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Death after Quadrivalent Human Papillomavirus (HPV) Vaccination:Causal or Coincidental? | OMICS International
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Death after Quadrivalent Human Papillomavirus (HPV) Vaccination:Causal or Coincidental?

Lucija Tomljenovic1* and Christopher A Shaw1,2,3
1Department of Ophthalmology and Visual Sciences, University of British Columbia, Canada
2Program in Experimental Medicine, University of British Columbia, Canada
3Program in Neuroscience, University of British Columbia, Canada
Corresponding Author : Lucija Tomljenovic
Neural Dynamics Research Group
828 W. 10th Ave.,Vancouver
BC, Canada, V5Z 1L8
Tel: 604-875-4111 (ext. 68375)
Fax: 604-876-4376
E-mail: [email protected]
Received September 13, 2012; Accepted October 02, 2012; Published October 04, 2012
Citation: Tomljenovic L, Shaw CA (2012) Death after Quadrivalent Human Papillomavirus (HPV) Vaccination: Causal or Coincidental? Pharmaceut Reg Affairs S12:001. doi: 10.4172/2167-7689.S12-001
Copyright: © 2012 Tomljenovic L, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract Background: The proper understanding of a true risk from vaccines is crucial for avoiding unnecessary adverse reactions (ADRs). However, to this date no solid tests or criteria have been established to determine whether adverse events are causally linked to vaccinations. Objectives: This research was carried out to determine whether or not some serious autoimmune and neurological ADRs following HPV vaccination are causal or merely coincidental and to validate a biomarker-based immunohistochemical (IHC) protocol for assessing causality in case of vaccination-suspected serious adverse neurological outcomes. Methods: Post-mortem brain tissue specimens from two young women who suffered from cerebral vasculitistype symptoms following vaccination with the HPV vaccine Gardasil were analysed by IHC for various immunoinflammatory markers. Brain sections were also stained for antibodies recognizing HPV-16L1 and HPV-18L1 antigen which are present in Gardasil. Results: In both cases, the autopsy revealed no anatomical, microbiological nor toxicological findings that might have explained the death of the individuals. In contrast, our IHC analysis showed evidence of an autoimmune vasculitis potentially triggered by the cross-reactive HPV-16L1 antibodies binding to the wall of cerebral blood vessels in all examined brain samples. We also detected the presence of HPV-16L1 particles within the cerebral vasculature with some HPV-16L1 particles adhering to the blood vessel walls. HPV-18L1 antibodies did not bind to cerebral blood vessels nor any other neural tissues. IHC also showed increased T-cell signalling and marked activation of the classical antibody-dependent complement pathway in cerebral vascular tissues from both cases. This pattern of complement activation in the absence of an active brain infection indicates an abnormal triggering of the immune response in which the immune attack is directed towards self-tissue. Conclusions: Our study suggests that HPV vaccines containing HPV-16L1 antigens pose an inherent risk for triggering potentially fatal autoimmune vasculopathies. Practice implications: Cerebral vasculitis is a serious disease which typically results in fatal outcomes when undiagnosed and left untreated. The fact that many of the symptoms reported to vaccine safety surveillance databases following HPV vaccination are indicative of cerebral vasculitis, but are unrecognized as such (i.e., intense persistent migraines, syncope, seizures, tremors and tingling, myalgia, locomotor abnormalities, psychotic symptoms and cognitive deficits), is a serious concern in light of the present findings. It thus appears that in some cases vaccination may be the triggering factor of fatal autoimmune/neurological events. Physicians should be aware of this association.

