TOULON Conference 2006
Poster PP 6.4
Chikungunya arthritis sequelae : Molecular homology between Lyme arthritis Borrelia Burgdorferi, Lymphocyte Functional Antigen-1 alpha (LFA-1 α) and Chikungunya virus gp1.

Tran M.K.G. 1*, Caprani A. 2*

1 University Paris V, corresp : 31 Avenue du Bois, Chatenay Malabry, France.
E-mail : mkg_tran@yahoo.fr
2 University Paris VII, France.
* Association Positifs, Paris, France.

INTRODUCTION

Chikungunya virus (“Chik”) is an arthropod-borne virus spread by the mosquito Aedes Albopictus. Chikungunya virus infection in the French island La Réunion (population : 770,000 inhabitants) was an epidemics of catastrophic amplitude : About (or probably more than) 244,000 patients were infected, with 203 deaths (mostly among elderly patients, but also young infants). Heavy after effects such as depression, invalidating asthenia impeding professional work and chronic painful arthralgias/arthritis (wrists, hands, phalanges, shoulders, ankles, knees) many months after are the main clinical sequellae, hence the Swahili name Chikungunya : “that which bends up”(because of arthralgias). This clinical picture mimics Ross River virus “epidemic polyarthritis”, which is an important public health problem in Australia (Harley D., 2001). Chik disease can be transmitted from pregnant mother to infant (Robillard P.-Y., 2006) : 10/84 children suffered of edema, thrombocytopenia, encephalitis, convulsions and intra-vascular disseminated coagulopathy. Severe myocarditis (Maiti C.R., 1978) and fulminant hepatitis (Schuffenecker I., 2006) has also been described.

OBJECTIVE

Until now no molecular biology research has been done on this virus, so we tried to discover why rheumatologic features were dominating the clinical picture of the chronic form in a subset of patients. Secondly, we extended our analysis to the mechanism of thrombocytopenia, because we have a good experience of this topic with HIV-1 and parvovirus B19 (Tran M.K.G., 2002a). Thirdly, as 123 cases developed meningoencephalitis and fulminant hepatitis (Schuffenecker I., 2006), we investigated also this problem by comparing Chik to Amanita Phalloides phallotoxin, a mushroom hepatotoxin inducing fulminant hepatitis which can be alleviated by the drug Silymarin (Legalon®) from Silybum Marianum (Tran M.K.G., 2002b).

METHOD

We reasoned by analogy with a well known disease, where arthritis followed infection : Lyme disease, caused by a spirochete (Borrelia Burgdorferi) and with a known rheumatologic susceptibility marker [the same as for rheumatoid arthritis (RA)] : The occurrence of "treatment-resistant" Lyme arthritis correlated with an increased frequency of rheumatoid arthritis-associated HLA-DR4 (= HLA-DRB1*0401) (Steere A. C., 2003).

Lyme disease
Lyme disease is a multisystem illness with dermatologic, neurologic, cardiac, and arthritic manifestations. Months after disease onset about 60% of untreated patients experience intermittent episodes of oligoarticular arthritis, or chronic synovitis, particularly of the knee. Most patients with Lyme arthritis respond favorably to antibiotherapy, but 10% have continuous joint inflammation for several months to years after the apparent eradication of the spirochete from the joint with antibiotic treatment. Such disease persisting for 12 months or longer was termed "treatment-resistant chronic Lyme arthritis." The synovial lesion in chronic Lyme arthritis is similar to that of other forms of chronic inflammatory arthritis, including rheumatoid arthritis . Hence, Lyme arthritis is an important model for understanding the basic mechanism(s) underlying the development of chronic inflammatory arthritis and autoimmune diseases.
In this case, there exists a molecular mimicry between leukocyte function
associated antigen-1 integrin LFA-1 α and Borrelia Burgdorferii Outer surface protein (OspA) (Trollmo C., 2001). We decided to screen all the alphavirus inducing arthralgias/arthritis with LFA-1 α. The amino acid sequences were aligned to find perfect matches (molecular mimicry or homologies). Were included also all the arthritis-inducing viruses [Hepatitis C Virus (HCV), HTLV-1 tax, Parvovirus B19, Visna (arthritogenic EV1) pol (sequence LEYKEGTR)] and bacteria Erysipelothrix Rhusiopathiae (RsA) of swine rheumatoid arthritis (sequence KKWVGG) (for the 2 last, to avoid extreme complexicity, results – positive- were not shown).

