MenB capsular vaccines for neonates. Their findings were widely disseminated with the paper being cited over 400 times. The same year, Finne et al discovered that long chain PSA was present on the embryonic form of neural cell adhesion molecule (NCAM) [2]. NCAM is a glycoprotein expressed primarily on the surface of neurons; it plays a role in cell–cell adhesion, neuron axon outgrowth, synaptic plasticity, learning, and memory [18]. Embryonic NCAM contains long chains of sialic acid up to 55 residues long, linked with alpha(2,8) glycosidic bonds. Though shorter in length, these polysialic acid chains are identical in structure to those found within MenB capsules (Figure 1) [19]. Other researchers including Rougon et al 1986 and Nedelec et al 1990 also found that antibodies to MenB capsules were cross-reactive with PSA-rich neonatal brain tissue [3][20]. Interestingly, the same antibodies were not cross-reactive with adult brain tissue. Investigation of this phenomena revealed that NCAM transitions from a heavily polysialyted neonatal form to the nearly PSA-free adult form early in childhood. Based on their findings, Nedelec et al 1990 warned “development of a vaccine against group B meningitidis should be considered with caution” [20]. Vaccine researchers have long known that protein conjugation is an effective method of breaking immunogenic tolerance toward polysaccharide antigens. Protein conjugation forms the basis of the pediatric Haemophilus Influenza B and Pneumococcal conjugate vaccines, converting non-immumogenic polysaccharides into potent antigens. Most isolated bacterial capsules are poorly immunogenic in infants, but do provoke an immune response later in childhood [21]. Protein conjugation provides a method to “jump start” this immune response, conferring disease protection at a much earlier age than a child could produce through natural exposure to the pathogen. This is achieved via a polysaccharide-protein T-cell dependent



immune response in the infant rather than the natural polysaccharide T-cell independent response seen in more mature children [21]. MenB capsules however, are different from other bacterial capsules as they do not induce an immune response even in mature children or adults [1]. It is tempting to consider this an evolutionary adaptation; the risk from anti-PSA antibodies exceeds the benefits these antibodies might provide in combating MenB. Following this same line of thinking, many have urged caution regarding MenB capsule conjugate vaccines as breaking immunological tolerance via protein conjugation or other techniques could entail significant risks. These warning have been heeded; all of the modern Neisseria meningitidis polysaccharide conjugate vaccines exclude MenB, thus being limited to the A, C, W-135, and Y meningococcal strains.

3. Consequences of Anti-Polysialic Acid Binding in the Neonatal Brain In the decades since Finne’s original findings, researchers have noted the transition from highly-polysialated embryonic NCAM to relatively PSA-free adult NCAM occurs in parallel with the central nervous system (CNS) development milestones. Suspecting a causal relationship, researchers further demonstrated premature PSA removal triggers substantial alterations in neonatal neuron growth. These observations are consistent and method-independent, comparable findings result from PSA genetic deletion, enzyme cleavage, or antibody-induced internalization [4][5][6][7][8]. It is now beyond doubt that PSA plays a

A B

Figure 1: Polysialic Acid Molecular Arrangement and Helical Structure A – Polysialic acid (PSA) chain linked to NCAM. PSA chain varies from few or none residues in adult NCAM to up to 55 residues in embryonic NCAM, all glycosidic bonds are alpha(2,8) linkage. Individual sialic acid residue is noted in brackets. PSA chain in the MenB capsule uses identical linkage with > 200 residues. B - Helical formation of PSA required for antibody binding from Hayrinen 2005 [19]. At least eight to ten residues are required to achieve the helical conformation and binding, helix pitch is variable, with nine PSA residues per turn being typical. IgG antibodies shown, same formation is needed for IgM antibody binding.



crucial role in temporally regulating embryonic and newborn mammalian brain development. Untimely PSA absence irreversibly changes CNS physiology. Antibodies toward MenB capsular polysaccharides do not differentiate MenB PSA from that within embryonic NCAM as the research of Finne, Rougon and others have demonstrated. Three later studies provide insight regarding the damaging effects of neonatal anti-PSA antibody proliferation. Cultured, neonatal neurons exposed to these same antibodies suffered dramatic growth alterations, particularly in regards to axon outgrowth and myelination. As will be shown, the documented changes found in these studies strongly resemble those abnormalities found in the autistic brain. Henke-Fahle et al 1996 and Monnier et al 2001 – Altered neuron axon outgrowth Although not fully understood by the authors at the time of its publication, Henke-Fahle et al 1996 demonstrated that anti-PSA IgM can dramatically alter neuron growth [6]. One IgM antibody studied (referred to as “mab10” in the paper) was found noteworthy for its striking effect on neuron axon outgrowth in cultured neonatal cells. Although the authors did not determine the specific antibody target in this paper, Monnier et al 2001 conclusively identified the target of mAb10 as PSA [7]. The Henke-Fahle et al findings are extremely significant as they reveal anti-PSA IgM antibodies alter neuron axon outgrowth and do so differently depending upon the neuron type (Figure 2). To summarize their findings:

Cortical axons extended on postnatal cortical membranes – the presence of mab10

(anti-PSA IgM antibodies) enhanced axon outgrowth to 139% of the control value (p > 0.01, n = 351 explants)

Thalamic axons extended on postnatal cortical membranes – the presence of mab10 (anti-PSA IgM antibodies) reduced axon outgrowth to 50% of the control value ( p > 0.001, n = 357 explants) [6]

Monnier et al 2001 also demonstrated in vivo antibody binding to PSA in chicken embryos and subsequent inhibition of optic nerve development [7]; an extremely important finding as some researchers contend the deleterious effects of anti-PSA antibodies are limited to in vitro conditions [22][23]. Monnier et al 2001 provides strong evidence that neuron changes documented to occur in vitro can also occur during embryonic and infant development.