HPV vaccines; Serious adverse reactions; Cerebral vasculitis; Vasculopathy; Autoimmunity; Molecular mimicry; Immune complexes; Autoantibodies
In the past several decades, there have been numerous studies and case reports documenting neurological and autoimmune adverse reactions (ADRs) following the use of various vaccines. Arthritis, vasculitis, systemic lupus erythematosus (SLE), encephalopathy, neuropathy, seizure disorders and autoimmune demyelinating disease syndromes are the most frequently reported serious adverse events [1-13]. Although a clear temporal relationship between the administration of a vaccine and the adverse event is sometimes observed, in the vast majority of cases no causal connection can be demonstrated. Thus, it is often concluded that, (i) the majority of serious ADRs that occur postvaccination are coincidental and unrelated to the vaccine [14] and, (ii) true serious vaccine-related ADRs (i.e., permanent disability and death) are extremely rare [15]. There are however several important reasons why causality is rarely established with regard to vaccination-associated ADRs. These include: the criteria for causality are poorly defined [6,16,17]; the latency period between vaccination and autoimmunity can range from days to years (individuals’ susceptibility factors most likely playing a role in determining the temporal onset, time course, and severity of symptoms) [6]; neurological outcomes, as in other neurological disorders may take considerable periods to manifest as obvious pathology [18]; post-vaccination adverse manifestations can be atypical and might not be compatible with a defined autoimmune or neurological disease [6]; individual susceptibility factors are not considered and a “one-size fits all” principle is assumed [19]; a triggering role of the vaccine in the adverse outcome is not considered [20].
Quadrivalent human papillomavirus (qHPV) vaccine Gardasil was licensed in 2006 by the U.S. Food and Drug Administration (FDA) following a fast track approval process [21]. In pre- and post-licensure epidemiological studies no autoimmune safety concerns associated with Gardasil vaccination were identified [22,23] and the vaccine has been considered to have a remarkably good safety profile [24]. However, a careful scrutiny of Gardasil safety trials shows evidence of significant flaws in study design, data reporting and interpretation [25-28]. Irrespective of these latter observations, it is important to note that epidemiological studies only test for “association” and not “causation”, thus providing unreliable estimates of true risks. On the other hand, data from numerous case reports documenting serious autoimmune and neurological complications following HPV vaccination continue to raise concerns [11,12,29-35]. Nonetheless, the precise etiology of these post-HPV vaccination associated phenomena has been elusive and hence causality remains unascertained.
We have recently developed an immunohistochemical (IHC) protocol based on analyzing two cases of sudden and unexplained death following vaccination with the qHPV vaccine Gardasil. This protocol has been developed for the purpose of determining whether the serious autoimmune and neurological manifestations reported following HPV vaccination are causal or merely coincidental.
Patients and Methods
Case 1
A 19-year-old female without a relevant medical history and taking no drugs expired in her sleep, approximately 6 months after her third and final qHPV vaccine booster and following exacerbation of initial vaccination-related symptoms. She had last been seen alive by her parents the previous evening. Her symptoms started after the first qHPV injection when she developed warts on her hand that persisted throughout the vaccination period. In addition, she suffered from unexplained fatigue, muscle weakness, tachycardia, chest pain, tingling in extremities, irritability, mental confusion and periods of amnesia (memory lapses). The autopsy was unremarkable and failed to determine the exact cause of death. Internal examination revealed some minor changes involving the gallbladder and the uterine cervix (both of which on further examination by microbiological studies and histology revealed no significant disease). After a full autopsy no major abnormality was found anatomically, microbiologically or toxicologically that might have been regarded as a potential cause of death. Histological analysis of the brain hippocampus, cerebellum and watershed cortex allegedly revealed no evidence of neuronal loss or neuroinflammatory changes. However, the autopsy report did not specify which immune antibodies and stains were used for histological investigations.
Case 2
A 14-year-old female with a previous history of migraines and oral contraceptive use developed more severe migraines, speech problems, dizziness, weakness, inability to walk, depressed consciousness, confusion, amnesia and vomiting 14 days after receiving her first qHPV vaccine injection. These symptoms gradually resolved. However, 15 days after her second qHPV vaccine booster she was found unconscious in her bathtub by her mother 30 minutes after she had entered the bathroom to have a shower. Emergency help was summoned and arrived quickly. Resuscitation efforts were attempted. The paramedic noted that the patient was found without a pulse. Upon arrival at the hospital and approximately 30 minutes later, the patient suffered cardiac arrest. Resuscitation was terminated approximately 40 minutes later and the patient was pronounced dead.
