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Vaccine Safety Conference: Evaluating the Science January 3-9 2010 Tryall Club West Indies Jamaica

Vaccine Safety Conference: Evaluating the Science January 3-9 2010 Tryall Club West Indies Jamaica
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Biopersistence and biodistribution of particles injected into muscle: relevance to vaccine Alum safety

Consumer Summary | by Romain Gherardi

Romain K. Gherardi, MD, Institut Mondor de Recherche Biomédicale, INSERM U955-E10, Univeristé Paris-Est, France. Email : romain.gherardi@hmn.aphp.fr

Alum adjuvant, empirically introduced in vaccines in 1927, is still widely used to boost the immune response to human and veterinary vaccines. French myopathologists have identified a condition called macrophagic myofasciitis (MMF), pointing to the lack of knowledge on the fate, systemic diffusion, and long-term safety of Alum particles injected into muscle. MMF was described in 1998 as a condition of unknown cause characterized by a stereotyped lesion at muscle biopsy detected in middle-aged adult patients with diffuse myalgias and fatigue. Macrophages, the major immune cell type in the lesion, enclosed agglomerates of nanocrystals in their cytoplasm, subsequently found to represent Alum from hepatitis B, hepatitis A and tetanus toxoid vaccines. MMF is now recognized to assess long-term persistence of Alum at site of previous i.m. immunization. Patients had received an average of 4.5 vaccine shots (92% against hepatitis B) and usually experienced first clinical symptoms about 1 year after the last immunization. Alum residence time is very long in affected individuals (mean delay from last vaccination to biopsy = 53mo, up to >12yrs). Several hundreds of patients with MMF have been identified in France, >250 of which are followed by the Paris-Est University hospital, Créteil, France. Almost all patients presented with diffuse arthromyalgias, chronic fatigue, and cognitive complaints, thus often fulfilling international criteria for chronic fatigue syndrome. A case control study conducted by AFSSAPS pointed out chronic fatigue as more frequent and more pronounced in patients with than without MMF in deltoid muscle. CNS involvement is assessed by cognitive dysfunction and, occasionally, overt multiple sclerosis (10%). Neuropsychological tests have shown cognitive alterations in almost all MMF patients. Compared to arthritis controls matched for pain severity and duration, depression and educational level, MMF patients displayed more severe and distinctive impairment of visual memory, working memory and dichotic listening, suggesting organic brain damage. Similar cognitive alterations occur in foundry workers exposed to inhaled Al fumes or powder. Both the “Gulf war syndrome”, which has been linked to exposure to multiple vaccinations, including the Alum-adjuvanted anthrax vaccine, and silicone implant-associated autoimmune conditions manifest by symptoms similar to those observed in MMF patients, forming the recently individualized Autoimmune/inflammatory Syndrome Induced by Adjuvants (ASIA). Alum safety crucially depends on whether the compound remains localized at site of injection or may diffuse and accumulate in distant organs. Macrophages are now recognized to avidly take up Alum agglomerates and, in so-doing, become long-lived cells. Interaction of nanomaterials with immune cells may strongly interfere with their biodisposition. We wondered if a proportion of particles injected into muscle could translocate to distant organs as part of a general mechanism linked to phagocytosis. Preliminary results are presented, confirming this view and suggesting implication of a MCP-1/CCL2 chemokine-dependant Trojan horse mechanism for translocation to the brain of nanomaterials injected into muscle.

Slide Summary | by Romain Gheradri

Slide 1

Biopersistence and biodistribution of particles injected into muscle: application to Alum safety

Slide 2

French myopathologists have identified in 1998 a condition called macrophagic myofasciitis (MMF), pointing to the lack of knowledge on the fate, systemic diffusion, and long-term safety of Alum particles injected into muscle. MMF was initially described as an emerging condition of unknown cause characterized by a stereotyped lesion at muscle biopsy detected in middle-aged adult patients with diffuse myalgias and fatigue.