Figure 2: Neuron Outgrowth After Exposure to Anti-PSA IgM Abs

From Henke-Fahle et al 1996 – Effect of mAb10 on thalamic and cortical axon outgrowth explants on postnatal cortical membranes after 4 days in vitro [6]. In Monnier et al 2001, mAb10 was proven to be anti-PSA IgM antibodies [7]. Charles et al 2000 – Accelerated myelination Myelin is an electrically-insulating material forming a layer around the axon of a neuron and is essential for the proper functioning of the nervous system. In 2000, Charles et al observed that neuron myelination in mice was inversely related to the quantity of polysialylated NCAM (PSA-NCAM) present [8]. They reasoned that PSA’s presence negatively regulates newborn myelination. To test this hypothesis they subjected cultured neonatal neuron specimens to anti-PSA IgM antibodies. Their hypothesis was confirmed as myelination increased at a 5.2-fold rate quantified by the number of myelinated internodes compared with control untreated cultures. Accelerated, uncontrolled myelination resulted from antibody binding the PSA chain on NCAM. Later researchers further confirmed the role of PSA in regulating myelination [24]. Charles et al further demonstrated that the addition of these antibodies caused neuron internalization of the resultant PSA-NCAM-antibody complex [8]. Additional tests showed no continuing presence of the IgM antibodies unless the neuron membranes had been first



permeabilized thus indicating complex internalization. The test was modified using the PSA-cleaving enzyme endoneuraminidase to further demonstrate that the accelerated myelination was caused by removal of the PSA from the neuron surface. Results were nearly identical with a 4.8-fold increase in myelination when measured in vitro. In vivo studies with Endo-N demonstrated a 1.6-fold increase although these tests used the optic nerve rather than the cultured forebrain neurons used in the in vitro studies. The authors speculated differing myelination patterns between the tissue types may have contributed the variation. Charles et al established that these antibodies can alter newborn cultured neuron myelination. In the same fashion as Henke-Fahle et al 1996, this was demonstrated with IgM anti-PSA antibodies that specifically recognize alpha(2,8)-linked PSA chains [8]. These antibodies match those used in the early PSA cross-reactivity studies of Finne et al [1] and Rougon et al [3], and are also found in sera of adults suffering from MenB [20].

Figure 3: Myelinated Internodes in Cultured Neurons

From Charles et al 2000 [8] - Anti-PSA-NCAM mAb or endo-N were added to the culture medium between 10 and 18 days in vitro (DIV) and myelination was quantified at 22–24 DIV by counting the number of MBP-positive myelinated internodes per coverslip. Controls of specificity of anti-PSA mAb were cultures treated with either K5-2 mAb, an irrelevant mouse IgM used at the same concentration as anti-PSA, or A2B5 mAb, an IgM that binds to axons, but not to PSA-NCAM, or anti-PSA mAb adsorbed with 1 mM colominic acid, a polymer of a2,8-linked sialic acid. Anti-PSA mAb and endo-N-treated cultures showed a 5.2- and 4.8-fold increase in myelination, respectively. Results are expressed as the mean 6 SEM of three experiments in quintuplicate. (**, P < 0.001). The Interaction of IgM Antibodies with Host Polysialic Acid An interesting aspect of the Henke-Fahle et al, Monnier et al, and Charles et al research regards the use of IgM antibodies. Literature discussing MenB vaccine development often includes broad statements that such IgM anti-PSA antibodies are poorly immunogenic. This



statement is a misconception, as it is rooted in the vaccine’s inability to produce high levels of lasting bactericidal antibodies and confer disease protection to the recipient. In truth, these experimental vaccines were strongly immunogenic in that they produced an immediate and sharp increase in IgM anti-PSA antibodies in human subjects [13][14]. But despite high titers these vaccines failed to engage the complement system, possibly due to autoimmunity protection mechanisms [25]. Thus the hypothesis requires that the deleterious effects of anti-PSA antibodies must arise from the mere act of antibody binding rather than a subsequent immune system attack on the neuron. The evidence supports this conclusion. Antibody-mediated clustering and internalization – Charles et al described internalization of the PSA–NCAM molecule by the neuron itself rather than phagocytosis or lysis from immune system components [8]. The IgM antibodies’ ten binding sites allow substantial cross-linking among PSA strands, facilitating NCAM molecule clustering into lipid rafts. Clustered, cross-linked NCAM would appear to be of little biological utility making it an ideal candidate for neuron surface clearing. As demonstrated in Diestel et al 2007, this same cross-linking and NCAM internalization phenomenon occurs in mice neurons exposed to NCAM-specific antibodies [26]. In the proposed hypothesis, antibody binding is among PSA strands rather than the NCAM protein itself, yet the same internalization results (Figure 4). Antibody-induced capping, clustering and subsequent neuron internalization of neuron surface molecules has been shown to cause encephalitis and neurological degeneration in humans [27]. On the other hand, IgG antibodies would likely facilitate less NCAM clustering with only two closely spaced sites available for binding. Based on PSA’s size and shape, it is conceivable many IgG antibodies would attach to only a single strand with no cross-linking. IgM’s pentameric shape would be more favorable to cross-linking, particularly among widely spaced binding sites. Charles et al also speculated IgM could be significant in this regard when discussing their findings [8]. This is important when considering the hypothesized sequence of events. The consequences of producing anti-PSA antibodies occur not from an immune system attack against the neuron, but from internalization of PSA-NCAM by the neuron itself after IgM antibody cross-linking. The mechanism is not “autoimmunity” as commonly perceived, though it is antibody induced.



Figure 4: PSA-NCAM Cross-linking and Neuron Internalization Visualization of PSA-NCAM internalization. A) IgM Anti-PSA antibodies cross-link PSA strands causing PSA-NCAM capping and clustering. Pentameric shape and widely spaced binding sites allows cross-linking of multiple PSA strands into a single complex. B) Vesicle formation and neuron internalization of antibody - PSA-NCAM complex via endocytosis.