Similar to Case 1, the autopsy failed to identify a precise cause of death. In particular, there were no anatomical, microbiological nor toxicological findings that could explain this case of death which was classified as “sudden and unexpected death”. Nonetheless, autopsy revealed cerebral edema and cerebellar herniation indicative of a focally disrupted blood-brain barrier. Although no specific antibodies to inflammatory markers were used in IHC analysis of brain sections, the autopsy reported that there was no evidence of inflammatory processes or microglial reactions in the patient’s brain. There were however acidophilic changes of the Purkinje cells in the cerebellum with vacuolation of the overlying molecular layer. According to the coroner, these changes were consistent with terminal ischemic-hypoxic encephalopathy. Neuropathological examination did not demonstrate an underlying structural brain disorder. In addition, the coroner’s report commented that the ischemic-hypoxic encephalopathy was terminal as was the cerebral edema and that either one could have been caused by the other. Based on the autopsy findings, the coroner was unable to establish a precise sequence of events and the specific etiology remained undetermined.
Tissue samples
Paraffin-embedded brain tissue specimens collected at autopsy from the two cases described above were used in this study. The tissues analyzed by IHC included the cerebellum, hippocampus, choroid plexus and watershed cortex (Case 1), and cerebellum, hippocampus, choroid plexus, portions of the brainstem (medulla, midbrain, pons), right basal ganglia, right parietal and left frontal lobes (Case 2). Sections were cut at 5 μm and mounted on glass slides.
IHC procedures
The paraffin brain sections were dewaxed, rehydrated and incubated for 15 min in methanol containing 3% H2O2 to block endogenous peroxidase activity. The sections were then incubated in 1% saponin in 1xPBS for 1 hr at room temperature (RT) and subsequently pre-treated by boiling for 20 min in an appropriate antigen retrieval solution (Table 1) to facilitate antigen retrieval and to increase membrane permeability to primary antibodies (Abs). The sections were then blocked in 1xPBS containing 0.1% Tween and 10% normal goat serum for 1hr at RT. Following the blocking step, the sections were incubated with appropriate dilutions of the primary Ab in the blocking solution (Table 1) overnight at 4oC in a humidified chamber.
Subsequently, sections were rinsed three times in 1xPBS and incubated with biotinylated, affinity-purified anti-immunoglobulin G (IgG) secondary Ab at 1:200 dilution (Vector Labs, Inc), and then with the avidin-biotin complex (ABC)-immunoperoxidase Vectastain Elite ABC kit (Vector Laboratories, Burlingame, Calif). The positive reaction was visualized by 3,3-diaminobenzidine (DAB) peroxidation according to standard methods. The sections were counterstained with methyl green, dehydrated, coverslipped, and observed on a Zeiss Axiovert microscope (Carl Zeiss Canada Ltd., Toronto, ON) connected to a computerized system with a photo camera. Images at 10x and 40x magnification were captured using AxioVision 4.3 software.
Results and Discussion
Principal findings
The results from our IHC examinations of brain tissue specimens from two young women who died following vaccination with the qHPV vaccine Gardasil showed strong evidence of an autoimmune vasculitis triggered by the cross-reactive HPV-16L1 antibodies binding to the wall of cerebral blood vessels (Figures 1 and 2). In addition, there was clear evidence of the presence of HPV-16L1 particles within the cerebral vasculature with some HPV-16L1 particles adhering to the blood vessel walls (Figures 1C, 2C and 2D). In contrast, HPV-18L1 antibodies did not bind to cerebral blood vessels nor any other neural tissues (Figure 1D).
Histopathologically, immune-mediated vasculitis is typified by excessive recruitment and vascular adhesion of T lymphocytes, increased complement and MHC II signaling and complementdependent deposition of immunoglobulin G–immune complexes to cerebral vasculature [5,36-40]. Increased expression of proteolytic matrix metalloproteinases (MMPs) also characterizes immune vasculopathic syndromes [41-43]. The MMPs play a major role in both the progression of inflammatory infiltrates and vessel destruction [41-43]. The ensuing vascular damage is manifested in hemorrhagic and ischemic brain tissue lesions. Notably, there was clear and consistent evidence of all these pathogenic immune processes in the brain tissue specimens from both young women (Figures 3-8).