Slide 3

Macrophages, the major immune cell type in the lesion, enclosed agglomerates of nanocrystals in their cytoplasm, subsequently found to represent Al hydroxide

Slide 4

These crystals corresponded to Alum derived from hepatitis B, hepatitis A and tetanus toxoid vaccines. It is now widely accepted that MMF is a specific histologic lesion assessing long-term persistence of Alum at site of previous i.m. immunization.

Slide 5

Alum residence time is very long in affected individuals with a mean delay from the last vaccination to biopsy of 53 months (up to >12years). Only a small minority of cases appear to represent chance association due to recent immunization in a patient with unrelated neuromuscular disease. In rats, experimental MMF shrinks progressively with time, and has very small size at 12 months. WHO has suggested that patients with longstanding post-vaccinal MMF could have individual inability to clear Alum out of the body.

Slide 6

This was supported by marked influence of the rat genetic background on the size of vaccine-induced experimental MMF.

Slide 7

About 1000 patients with MMF have been identified in France. Adult patients with MMF collected in our Center (>250), usually presented with diffuse arthromyalgias and chronic fatigue. A case control study conducted by AFSSAPS pointed out chronic fatigue as more frequent and more pronounced in patients with than without MMF in the deltoid muscle.

Slide 8

Adult MMF patients with diffuse arthromyalgias, chronic fatigue, and cognitive complaints, often fulfilled international criteria for chronic fatigue syndrome. They usually lack fibromyalgic tender points. About 10% have concurrent demyelinating CNS involvement similar to multiple sclerosis.

Slide 9

Neuropsychological tests have shown cognitive alterations in almost all MMF patients. Compared to arthritis controls matched for pain severity and duration, depression and educational level, MMF patients displayed more severe and distinctive impairment of visual memory, working memory and dichotic listening, suggesting organic brain damage. Similar cognitive alterations occur in foundry workers exposed to inhaled Al fumes or powder.

Slide 10

In contrast to arthritic controls, most MMF patients had some altered neuropsychological tests reaching the dementia threshold.

Slide 11

In 2003 we observed that the “Gulf war syndrome”, which, in UK, has been linked to exposure to multiple vaccinations, including the Alum-adjuvanted anthrax vaccine, had symptoms strikingly similar to those observed in our MMF patients. Similarity is also obvious with clinical manifestations in silicone implant-associated autoimmune conditions. This recently led to individualization by Yehuda Shoenfeld of the so-called Autoimmune/inflammatory Syndrome Induced by Adjuvants (ASIA).

Slide 12

Although a set of clinical manifestations (ASIA) is typically observed in adult patients with longstanding MMF lesion in the deltoid muscle, significance of the association remains unclear as nobody has characterized the biodistribution pattern of particles injected into muscle. Admittedly, Alum safety concerns crucially depend on whether the particulate compound will entirely remain localized at site of injection or can diffuse and accumulate in distant organs,

Slide 13

Nanosized particles (NSPs) have various innovative medical applications in fields such as, imaging contrast fluids, topic antimicrobials, surgery tools, and drug or gene delivery, and vaccine. In balance with these promising applications, safety issues need to be very carefully assessed. Due to the rapidly growing number of novel compounds and formulations, questions relative to biodistribution, persistence and toxicity of most nanomaterials have not been thoroughly explored, and long-term data are lacking. Therefore, the understanding of general mechanisms that may underlie beneficial/adverse effects of NSPs, especially those interacting with immune cells, is mandatory.

Slide 14

Alum is nano-crystaline particle composed of Aluminium hydroxide that has been introduced in vaccine for its immunologic adjuvant effect in 1927. Alum remains the most commonly used vaccine adjuvant although mechanisms by which it stimulates immune responses remain incompletely understood. Since biodistribution of particles injected into muscle has not been previously reported, biological likelihood of a cause to effect relationship between Alum i.m. injection and systemic manifestations remains elusive. Macrophages are now recognized to avidly take up Alum agglomerates and, in so-doing, become long-lived cells. Interaction of nanomaterials with immune cells may strongly interfere with their biodisposition. We wondered if a proportion of particles injected into muscle could translocate to distant organs as part of a general mechanism linked to phagocytosis.