B

A



4. A Potential Role of Anti-PSA Antibodies in Autism The CNS growth alterations found in the research of Henke-Fahle et al 1996, Monnier at al 2001 and Charles et al 2000 strikingly resemble that seen in the brains of autistic individuals via post-mortem examinations and MRI studies. The inappropriate neonatal presence of anti-PSA antibodies is a biologically-plausible mechanism that could explain the unusual growth patterns found in the autistic brain. Accelerated head and frontal white matter growth Large head size (macrocephaly) has been associated with autistic individuals since the days of Kanner. In his 1943 paper Autistic disturbances of affective contact, he observed five of his eleven subjects had notably large heads [28]. Recent studies have focused these observations; consistently finding unusual infant head circumference growth patterns as opposed to general macrocephaly. As shown in Contantino et al 2010, head circumference growth rates exceed norms during the first year of life then drop below norms [29]. Courchesne, Pierce and others have published extensively on the neurology of autism over the last decade. In agreement with head growth patterns, they found a majority of children with autism exhibited accelerated brain growth in the first year of life focused primarily in the frontal and temporal cortex regions. A pronounced growth deceleration occurred in the toddler years [30][31]. The overgrowth has been attributed to both white and gray matter with excessive myelination and accelerated axon outgrowth both suggested as possible causes [32]. Intriguingly, areas such as the occipital cortex that develop very early show little or no signs of overgrowth [33]. Courchesne and Pierce further hypothesized that this accelerated neuron growth would most affect the higher order-functions typically impaired in autistic individuals. They discussed the potential fallout from this accelerated cortex neuron growth:

“Large integrative and projecting pyramidal neurons that normally require many years of slow growth, such as those in frontal cortex, would be maldeveloped which may result in a reduction in long-distance connectivity and topdown control signaling, while abnormal increases may occur in local and short-distance connectivity and processing. Maldevelopment of large frontal interneurons, such as chandelier cells, could undermine the development of selective inhibitory control.” [32] (emphasis added)

The cortex overgrowth patterns described in the literature are consistent with that documented in cultured cortex neurons exposed to anti-PSA antibodies. The cortical neuron axon accelerated outgrowth (rather than “years of slow growth”) demonstrated by Henke-Fahle et al 1996 matches that hypothesized by Courchesne and Pierce that may affect higher-order cognitive capabilities. Curtailed thalamus growth and connectivity in Autism In contrast to that seen in the cortex, post mortem and MRI studies of those with autism indicate thalamic growth curtailment. Thalamus volume has been consistently shown to be a



smaller proportion of overall brain volume than that seen in controls [34][35][36]. Sensory disturbances are a characteristic feature of autism. Sensory input is primarily routed through the thalamus, suggesting altered connectivity may play a role. Studies using MRI on those with autism reveal reduced activity within the thalamus [37] and diminished thalamocortical connectivity affecting sensory stimulus and motor processing [38][39]. Again anti-PSA antibody binding can explain a hallmark feature seen in the autistic brain; smaller thalamus volume and decreased thalamocortical connectivity. In contrast to their findings in cortical neurons, Henke-Fahle et al 1996 demonstrated anti-PSA antibodies inhibit the growth of thalamic axons. These findings led them to a very clear description regarding the role of PSA in thalamocortical connectivity:

Moreover, the spatiotemporal distribution of the carbohydrate epitope defined by mAb10 (Ed: PSA), together with its action on growing thalamic axons, suggests that it might play an important role in regulating the timing of thalamocortical innervation. [6]

Their demonstration that anti-PSA antibodies inhibit thalamic neuron axon outgrowth would appear to closely describe conditions found in the autistic brain. Advanced myelinated white matter maturity The past five years has seen a flurry of studies using MRI technology to examine the brain structure of autistic individuals. Many of these studies have focused on white matter development as autistic individuals appear to have atypical development patterns [40]. A research group from Israel has published two studies that are particularly noteworthy as they examined children with autism as young as 18 months of age; whereas most other studies examined older children and adults. If premature myelination is present in autism, discriminating indicators should be evident at a very early age. Data from older children or adults includes additional brain growth that could make the precipitating event more difficult to discern. The studies indicate myelinated white matter from the frontal lobe, corpus callosum, left superior longitudinal fasciculus and right and left cingulum in autistic individuals exhibits an advanced level of maturity in even the youngest (18 month old) test subjects [40][41]. Indications of advanced myelinated white matter maturity included increased fractional anisotropy and corresponding decreased radial diffusivity. Maturation trends between 18 months and five years were slower than that of controls [41]. Extrapolating this data back, a maturity surge of the myelinated white matter must occur prior to 18 months of age assuming normalcy at birth. The authors suggest causes could be accelerated myelination patterns, altered axon growth, or the presence of crossing fiber pathways. The authors further suggested that abnormal structure that has been observed in the gray matter of autistic individuals may be related to malformations of the white matter. In early 2012 an elegant study from Wolff et al supported the findings of those from Israel and further bolstered suspicions that autism is related to advanced myelinated white matter maturity occurring early in life. Wolff et al performed MRI studies on the brains of infants at



higher risk for autism based on incidence in immediate family members [42]. This clever methodology allowed MRI data collection on a large number of infants prior to an autism diagnosis. MRIs were conducted at 6, 12, and 24 months of age; results from infants later diagnosed with autism were compared to those that did not become autistic. Increased white matter fractional anisotropy was found in those later diagnosed with ASD, indicating advanced myelination maturity comparable to that seen in the Israeli studies. However, in this case it was present in infants as young as six months of age. For these infants, accelerated white matter maturity must start in the first few months of life, again assuming normalcy at birth. More studies are required, but the abnormalities noted may be caused by accelerated myelination brought on by premature PSA removal. Accelerated cortical neuron outgrowth may also contribute to the observed maturity trends. Early Myelination and Purkinje Cell Development in Autism Purkinje cells are large neurons found in the cerebullar cortex of the brain that contribute to motor control. They are characterized by cell bodies with numerous branching dendrites, and by a single long axon. Multiple researchers have found Purkinje cells abnormalities during post-mortem exams on the brains of autistic individuals with Purkinje cells counts and proliferation consistently lower than that of controls [43][44]. Bauman and Kemper 2005 put it succinctly:

“the presence of reduced numbers of Purkinje cells is the most reproducible pathological observation in the autopsied autistic brain” [45]

Research from the last decade has found a strong relationship exists between myelination and Purkinje cell development and regenerative capabilities. More specifically, the myelin-associated protein Nogo-A controls gene expression linked to Purkinje cell growth [46]. Gianola and Rossi have several publications probing the relationship between myelination and Purkinje cell development. In their 2006 work, they made several made several connections between Purkinje cell maturation and myelination. Unlike some other neuron types, adult Purkinje cells entirely lose regenerative capability that is present during embryonic and newborn development stages [46]. Gianola and Rossi noted this transition happens in parallel with the myelination process and concluded myelin-associated proteins regulate development and shape cerebellar neuron connectivity. Myelin acts as a regulator and stabilizing factor in Purkinje cell plasticity and growth [9]. With an understanding of the link between Purkinje cell development and myelin a potential explanation for arrested Purkinje cell growth in the autistic brain takes shape. As shown in the Charles et al research, anti-PSA antibody binding with PSA causes accelerated myelination. As Purkinje cells are prematurely myelinated, growth ceases and regenerative capability is lost before normal neuron connectivity patterns are in place. In effect, the Purkinje cell development “off switch” is thrown far too early in the infant’s path to neurological maturity.