The finding of HPV-16L1 particles in cerebral blood vessels and adhering to the wall of these vessels in brain tissue specimens from both cases (Figures 1,2,5 and 6) is of significant concern as it demonstrates that vaccine-derived immune complexes are capable of penetrating the blood-brain barrier. Gardasil is a recombinant vaccine and contains virus-like particles (VLPs) of the recombinant major capsid (L1) protein of HPV types 6, 11, 16, and 18 as active substances (note that the bivalent HPV vaccine Cervarix contains only HPV-16L1 and HPV-18L1 VLPs). The HPV VLPs in Gardasil (including HPV-16L1), are adsorbed on amorphous aluminium hydroxyphosphate sulfate adjuvant and recent studies in animal models show that aluminum adjuvant nanoparticles, taken up by monocytes after injection, first translocate to draining lymph nodes, then travel across the blood-brain barrier and eventually accumulate in the brain where they may cause significant immune-inflammatory adverse reactions [44]. Thus, the presence of HPV-16L1 particles in cerebral vasculature in brain tissue specimens from both young women vaccinated with Gardasil may be explained by a “Trojan horse” mechanism dependent on circulating macrophages by which these particles adsorbed to aluminum adjuvant gain access to brain tissue.
It should also be noted that accumulation of immune complexes and immune cells in brain tissues does not normally occur unless there is either: 1) a direct brain infection, 2) brain trauma or alternatively, 3) an excessive stimulation of the immune system, such as through vaccination. It would appear from the autopsy results and the medical histories (see Methods) that only the latter applies in both cases described.
The finding of a cerebral edema following autopsy in Case 2 is strongly suggestive of a focal blood-brain barrier breakdown [10,45]. Additionally, H&E staining showed clear evidence of hemorrhages (Figure 8), which were also present in Case 1 (Figure 7). Disruption of the blood-brain barrier manifesting in hemorrhagic tissue lesions could have resulted from both vasculitis and deleterious effects of excessive levels of glia-derived inflammatory cytokines. Although the autopsy failed to show evidence of microglial and inflammatory reactions in both cases, this was likely because no glia-specific markers were used in the histopathological analyses of brain tissue specimens. In contrast, results from our IHC analysis using micro- and astroglia specific markers (Table 1) showed exceptionally intense micro- and astrogliosis in all brain tissue sections examined from both Case 1 and Case 2 (Figures 9 and 10). Microglia are the brain’s resident immune cells and their excessive activation can lead to irreversible neurodestructive and pro-inflammatory processes in the brain [46,47].
It is also well known that activated microglia increase the permeability of the blood-brain barrier to other inflammatory factors and to trafficking lymphocytes [48]. Moreover, microglial aggregation in the brain is also recognized as a marker for hypoxic-ischemic brain injury [49], the latter diagnosed by the coroner in Case 2. Both microglia and astroglia are activated by a variety of immune insults, including aluminum vaccine adjuvants which are present in both HPV vaccines Gardasil and Cervarix. In addition, animal experiments show that only two injections of aluminum adjuvants at concentrations comparable to those used in human vaccines are sufficient to cause highly significant activation of the microglia that persists up to 6 months post-injection [50].
Autoimmune vasculitis and vaccinations
Vasculitis encompasses heterogeneous disorders whose central feature is inflammatory destruction of the blood vessel wall with resultant hemorrhagic lesions and tissue ischemia. Brain edemas may also be present due to compromised integrity of the cerebral vasculature [36,38,51-53]. Because involvement of blood vessels is intrinsic to inflammation of various types, vasculitis may underlie diverse diseases, making its diagnosis difficult [37,39]. Vasculitis can affect blood vessels including arteries and veins of all sizes in all areas of the body, resulting in a variety of clinical neurological manifestations [37,39].