Slide 15

We recently observed that muscle envelopes host most of the so-called resident muscle macrophages

Slide 16

These macrophages differ from exudate macrophages by their abundant production of KC and MCP-1, two potent chemokines attracting neutrophils and circulating monocytes, respectively.

Slide 17

Thus, upon muscle injury or other danger signal, resident MPs first accumulate in fascia, then release chemokines, thus eliciting huge muscle infiltration by exudate MO/MPs. Phagocytosis of debris, is followed by a phenotypic switch of MO/MPs into alternatively activated MPs, helping myorepair, and migration to lymphoid organs

Slide 18

These migrating cells derived from circulating MOs are known as ‘inflammatory’ dendritic cells, and substitute to classical migrating dendritic cells present in tissues other than muscle (such as dermis).

Slide 19

After i.m. injection of Alum-containing vaccines in mice, muscle Al content decreases by 50% within 4 days after injection and then remains stable once the granuloma of Alum-loaded phagocytes is formed. To examine if and how particles injected into muscle can also translocate to distant sites, we used two types of fluorescent particles: exploratory polychromatic fluorescent latex beads (FLBs), and a confirmatory Alum-relevant nanohybrid (Al-rho) in which Al(OH)3 is coupled with rhodamine.

Slide 20

Our preliminary results indicate that : -After i.m. injection particles are captured by phagocytes(MO/MPs) within hours. -Some particle-loaded cells form stable local granuloma in fascia; other ones migrate through lymphatics to the draining lymph nodes (initial lymphatic biodistribution). -Migratory particle-loaded cells then exit LNs via the thoracic duct, gain access to the blood stream and distribute to distant organs (secondary hematogenous distribution). -Incorporation into brain is delayed, and slowly accumulative. -Neurodelivery is achieved through a MCP-1-dependant Trojan horse mechanism. -A similar elementary mechanism has been previously shown for neurodelivery of viral particles (HIV, HCV, possibly EBV). -This might constitute a common pathway at play in chronic fatigue syndrome / ASIA syndrome induced by a variety of infectious or mineral particles, including vaccine adjuvants such as Al(OH)3.

Implication of this mechanism of particle neurodelivery in pathophysiology of neuroinfllamatory (MS) and neurodegenerative (AD) diseases desserves investigation.

Slide 21

References Cited in Presentation

Gherardi RK, Coquet M, Chérin P, Authier FJ, Laforêt P, Bélec L, Figarella-Branger D, Mussini JM, Pellissier JF, Fardeau M. Macrophagic myofasciitis: an emerging entity. Groupe d'Etudes et Recherche sur les Maladies Musculaires Acquises et Dysimmunitaires (GERMMAD) de l'Association Française contre les Myopathies (AFM). Lancet. 1998 Aug 1;352(9125):347-52.

Chérin P, Authier FJ, Gherardi RK, Romero N, Laforêt P, Eymard B, Herson S, Caillat-Vigneron N. Gallium-67 scintigraphy in macrophagic myofasciitis. Arthritis Rheum. 2000 Jul;43(7):1520-6.

Authier FJ, Cherin P, Creange A, Bonnotte B, Ferrer X, Abdelmoumni A, Ranoux D, Pelletier J, Figarella-Branger D, Granel B, Maisonobe T, Coquet M, Degos JD, Gherardi RK. Central nervous system disease in patients with macrophagic myofasciitis. Brain. 2001 May;124(Pt 5):974-83.