Reduced NCAM-180 in the brains of those with autism Adding to evidence supporting a link between PSA-NCAM internalization and autism, Purcell et al 2001 found expression of NCAM-180 was significantly lower in post-mortem cerebellar cortex samples of autistic subjects when compared to controls [47]. This finding is consistent with antibody-induced internalization of the cross-linked antibody-PSA-NCAM complex described in Charles et al 2000. Purcell et al further found mRNA expression of NCAM was not altered in the autistic brains, though the sample size was small with considerable variability. Still, this suggests an environmental rather than a genetic cause [47]. The results need to be interpreted with caution as the older average age of the test subjects (19.0 years) may have included subsequent brain changes that masked the original conditions seen in childhood. Addition links between Polysialic Acid and Neurological Disorders Betancur et al 2009 noted that a high proportion of genetic copy number variations and rare point mutations found in autistic individuals relate to Synaptic Cell Adhesion Molecule (SynCAM) pathways [48]. Although not discussed in detail in this hypothesis, a small percentage of the PSA within the brain is expressed by SynCAM [49]. Anti-PSA antibody binding of PSA-SynCAM could be another mechanism at work in autism. The presence of these variants, coupled with a genetic predisposition toward antibody generation, may play a role in autism susceptibility for infants with anti-PSA antibodies. Alternatively, they may represent a purely genetic autism mechanism using comparable pathways to that of externally-induced PSA internalization via anti-PSA antibodies. In a further link between PSA-NCAM and neurological disorders, Hildebrandt et al 2009 noted that disrupted brain fiber tract connectivity brought on by PSA deletion resembled that seen in schizophrenic patients. They proposed that incomplete polysialylation of NCAM could be a predisposition candidate for schizophrenia [50]. Brennaman and Maness 2010 observed that NCAM appears to play a role in multiple neuropsychiatric disorders in addition to schizophrenia including bipolar disorder, depression, and anxiety disorder, as well Alzheimer’s disease [51]. Polysialic Acid and Gut Dysfunction Seen in Autism Just as autoimmunity toward PSA-NCAM may explain brain development abnormalities and neurological manifestations seen in autistic individuals, it may also explain frequently comorbid gastrointestinal tract dysfunction. The hypothesized mechanism is the same for both cases; dysregulated nervous system development brought on by antibody-mediated internalization of PSA-NCAM. However, the causes of gut dysfunction are anomalies within the enteric nervous system (ENS) rather than the central nervous system. The ENS, sometimes referred to as “the second brain” is the only part of the peripheral nervous system where neural circuits act autonomously. It detects conditions within the gastrointestinal tract, processes information, and provides stimulatory signals to control gut movement, lumen secretion, and blood flow [52]. It has a critical role in coordinating activities crucial to proper GI function. In studying Hirschprung’s Disorder, Horigome et al 2007 found the ENS ganglions of control rats expressed PSA in a developmentally-controlled



manner comparable to that seen in the brain [53]. Faure et al 2007 determined PSA played a vital role in directing ENS development. The concluded:

Observations thus support the hypotheses that polysialylation of NCAM is required for the remodeling of chains of neurons into the mature patterns of ganglia and connectives of the enteric plexuses and that BMPs (Ed: Bone Morphogenetic Proteins) may participate in the sculpting of enteric plexuses by regulating clustering and the polysialylation of NCAM. [54] (emphasis added)

Unlike the central nervous system, few detailed studies on the ENS of autistic individuals exist. There are many studies describing the symptoms and frequency of gut dysfunction within autism, but the wide variety of seemingly disparate manifestations has created controversy. There has been no obvious common thread, no unifying mechanism to explain why so many bowel functions appear “out of whack”. This inconsistency has created disagreement within the research community leading some autism researchers to reject the notion of a link between autism and gut dysfunction entirely. As within the brain, anti-PSA antibody-mediated ENS malformation provides this common thread. Gut dysfunction needs to be viewed as a systemic problem, rather than a failure of a single, localized function. In the proposed hypothesis, the ENS continues to operate in a suboptimal and disconnected manner comparable to impairment seen in the central nervous system of those with autism. Disordered ENS connectivity may lead to many seemingly unrelated disorders; peristalsis synchronization problems, inefficient digestive enzyme secretion, difficulties in fluid transfer, inappropriate bowel immune system activation, and potentially other specific malfunctions not yet explored. Again, the proposed internalization of PSA-NCAM neatly explains numerous contradictory indications. Further study is warranted.

5. The HIB-OMPC Vaccine and Anti-PSA Antibodies If anti-PSA antibodies play a role in autism, what might trigger a proliferation during the critical period prior to the transition to adult NCAM? Evidence suggests a possible candidate is a specific variant of the Haemophilus Influenza B (HiB) conjugate vaccine administered routinely to infants in the United States and other countries starting at 2 months of age [55]. HiB conjugate vaccines were first introduced in the late 1980’s and recommended for universal infant use shortly thereafter. There are several conjugate variants using diverse protein carriers; a popular product from Merck and Company (USA) uses the MenB outer membrane protein complex (OMPC) as the conjugate. In light of potential embryonic NCAM cross-reactivity, the choice of MenB OMPC in a pediatric vaccine raises some disconcerting questions. Presumably, safety concerns would dictate the OMPC be purified of all capsular PSA content prior to vaccine use. There is no public evidence such concerns were considered when choosing MenB as the conjugate carrier for the HIB-OMPC vaccine despite the early warnings regarding PSA autoimmune potential. A review of available literature indicates that the HIB-OMPC vaccine contains significant residual PSA, the PSA can spontaneously complex with the OMPC forming a potent antigen, and that the levels an infant might receive during routine vaccination are sufficient to induce a strong immune system response.