The brain is particularly sensitive to ischemia. Vasculitis of the nervous system is thus of paramount importance to clinicians as it almost inevitably leads to permanent injury and disability when unrecognized and left untreated [37]. Cerebral vasculitis is thought to be rare [40]. However, because clinical presentation of cerebral vasculitis is highly variable with fluctuating signs and symptoms [40], and because it frequently underlies many diverse inflammatory diseases (i.e., SLE [41] and bacterial meningitis [43]), it is probable that many cases of cerebral vasculitis remain undiagnosed and/or misdiagnosed. Typical symptoms of cerebral vasculitis include severe headaches, orthostatic dizziness, syncope, seizures, tremors and tingling, weakness, locomotor deficits, cognitive and language impairments [39,51,52]. Note that the vast majority of these symptoms were experienced by both Case 1 and Case 2 post- qHPV vaccination (Table 2).
Vasculitis has long been recognized as a possible severe ADR to vaccination [5,54-57]. For example, Carvalho and Shoenfeld described a case of polyarteritis nodosa, a rare, life-threatening, necrotizing vasculitis that affects medium-sized arteries following the administration of the hepatitis B vaccine in a 14-year-old boy who had no relevant previous history and who was not taking any drugs [5]. There have been numerous other case reports of vasculitis posthepatitis B vaccination. In IHC examinations of these cases, no virus B antigen was detected in the blood vessels and no proof of cause and effect was established [55,57]. However, the possibility that the vaccine-derived hepatitis B antigen might have caused autoimmunity by molecular mimicry was not investigated. In fact, to the best of our knowledge, our study is the first to show direct evidence of a vaccine antigen-induced autoimmune vasculitis in the human central nervous system (CNS).
It is worth emphasizing that molecular mimicry (whereby the vaccine antigen resembles a host antigen) is generally accepted as a mechanism by which vaccines can trigger autoimmune diseases. Thus, antibodies and T cells that are produced to destroy the vaccine antigen also attack structurally similar self-antigens in different tissues (i.e., the wall of blood vessels) [5,6,35]. The fact that vaccination is often intended to prevent a disease and thus carried out in the absence of an active infection in the host, implies that the risk of autoimmunity may be exacerbated if there is structural similarity between the vaccine antigen and the host tissue. The reason for this is two-fold. Firstly, vaccination produces a much higher and sustained level of antibodies compared to natural infection (i.e., Gardasil-induced HPV-16 antibody titers are 10-fold higher than natural HPV infection titers [58]). Secondly, in the absence of an actual infectious agent (i.e., HPV-16 virus), the vaccineinduced antibodies are likely to preferentially bind to host antigens.
Vasculitis and HPV vaccination
Recently two cases of vasculitis affecting young teenage girls following HPV vaccination have been reported. In both cases there was no history of preceding infection, and the strong temporal relationship between the administration of the bivalent HPV vaccine (Cervarix) and the development of vasculitis was notable [32].
Our search of the Vaccine Adverse Event Reporting System (VAERS) internet database [59] revealed numerous reports of post- HPV vaccination-associated vasculitis. An analysis of these cases shows that post-vaccination vasculitis-related symptoms most typically manifest within the first three to four months of vaccination (Table 3), as was also reported in Case 1 and Case 2 described herein. Moreover, we noted a striking similarity between the vasculitis-related symptoms reported to VAERS and those experienced by Case 1 and Case 2 (Table 2). Although a report to a passive vaccine safety surveillance system such as VAERS does not by itself prove that the vaccine caused an ADR, it should be noted that many VAERS reports also include detailed records of diagnostic laboratory analyses and clinician’s follow-ups and their expert diagnosis. Notably, some of these reports include medically confirmed cases where the diagnosis of immune-mediated vasculitis was ascertained (i.e., VAERS ID# 425345-1, 436679-1; Table 3).