Gherardi RK, Coquet M, Cherin P, Belec L, Moretto P, Dreyfus PA, Pellissier JF, Chariot P, Authier FJ. Macrophagic myofasciitis lesions assess long-term persistence of vaccine-derived aluminium hydroxide in muscle. Brain. 2001 Sep;124(Pt 9):1821-31

Authier FJ, Sauvat S, Champey J, Drogou I, Coquet M, Gherardi RK. Chronic fatigue syndrome in patients with macrophagic myofasciitis. Arthritis Rheum. 2003 Feb;48(2):569-70

Gherardi RK. [Lessons from macrophagic myofasciitis: towards definition of a vaccine adjuvant-related syndrome]. Rev Neurol (Paris). 2003 Feb;159(2):162-4.

Bassez G, Authier FJ, Lechapt-Zalcman E, Delfau-Larue MH, Plonquet A, Coquet M, Illa I, Gherardi RK. Inflammatory myopathy with abundant macrophages (IMAM): a condition sharing similarities with cytophagic histiocytic panniculitis and distinct from macrophagic myofasciitis. J Neuropathol Exp Neurol. 2003 May;62(5):464-74.

Gherardi RK, Authier FJ. Aluminum inclusion macrophagic myofasciitis: a recently identified condition. Immunol Allergy Clin North Am. 2003 Nov;23(4):699-712. Review

Authier FJ, Sauvat S, Christov C, Chariot P, Raisbeck G, Poron MF, Yiou F, Gherardi R. AlOH3-adjuvanted vaccine-induced macrophagic myofasciitis in rats is influenced by the genetic background. Neuromuscul Disord. 2006 May;16(5):347-52.

Exley C, Swarbrick L, Gherardi RK, Authier FJ. A role for the body burden of aluminium in vaccine-associated macrophagic myofasciitis and chronic fatigue syndrome. Med Hypotheses. 2009 Feb;72(2):135-9.

Couette M, Boisse MF, Maison P, Brugieres P, Cesaro P, Chevalier X, Gherardi RK, Bachoud-Levi AC, Authier FJ. Long-term persistence of vaccine-derived aluminum hydroxide is associated with chronic cognitive dysfunction. J Inorg Biochem. 2009;103(11):1571-8.

Brigitte M, Schilte C, Plonquet A, Baba-Amer Y, Henri A, Charlier C, Tajbakhsh S, Albert M, Gherardi RK, Chrétien F. Muscle resident macrophages control the immune cell reaction in a mouse model of notexin-induced myoinjury. Arthritis Rheum. 2010 Jan;62(1):268-79.

Slide 22

Previous presentations of the preliminary experimental data

Khan Z, Chrétien F, Gherardi RK, Cadusseau J. Nanoparticle neurodelivery increase in a model of chronically altered BBB. Euroglia, Paris, France, 2009.

Cadusseau J, Khan Z, Itier V , Tillement O. Gherardi RK. Nanoparticle translocation from muscle to brain. Society of Neuroscience, Chicago, October 2009

Khan Z , Christov C, Lux F, Itier V, Tillement O, Authier FJ, Gherardi, R.K., Cadusseau J. Bio-distribution of nanosized aluminic particles after i.m. injection in the mouse, Nanotoxicology Meeting, Edinburgh, UK, 2010.

Gherardi, R.K., Khan Z Tillement O, Cadusseau J. Nanohybrid particle distribution after i.m. injection : relevance to vaccine safety. Nanohybrids Congress. Porquerolle, France, may 2010.

Khan Z., Tillement O, Cadusseau J, Gherardi R.K Nanohybrid particle distribution after i.m. injection : relevance to vaccine safety. First meeting of the DIM SEnT, Versailles, France, 2010.

Khan Z, FJ Authier, Cadusseau J Gherardi RK. Full session on Biopersistence, Biodisctribution and Biotoxicity of nanoparticles injected into muscle. NanoBioMed meeting, Creteil, France, June 2010

Gherardi, R.K., Khan Z, Itier V, Authier FJ, Tillement O, Cadusseau J. Biodistribution des particules injectées dans le muscle : neurodélivrance accrue chez la mdx et rôle de la chimiokine MCP-1. Societé Fraçaise de Myologie, Nice, France, november 2010