The OMPC extraction process Supporting this contention are published patents from the vaccine manufacturer documenting the production process. Two patents filed January 31, 1983 and April 4, 1985 give considerable insight into the vaccines composition and OMPC extraction process. The first patent (number 4459286) briefly describes the invention [11] while the second patent (number 4695624) amplifies the claims of the first patent, provides additional detail on the conjugation process, and also extends the patent claims to other conjugate vaccines beyond HiB [12]. Comparison with the Merck PedvaxHiB and Comvax package inserts clearly indicates the patents form the basis of those commercial products [10][56]. These patents show the final OMPC product used in the vaccine contains substantial residual PSA. The first of these patents was filed just months before the Finne et al 1983 publication detailing the Cross-reactivity between MenB capsule antibodies and embryonic NCAM (Figure 5).

Figure 5: HiB-OMPC Hypothesis Timeline

Timeline of important events in the HiB-OMPC conjugate vaccine hypothesis. Dates shown are that of publication of key findings, HiB-OMPC patent filings, FDA licensing, and ACIP vaccine recommendation (see text). The Patents describe a multi-step process to extract the OMPC final protein product from the MenB organism; fermentation, harvest and inactivation, washing of the resultant cell paste, extraction of the outer membrane, concentration by ultra-filtration, washing the resultant serotype protein, collection of the final product, and chemical assay. They include considerable detail on the process, but provide little explanation as to the specific intent of each step. None of the steps are particularly novel, instead using long-established separation and purification techniques. While the process was successful at extracting the OMPC, the later filtration and ultra-centrifugation steps would not be effective at removing intact capsular polysaccharides. MenB outer membrane protein molecular weights range from 28 to 46 kDaltons, whereas the



MenB polysaccharides are larger. The process contains no steps to hydrolyze the polysaccharide glycosidic bonds rendering them smaller and separable after the OMPC extraction. Disassociated lipooligosaccharides (molecular weight ~6 kDa) could be separated by the process, but not intact capsular polysaccharides.

Figure 6: The Meningococcal Outer Membrane and Capsule The HiB-OMPC vaccine uses the protein complex extracted from the MenB outer membrane as the protein conjugate. Note proximity of the capsule (light blue) to the outer membrane. The MenB capsule is composed of long-chain PSA anchored to the outer membrane phospholipids via phosphodiester linkage [57]. Patent information indicates the OMPC extraction process was ineffective in fully removing residual PSA. Measurement artifacts found in final product testing would cause inaccurate PSA content analysis (see text).

Residual sialic acid content in the OMPC product Both patents contain results from the chemical assays of the final protein products that have been reproduced herein (Table 1). Results are included for protein content, nucleic acids, neutral sugars, sialic acid and molecular weight. Protein is expressed in mg/ml using the Lowry method; nucleic acids, neutral sugars, and sialic acid are listed as a percentage of the protein weight. Note that sialic content is measured using the colorimetric technique of Svennerholm [58], also referred to as the recorcinol-HCl method. Sialic acid content is a



surprisingly high at 2.4% (1983 filing) and 3.0% (1985 filing) of the protein weight using the Svennerholm method. The importance of this finding will become evident.

Patent 4459286 (1/31/1983 filing)

Patent 4695624(4/4/1985 filing) Unit

ProteinLowry method 5.4 4.1 mg/mlUV method 3.8 NA mg/mlAdsorb. 280 nm/260 nm 1.01 NA (unitless)

Nucleic AcidUV method 2.0 NA % (wt / protein wt - Lowry)Bial (RNA) 2.1 1.8 % (wt / protein wt - Lowry)Diphenylamine (DNA) 0.1 0.6 % (wt / protein wt - Lowry)

Neutral Sugars 0.9 1.05 % (wt / protein wt - Lowry)Sialic Acid 2.4 3.0 % (wt / protein wt - Lowry)Molecular Weight 41,000 40,000 Daltons

Notes:

N/A - value not listed in patent

Assay

Bial - orcinol reactionNeutral Sugars - anthrone colorimetric testSialic Acid - resorcinol-HCl method (Svennerholm)Molecular weight - SDS polyacrylamide gel electrophoresis (SDS-PAGE)

Table 1: Assay of Final OMPC Protein Product (Patents 4459286/4695624)

Yu-Ip and Miller 1993 [59] describes a novel method for assessing process control of the OMPC and HiB polysaccharide capsule products. The authors obtained samples of the Merck products at various stages of production, the samples were hydrolyzed in trifluoroacetic acid and subjected to high-pH anion-exchange chromatography followed by pulsed amperometric detection. Their results (Table 2) reveal a large discrepancy with those assays published in the 1983 and 1985 patent filings. All three data sets contain neutral sugars and sialic acid assays of the final protein product. While there is general agreement on the neutral sugar content, the sialic acid content shown in the patents is at least twenty-fold higher than that shown in final product of Yu-Ip and Miller 1993 (Table 3). Perhaps not widely understood at the time of Yu-Ip and Miller 1993, PSA resists acid hydrolysis instead forming lactones under even mildly acidic conditions. Lactonization occurs between linked sialic acid residues preventing acid-catalyzed cleavage of the glycosidic bond. Even mildly acidic conditions produce extensive lactonization. At pH < 1 lactonization is complete [60]. Yu-Ip and Miller utilized 0.5 M trifluoroacetic acid as their reagent (pH 0.5); therefore lactones would have been widespread. Within MenB, sialic acid is found in two forms; the alpha(2,8)-linked polymer of the capsule (i.e. polysialic acid), and as an alpha(2,3)-linked terminal structure of the lipooligosaccharide (LOS). Yu-Ip and Miller commented that the tests found sialic acid, 2-keto-3-deoxyoctonic acid, and neutral sugars in molar quantities roughly equal to their known presence in the LOS of MenB. The authors concluded their methodology provided useful information regarding the composition of the LOS within the OMPC protein product. While this conclusion was



correct in a strict sense, it was incomplete as it did not consider nor address PSA lactonization. Assay Intact

Cells1st TED extract

2nd TED extract

3rd TED extract

4th TED extract

Final Product Unit

Neutral Sugars 2.22 1.89 2.29 3.32 2.17 1.48 % (wt / protein wt)Glucosamine 0.95 1.21 1.12 1.19 0.67 0.57 % (wt / protein wt)Galactose 0.34 0.32 0.52 0.89 0.54 0.5 % (wt / protein wt)Glucose 0.67 0.27 0.40 0.78 0.58 0.41 % (wt / protein wt)Mannose 0.26 0.09 0.25 0.46 0.38 < 0.01 % (wt / protein wt)