The precise etiology and the role of HPV vaccination in vasculitis cases reported to VAERS remained undetermined. However, we note that the histopathological examinations when conducted were very limited in scope (Table 3). Specifically, the possibility of HPV vaccine-induced autoimmunity via molecular mimicry due to cross reactivity between vaccine antigens and host vascular structures was neither investigated nor considered. The reason for such omissions in histopathological analyses of vaccine-suspected autoimmune pathologies is unclear, especially since medical scientists generally accept molecular mimicry as a plausible mechanism by which vaccines may trigger autoimmune diseases [5,6,35].
The pathophysiology of autoimmune vasculitis in light of present findings
Although most types of vasculitis are associated with immunologic abnormalities, the primary immunopathogenic events that initiate vascular inflammation and ensuing vascular damage are still far from clear. Nonetheless, it is recognized that the generation of autoantibodies and deposition of immune complexes in vascular tissues play a central role in immune vasculopathies [39,60,61]. As shown in Figures 1-6, there appears to be clear evidence of both of these immunopathogenic processes in brain tissue specimens from both cases reported herein. Namely, numerous HPV-16L1 positive cells are found adhering to the walls of cerebral vessels and additionally, the vascular wall itself shows remarkably prominent immunoreactivity for HPV-16L1 (Figures 1-6).
Circulating immune complexes can result from either, response to infection, tissue injury or vaccination. The fact that vaccines are designed to hyper-stimulate antibody production (thus producing much higher antibody levels than what occurs following natural infection), suggests that vaccination may carry a much higher risk for immune vasculopathies. As noted above, Gardasil induces sustained antibody titers for HPV-16 that are more than 10-fold higher than natural HPV infection titers [58]. Notably, elevated levels of circulating immune complexes are typically observed in human vasculitis syndromes [61].
Deposition of immune complexes is a potent and rapid trigger for inducing vascular inflammation as it has the capacity to activate the complement pathway [5,39,53,61-63]. Indeed, inappropriate activation of the complement (i.e., in neurons and cerebral vasculature) is frequently observed in inflammatory neurodegenerative and neuroimmune diseases with underlying vascular dysfunction [53,61,63,64]. In particular, activation via the classical antibodydependent complement pathway has long been recognized in immune complex-mediated diseases including vasculitis and SLE [37,39,65].
Vascular deposition of immune complexes and leukocyte recruitment to the vascular wall are themselves dependent on activation of the antibody-dependent complement pathway. In particular, abnormal expression of C1q by the vascular smooth muscle cells and vascular endothelium facilitates binding of T-lymphocytes and immune complexes to the vascular wall, resulting in vascular damage, ischemic lesions and brain edemas [53,62,66]. The demonstration of the crucial role of C1q activation in secondary brain edema due to compromised cerebral vasculature [53] is of particular relevance to Case 2 described in this report, where cerebral edema with bilateral uncal notching and early cerebellar tonsillar herniation was revealed on autopsy. Moreover, the autopsy of Case 2 also revealed ischemic changes affecting the Purkinje cells in the cerebellum which, according to the coroner’s report, were indicative of a terminal ischemic-hypoxic encephalopathy.
It has further been demonstrated that both vascular endothelial and smooth muscle cells express MHC II and thus by facilitating T-cell recruitment essentially operate as competent antigen-presenting cells [36,67,68]. In this regard it is important to note that lymphocyte trafficking through the CNS is normally limited and that lymphocyte adhesion to brain endothelium is very low (less than 5% compared with 15%-20% in other organs) [39]. Furthermore, although cerebral vascular endothelial cells are capable of expressing MHC class I and II molecules (which are crucial for antigen presentation to the T lymphocytes), this occurs less often than in endothelium of the systemic vasculature [39].
Thus, the prominent and consistent HPV-16L1, Ig-complex, CD3+ T-cell, MHC II, C5b-9 and C1q vascular staining patterns in the brain tissue specimens described here (Figures 3-6), strongly point to a vasculitic neuropathy, triggered by an aberrant hyper-active immune response, as a plausible explanation for the fatal outcomes post-HPV vaccination in these two cases. Specifically, all six markers prominently stain numerous immune cells adhering to the walls of cerebral blood vessels (Figures 1-6). In addition, C1q and HPV-16L1 prominently stain the vascular endothelium and smooth muscle cell layer (Figures 3 and 4). Collectively, these findings suggests the involvement of both immune complexes and cross-reactive HPV-16L1 antibodies (binding to vascular walls) in triggering vascular damage.