Sialic acid 0.71 0.81 0.85 0.39 0.15 0.12 % (wt / protein wt)KDO 0.25 0.31 0.33 0.37 0.37 0.27 % (wt / protein wt)

Step in Figure 1

Notes:

1st and 2nd TED extraction - extraction in 0.1M Tris-HCl, pH 8.5, 0.01 M EDTA and 0.5% sodium deoxycholate

Intact cells - After harvest of N. meningitidis serogroup Β fermentation and washing with saline

Method - high-pH anion-exchange chromatography with pulsed amperometric detection after acid hydrolysis

KDO = 2-keto-3-deoxyoctonic acid

Neutral sugar totals are sum of glucosamine, galactose, glucose, and mannose assays

1 2 3 4 5 6

Table 2: Assay of the OMPC Product Monosaccharides (Yu-Ip and Miller 1993)

Patent 4459286 1/31/1983 filing

Patent 46956244/4/1985 filing

Ip and Miller 1993final OMPC product Unit

Neutral Sugars 0.9(anthrone)

1.05(anthrone)

1.48(acid hydrolyzed) % (wt / protein wt)

Sialic Acid 2.4(Svennerholm)

3.0(Svennerholm)

0.12(acid hydrolyzed) % (wt / protein wt)

Notes on analysis methods indicated in parenthesis:

Acid hydrolyzed - hydrolyzed in trifluoroacetic acid and subjected to high-pH anion-exchange chromatography followed by pulsed amperometric detection

Assay

Anthrone - anthrone colorimetric testSvennerholm - resorcinol-HCl colorimetric test

Table 3: Comparison of Common Analytes - Patents vs. Yu-Ip and Miller 1993 Analysis method for each measured value is shown in parenthesis. In hindsight, these test results may have provided a false sense of security regarding the purity of the OMPC. The authors and subsequent reviewers may have assumed the small quantity of sialic acid in the OMPC final product (0.12% of the protein weight) indicated the OMPC was nearly free of capsular PSA. With knowledge of PSA lactonization a different conclusion emerges: the low quantities of sialic acid found using acid hydrolysis indicates the overwhelming majority of residual sialic acid in the OMPC is in the polysialic form. Combining the results of the Svennerholm technique and acid hydrolysis measurements



reveals that >95% of the sialic acid noted in the patents must be residual material from the polysaccharide capsules rather than LOS terminal residues. PSA Spontaneously Complexes with OMPC forming a Potent Antigen In and of itself, the simple presence of residual PSA within the vaccine would not necessarily be cause for concern as pure PSA is not immunogenic in mammals due to self tolerance. The warnings of Finne, Rougon, and others regarded manipulative attempts to break this immunogenic tolerance thus bypassing the evolutionary mechanisms in place to prevent autoimmunity. A proven method of increasing capsule immunogicity is to combine the antigen with a protein. It is this very technique that makes the HiB-OMPC vaccine effective since HiB capsules themselves are not immunogenic in infants [21]. Importantly, considerable research demonstrates PSA spontaneously bonds with the OMPC thus rendering the non-immunogenic capsule highly immunogenic. It is this protein-bonding phenomenon that may make PSA within the HIB-OMPC vaccine hazardous to infants. A review of the literature found multiple experimental MenB vaccines that complexed capsular PSA with the OMPC to break immunogenic tolerance indicating the feasibility of such an arrangement. Unlike the HiB polysaccharide where the capsule is bonded covalently to the OMPC, the MenB capsules complex via hydrophobic bonds. These bonds form spontaneously without requiring specialized catalysts or spacers [15]. Zollinger et al 1979 was one of the first of these experimental vaccines, being particularly noteworthy as in addition to animal tests it also included tests on eight human subjects. Six of the eight volunteers produced large increases in antibodies toward the MenB strain after vaccination with the complex. (The two who did not respond had high preexisting titers.) The study demonstrated that antibodies were present toward both capsular PSA and the OMPC itself [13]. Capsular antibodies were predominantly of the IgM isotype, not surprising as capsular antibodies produced by naturally-acquired MenB are also predominantly IgM [3]. A second vaccine dose was administered at five weeks with modest increases in IgM noted. At fourteen weeks titers had dropped somewhat though the average value was still six-fold higher than pre-immunization levels. They concluded with the definitive statement that MenB Capsule PSA and the outer membrane protein are immunogenic in man when presented as a complex [13]. Further experimental MenB capsule – OMPC complex vaccines were studied by Frasch and Peppler [61] and Moreno et al [15]. Although these tests were limited to animal subjects the same conclusions resulted; the OMPC and capsules readily complex together and IgM antibodies toward both the protein and capsule were induced. Again it was noted that the capsule itself was an extremely poor immunogen, but when linked to OMPC capsule IgM antibodies were invariably generated [15][61]. In 1991 Lifely et al conducted tests on twenty-five adult human subjects using the MenB capsule – OMPC complex vaccines at various dosages [14]. Each subject was vaccinated three times at four week intervals with adverse reactions resulting in the withdrawal of three participants. Vaccine dosages were 50, 100, and 150 mcg, with capsule antibody titers increasing 2.0-fold, 5.9-fold, and 3.9-fold respectively. Antibodies were again of the IgM



isotype, and remained at least twice the level of pre-vaccination titers twelve months after the initial vaccination. Comparable Levels of Polysialic Acid in the HiB-OMPC Vaccine Zollinger et al 1979 and Lifely et al 1991 include details regarding the polysialic acid and OMPC content of the experimental vaccine, in addition to the dosage used in the human trials. This information, along with sialic acid content from the HiB-OMPC patents, and the OMPC content from the commercial product package insert was used to compare potential capsule-OMPC content between the study test subjects and infants on the pediatric vaccine schedule. The results indicate that infants could receive doses per kilogram of body mass nearly identical to that which induced the strong IgM antibody response in the Zollinger et al 1979 and Lifely et al 1991 test subjects (Table 4). Also note that the inadvertent PSA content could be up to half that of the HiB capsule content, the intended vaccine antigen. A possible amplifying factor not considered is the use of an aluminum hydroxide adjuvant in the pediatric vaccine [10][56], whereas the experimental vaccine given the test subjects contained no adjuvant. This could further intensify the pediatric immune response as aluminum hydroxide-adjuvated MenB OMPC vaccines consistently induced stronger immune responses than unadjuvated OMPC vaccines in animal tests [62]. As discussed previously, Finne and Rougon demonstrated unequivocally that MenB capsule antibodies were cross-reactive with PSA in embryonic NCAM. Henke-Fahle, Monnier, and Charles demonstrated exposure to these same IgM antibodies significantly alters neuron development in cultured neonatal tissue. The results from these experimental vaccines support the hypothesis that the pediatric vaccine, containing residual PSA spontaneously complexed to the OMPC carrier, could produce the very same anti-PSA IgM antibodies when administered to infants.



Table 4: Potential Sialic Acid Content as a Function of Body Mass Experimental MenB PS Vaccines vs. HiB-OMPC Pediatric Vaccine

6. Autism Prevalence and the HIB-OMPC Vaccine Furthering supporting this hypothesis, the introduction of the HiB conjugate vaccine corresponds to initial increases in autism spectrum disorder. Due to mandates within the Lanterman act of 1969, California has comprehensive autism diagnosis and caseload statistics that predate those of most jurisdictions by two decades. McDonald and Paul’s 2010 analysis of California birth year data indicates an autism rate change point occurred between the birth



year cohorts of 1987 and 1988. A similar trend was noted in autism data from Denmark during this same period [16]. Richmand 2011 noted that this change point corresponded with the birth year cohort of the HiB conjugate vaccine in the US and other countries [17]. Using caseload data from the U.S. Department of Education and the California Health and Human Services Department, California autism rates were compiled for the 1970 through 2003 birth year cohorts (Figure 7) [63][64]. Between 1970 and 1987 birth year cohorts autism rates increased modestly with an average yearly increase of 0.56 cases per 10,000 births. Between the 1988 and 2003 birth year cohorts the autism rate increased ten-fold faster than the prior period with an average yearly increase of 5.5 cases per 10,000 births.

California Autism Rate and US Vaccine Introductionby Birth Year Cohort (2009 Data)

Source: US DOE, California HHSA, and US CDC

0

20

40

60

80

100

120

1970 1975 1980 1985 1990 1995 2000Birth Year Cohort

ASD

rate

per

10,

000

birt

hs

HIB CV using OMPC conjugate added, all three changed to 15+ mos (4/90)

HIB CV using any of the three conjugates added at 2+ months (1/91)

Arrows show birth year cohort for vaccine introduction. Dates in brackets are that of the CDC recommendation, birth year cohorts are earlier and

based on intended recipient age group.

Dashed lines are ASD rate linear trends. ASD Rate change points are evident

Rate increase from 1970 to 1987 = 0.56 / yearRate increase from 1988 to 2003 = 5.54 / year

HIB CV using diphtheria toxin conjugate added at 18+ months (1/88)

1987.5 Change Point

HIB CV using diphtheria CRM197 conjugate added at 18+ months (1/89)

Figure 7: California Autism Rates and HiB Vaccine Introduction

Autism rates by birth year cohort using California autism caseload data and birth year vital statistics. Autism rate methodology differs from that of McDonald & Paul 2010 [16] yet 1987.5 change point remains. Birth year cohorts for HiB CV introduction are shown. The HiB-OMPC variant was the last of three HIB conjugate vaccines launched and recommended by the CDC during the introductory period (Figure 7 and Table 5). The December 1989 licensing and April 1990 recommendation for toddlers 15 months of age an older corresponds to birth cohort of late 1988. California autism rates indicate a small uptick in 1988 followed by a larger uptick in the 1989 birth year cohort. In 1990 a smaller increase



follows with larger increases in 1991 and 1992. This is consistent with the April 1991 CDC recommendation of universal HiB vaccination for all toddlers two months of age or older.

Date HiB Vaccine Variant Action Age Group Birth Year Cohort

1/22/1988 [65] HiB - diphtheria toxin CDC recommended 18 months plus 1986

1/13/1988 [66] HiB - diphtheria CRM197 CDC recommended 18 months plus 1987

4/13/1990 [67]

HiB - Outer Membrane Protein Complex (OMPC) CDC recommended 15 months plus 1988

4/13/1990 [67]

HiB - diphtheria toxin HiB - diphtheria CRM197

CDC recommended age revised 15 months plus 1988

1/11/1991 [68]

HiB - diphtheria toxin HiB - diphtheria CRM197 HiB - OMPC

CDC recommended age and number of doses revised

2, 4, 6* and 15 months 1990-1991

Table 5: HiB Conjugate Vaccine Introduction Timeline * Note that the HiB-OMPC CV recommendation was for three doses and did not include the dose at 6 months of age recommended for the other HiB CV variants. Potential Role of the Blood Brain Barrier in Protecting Vaccine Recipients The HiB-OMPC vaccine variant held up to half the market share in the United States in 2007 [69]. The most recent CDC Autism and Developmental Disabilities Monitoring Network study estimated the 2006 autism prevalence among eight-year olds at 1 in 110 [70], consistent with levels seen in the California caseload data. The overwhelming majority of those that have received the vaccine did not become autistic. The protection afforded by the blood brain barrier (BBB) may be a factor in this divergence. Under normal physiological conditions, IgM antibodies do not cross the BBB. If an infant were to prematurely develop high anti-PSA antibody titers this barrier should provide protection for the developing nervous systems. The hypothesis requires both high anti-PSA IgM titers and breach of the diffusion barrier concurrently. The HiB-OMPC vaccine may routinely induce these antibodies, but only those infants that suffer a BBB breach prior to the transition from embryonic to adult NCAM would subsequently suffer the nervous system development anomalies that may cause autism. There are many methods of increasing BBB permeability, though tight junction disassembly brought on by inflammatory pain is a likely candidate. Huber et al 2001 studied the role of peripheral inflammatory pain in modulating BBB permeability in mice. Using three different mediums to induce acute, short term, and long term pain models they found all three significantly increased BBB permeability over that seen in controls; further tests demonstrated the mechanism was increased paracellular transport.