The dense and prominent immunoreactivity for the membrane attack complex (MAC) component C5b-9 in perivascular deposits (Figure 5) provides further corroborative evidence for a vasculopathy resulting from an abnormal activation of the classical antibodydependent complement pathway. The formation of MAC is a terminal step in the classical pathway and its activation results in a lytic destruction of target cells [64,69]. In immune vasculopathies, C5b-9/ MAC activity is associated with destruction of both perivascular tissues and cells within the vessel wall [37,39]. MAC is normally activated by immune triggers, including vaccinations [70,71]. Activation of MAC in the brain in the absence of an active brain infection due to an invading foreign infectious agent (i.e., virus or bacteria) thus indicates an abnormal triggering of the immune response in which the immune attack is directed towards self-tissue. Such activation of the MAC in brain tissue specimens from Case 1 and Case 2 can therefore plausibly be explained by the receipt of the qHPV vaccine given that in both cases the autopsy examinations found no evidence of an alternative microbiological insult to explain the fatal outcomes. Moreover, prominent MAC immunoreactivity was also detected in cerebellar Purkinje cells and neurons in various areas of the brain, including the hippocampus. These results will be presented and discussed in greater detail in a separate publication.
In addition, we detected intense MMP-9 immunostaining in the vascular wall and the perivascular extracellular matrix (ECM) in brain tissue specimens from both cases. The vascular wall MMP-9 immunostaining pattern closely coincided with that of HPV-16L1 and C1q (Figures 3 and 4). This finding again corroborates our suggestion of an immune-triggered vasculopathy as an underlying cause for the fatal outcomes in both cases following qHPV vaccination. Indeed, MMP-9 is one of the key proteolytic enzymes involved in progression of inflammatory infiltrates and vessel and ECM destruction in vasculitis [41-43]. Elevated MMP-9 expression is strongly associated with neuroimmune vasculopathies and diverse severe inflammatory nervous system pathologies, including ischemic nerve damage in SLE [41], autoimmune demyelinating syndromes, ischemia and stroke [72,73]. MMPs are also prominently elevated in neuropathic pain syndromes such as migraine headaches [74,75]. In this regard, we note that headaches are one of the most frequently reported symptoms post-HPV vaccination (Table 4) and could be due to an underlying undiagnosed or misdiagnosed cerebral vasculitis.
Moreover, MMP-2 and MMP-9 have been shown to induce bloodbrain barrier breakdown and to facilitate leukocyte extravasation in experimental bacterial meningitis [76] and other models of neuroinflammation [77,78] of the central and peripheral nervous systems. In particular, in bacterial meningitis, acute breakdown of the blood-brain barrier, intrathecal production of pro-inflammatory cytokines and accumulation of blood derived leukocytes in the cerebrospinal fluid lead to brain edema, cerebral vasculitis, and ultimately permanent neuronal injury. An overactive immune response of the host, rather than the bacterial pathogen per se, is thought to be the cause of neuronal injury, resulting in permanent neurological sequelae [43,79]. In this regard, it is important to re-emphasize the fact that vaccines, designed to hyper-stimulate the immune response (owing to the action of immune adjuvants), appear to carry an inherent risk for serious autoimmune disorders affecting the CNS [17,80-82]. Based on exhaustive investigations of post-vaccination induced autoimmune phenomena, Cohen, Carvalho and Shoenfeld have concluded that, “it seems that vaccines have a predilection to affect the nervous system” [1,83].