Proteins associated with tight junction integrity were altered by inflammatory pain with decreases in occludin and corresponding increases in zonulin [71]. Most pediatric vaccine adverse reactions are relatively benign, but all vaccines include a subset of recipients that suffer substantial injection site swelling, fever, and/or prolonged crying. All can be indications of inflammatory pain, though it is obviously impossible to correlate levels with those produced in the research of Huber et al. The use of aluminum salt adjuvants in pediatric vaccines may also play a role in BBB permeability. Kaya et al 2003 [72] and Song et al 2008 [73] both studied the effect of intraperitoneal aluminum administration on rat BBB permeability. Both studies consistently demonstrated aluminum not only entered the brain, but also caused the increased BBB permeability that facilitated aluminum brain access. Song et al further demonstrated that aluminum decreased occludin [73] also seen in the Huber et al 2001 inflammatory pain BBB study. Due to numerous additions to the infant vaccine schedule, cumulative aluminum content during routine immunization more than doubled between 1986 and 1999 [74][75]. This factor, coupled with increased collective adjuvanticity, may have rendered more infants susceptible to anti-PSA antibody generation and binding as the number of administered vaccines increased. These mechanisms also provide a possible explanation for late onset, regressive autism temporally delayed from administration of the HiB-OMPC vaccine. Anti-PSA IgM antibodies may be induced in infancy with a subsequent BBB breach occurring later when antibody titers are still elevated. This delayed stimulus could be ensuing vaccine administration unrelated to HiB-OMPC, illness, or other causes of inflammation.

7. Other potential triggers and future trends The hypothesis put forth is not meant to be the sole explanation for all cases of autism. As an autism diagnosis is based on selective examples of general behavior patterns, it is not uncommon for two individuals both diagnosed as being “on the spectrum” to have different particular manifestations. This suggests autism may be multifactorial, possibly involving numerous biochemical pathways. Another complication involves increased awareness; although the MIND studies made clear the increase in autism incidence is real, some of the increase is almost certainly the inclusion of less severe disorders that may have been missed in the past. These perspectives need to be kept in mind as it is unlikely a single precipitating event is responsible for the entire autism caseload increase. Other potential triggers of premature neonatal PSA removal Natural MenB exposure - A possible candidate might be from natural newborn exposure to MenB bacteria. In working with their experimental MenB OMPC complex vaccine, Moreno et al suggested the complexed polysaccharide induced antibodies as its structure was similar to that of the bacteria [15]. Neonatal antibodies toward MenB polysaccharides could cause PSA autoimmunity regardless of their genesis. It should also be noted that Escherichia coli K1 also contains PSA capsules, natural exposure to that bacteria could conceivably induce anti-PSA antibodies [1].



Neisseria meningitidis C vaccine administration – Other researchers have noted that the Neisseria meningitidis type C (MenC) conjugate vaccine may inadvertently produce antibodies cross-reactive against MenB capsules in a fraction of recipients [23]. Both MenB and MenC contain capsular sialic acid chains, though MenB uses alpha(2,8) linkage (identical to that in embryonic NCAM), while MenC uses alpha(2,9) linkage. Shin et al 2001 observed that one of twelve IgM hybridoma secreting monoclonal antibodies obtained from mice injected with Escherichia coli K1 was reactive with both MenB and MenC [76]. Brandt et al 1972 also observed unexpected MenB/MenC cross-reactivity during work at Walter Reed Army Institute of Research [77]. MenC is not included in the current United States pediatric immunization schedule so it is unlikely this played a role in autism caseload statistics cited [65]. Nonetheless, MenC is a routine pediatric vaccine in other countries, including eleven countries in Europe. In particular, Greece, Ireland, Portugal, Spain, and the United Kingdom all administer MenC routinely to infants between two and four months of age, with the UK doing so since 1999 [78]. In addition, MenC is routinely administered in Canada at twelve months of age, with some provinces administering additional doses as early as two months of age [79]. The MenC vaccine may play a role in autism increases seen outside the United States. Although it was not a focus of this paper, it warrants further examination. Environmental exposure - Other prospective triggers could be antibody independent, such as environmental exposure to chemical compounds. Dey et al 2000 found that methylmercury exposure perturbed NCAM polysialation in rat pups, with little effect on mature rats. They concluded:

“Toxic perturbation of the developmentally-regulated expression of polysialylated NCAM during brain formation may disturb the stereotypic formation of neuronal contacts and could contribute to the behavioral and morphological disturbances observed following MeHg poisoning.” [80]

Organic mercury products began to see widespread use in industrial, agricultural, and medical products in the 1930’s. Kanner’s seminal treatise on autism was published in1943, noting the disorder was first observed in 1938 [28]. Future Trends With clear warnings in the literature regarding the potential for anti-PSA antibody cross-reactivity with embryonic NCAM, it would be reasonable to expect this would have been addressed prior to the HiB-OMPC vaccine being licensed for pediatric use. Publicly-available studies appear to show it was not; other research may exist that is not in the public domain. If this is the case, this matter may be resolved quickly if the data were released for independent review. In the event of data unavailability, studies to test the hypothesis take on an added sense of importance. Trends indicate future vaccines may also pose a risk of anti-PSA antibody induction apart from that of the HIB-OMPC vaccine. If the proposed link between anti-PSA antibodies and autism is genuine, it raises several points worthy of consideration:



Children can still be protected from HiB without using the HiB-OMPC vaccine. Other

HiB conjugate vaccines that do not use MenB OMPC are currently licensed in the United States.

Two published papers from 2006 and 2011 strongly advocate for pediatric clinical trials of a MenB capsule conjugate vaccines, arguing that despite in vitro anti-PSA binding, binding does not appear to occur in vivo [22][23]. (Note that Monnier et al 2001 shows this assertion is erroneous as their work does demonstrate in vivo anti-PSA binding. [7].)

A non-capsular MenB vaccine from Novartis (4CMen) is currently completing clinical trials in anticipation of future regulatory approval; the intended market includes infants as young as two months of age. The use of MenB outer membrane proteins as one of the four antigens raises questions regarding residual PSA, in a comparable manner to that seen in the HiB-OMPC patents [81].

The FDA recently approved the Sanofi-Pasteur’s Menactra Neisseria meningitidis capsule conjugate vaccine for infants as young as nine months of age, along with CDC recommendation for infants in certain high risk groups [65][82]. Menactra does not include MenB capsules, as it is limited to the A, C, W-135, and Y meningococcal strains. As previously discussed, the presence of sialic acid and similar structure between the MenB and MenC capsules might cause cross-reactivity in some fraction of recipients.

Looking forward, vaccine trends indicate that the likelihood of generating anti-PSA antibodies will increase, rather than decrease. In light of the evidence presented supporting the hypothesis, the implications are troubling. A well-designed, independent study testing the hypothesis is urgently needed.

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