The obvious limitations of our study are that the tissues examined represent two individuals against which there were no control samples. For this reason, we could not obtain a quantitative measure of immunoreactivity. We aim in the future to further corroborate our findings by examining brain tissues from other cases of sudden and unexplained death following HPV vaccination, as well as control brain tissue from age-matched individuals who clearly died from nonvaccination related causes. Nonetheless, the marked resemblance in immunostaining patterns for all immunohistological markers in brain tissue specimens in the present two cases (i.e., compare Figures 1-4), as well as the similarity between their symptoms and those noted on VAERS reports related to post-HPV vaccination vasculopathies (some of which were medically ascertained cases; Tables 2 and 3), strongly support our present conclusions.
Any medicinal product (including vaccines) carries some risk of adverse effects. However, unlike most medicinal products, vaccines are often administered to otherwise healthy individuals and this fact, according to the FDA, places significant emphasis on their safety [84]. The proper understanding of a true risk from vaccines is thus crucial for avoiding unnecessary ADRs [19]. In this regard, the fact that to date no solid tests or criteria have been established to determine whether adverse events are causally linked to vaccinations [6,16] should be a cause for concern.
In recent years it has become increasingly clear that vaccines may be a triggering factor for severe neurological manifestations of autoimmune etiology [1-13]. Some of these autoimmune phenomena may be explained by molecular mimicry whereby an antigen of a recombinant vaccine (i.e., HPV or hepatitis B vaccine) or of a live, attenuated virus (i.e., MMR) may resemble a host antigen and trigger autoimmunity [5,6,83]. Owing to their structural resemblance, antibodies and autoreactive T cells not only destroy the invading pathogen but also attack the host tissue. The data from the present study not only validate the molecular mimicry hypothesis of vaccine-induced autoimmune diseases, but also further expand on the proposed pathway, which most likely begins with the passage of vaccine-derived HPV-16L1 antibodies across the blood-brain barrier and the choroid plexus. Once in the CNS, some of these antibodies bind to neuronal host-antigen(s) due to molecular mimicry, such as vascular endothelial and/or smooth muscle cells. This further leads to a classical antibody-dependent complement pathway activation (C1q and MAC), resulting in destruction of blood vessel integrity, hemorrhages and ischemic tissue injury. The resultant blood-brain barrier breakdown allows further nondiscriminatory passage of immune cells and vaccine-derived immune complexes into the brain thus perpetuating the HPV vaccine triggered neurodestructive autoimmune process. The HPV-16L1 VLPs appear to contribute to this aberrant immune process by invading the CNS (most likely via a macrophage-dependent Trojan horse mechanism), and depositing on the walls of cerebral blood vessels.
Cerebral vasculitis is a serious disease which typically results in fatal outcomes when undiagnosed and left untreated [37]. The fact that many of the symptoms reported to VAERS following HPV vaccination are indicative of cerebral vasculitis, but are unrecognized as such (i.e., intense persistent migraines, syncope, seizures, tremors and tingling, myalgia, locomotor abnormalities, psychotic symptoms and cognitive deficits; Table 4) is thus a serious concern in light of the present findings. In particular, the fact that positive cerebral blood vessel wall immunoreactivity was observed with HPV-16L1 and not with HPV- 18L1 antibody (Figure 1) suggests that HPV vaccines containing HPV-16L1 VLPs (including Gardasil and Cervarix) pose an inherent risk for triggering potentially fatal autoimmune vasculopathies and are therefore inherently unsafe for some individuals. Exactly which individuals might be more prone to developing a serious ADR following HPV vaccination is currently unknown. It is also unknown whether HPV vaccination can actually prevent cervical cancer cases since the current optimistic surrogate-marker based extrapolations have not been validated and appear to have arisen primarily from significant misinterpretation of existing data [25,85]. Because the HPV vaccination programme has global coverage, the long-term health of many women may be at risk against still unknown vaccine benefits. In conclusion, any case of sudden unexpected death occurring after HPV or other vaccinations should always undergo an exhaustive immunohistochemical study according to the methods presented in this report.
This work was supported by the Dwoskin, Lotus and Katlyn Fox Family Foundations. We are very grateful to Ingrid Barta from the University of British Columbia Department of Pathology and Laboratory Medicine for technical support.

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