RESULTS

1°) IN RHEUMATOLOGY : They were exactly as expected. Borrelia Burgdorferi OspA sequence SYVLEGT was identical to Chikungunya virus gp1 SY-LEGT excepted one gap. OspA has a second cross-reactive epitope 214-AAWNSGT with LFA-1α (Kamradt T., 1996). This nucleus alignment allows to align correctly all the arthritis-inducing arbovirus family (Chickungunya, Ross River, Sindbis, Barmah Forest, Mayaro, O Nyong Nyong) (McGill PE, 1995) with LFA-1 α :

EBV EA-D (BMRF1) TYTSE GAcLTLD
HTLV-1b (Ratner L, 1985) Tax VYVFDG cVQGD LGSQWAT
HCV E1 MYVLDG c VG NWA
Parvovirus B19 YVLGQG TETNWAG
Borrelia Burgdorferii OspA SYVLEGT 220-TGSNWAA -214
Chikungunya virus gp1 1532SY- LEGT- R1538 TNWA
LFA-1 α human KIYVIEGTSKQ VGAKDWAG
LFA-1 α mouse R
Ross River virus HSY -LEGT-R TNWA
Sindbis virus Y V EGT-RD TNWA
Barmah Forest virus SY- LEGT-K TNWA
Mayaro virus SY- MEGT-K TNWA
O nyong nyong virus LYALEG SIE STNWA

Letters in Italics and underlined are read in reverse sense.

A nice confirmation of the homology significance is the finding of Parvovirus B19 capside in the list. Parvovirus B19 may be confounded clinically with rheumatoid arthritis or lupus : Parvovirus B19 arthritis often meets clinical diagnostic criteria for RA, can be erosive, is not infrequently followed by development of rheumatoid factor, and B19 DNA may be detected in synovial fluid, cells, and tissue of affected joints. The RA associated HLA-DR4 antigen was present in 12 of 18 patients with B19 associated arthropathy in one study (Klouda P.T., 1986) and was associated with prolonged arthritis after B19 infection (more than one year) in another (Kerr J.R., 2000).
The 3 viruses [HTLV-1 tax (Lelièvre J.D., 1996), Hepatitis C virus HCV E1 (NP_056766), Epstein-Barr virus EBV] found in Sjögren’s disease (Mariette X., 1999) can be aligned. They differed slightly by the presence of a cysteine C.

2°) CHIK AUTO-IMMUNE THROMBOCYTOPENIA : A molecular homology exists between platelet gpIb α (P07359), integrin gpIIb IIIa (the 2 targets of idiopathic thrombocytopenic purpura) (He R., 1995) and Chik :

platelet gpIb α 351-EQTTFPP-357
Chik NE -TFPI
Chik SETFPIT
platelet integrin gpIIb IIIa SIEFPVS
Dengue virus type 2 ns2b SGLFPVS
HIV-1 protease clivage site TLNFPVS
Parvovirus B19 TFDFPGI

All thrombopenia inducing viruses (Dengue virus-2, HIV-1 and Parvovirus B19) could be aligned by centering on the FP prominent dipeptide. Proline is a strong immunogenic amino acid, because it points in the space.

3°) CHIK FULMINANT HEPATITIS : Molecular homology centered on the “WSP hepatocyte motif” with thrombospondin, Plasmodium Falciparum TRAP and Circumsporozoite CS, complement properdin and phallotoxin of Amanita phalloides (causing a fulminant hepatitis) (Tran M.K.G., 2002) :

“ Hepatocyte motif ” WSP
Complement properdine WSPCS
Plasmodium Falciparum TRAP & CS TEWSPCS
Chik SDWSTCS
Thrombospondin 386-SEWTSCS-392
Phallotoxin (covalent bridge between W and C) WA*PCT

Phallotoxine Alanine A is hydroxylated by proximity (OH-Proline OH-P or *P) and becomes equivalent to Serine S.
(Hence the interest of Silymarin (Legalon® per os) (Madaus Laboratory documentation), an emergency high doses intraveinous treatment of Amanites Phalloides intoxication, as an hepatoprotective non toxic, well tolerated treatment of Chik hepatitis).

CONCLUSION

By analogy with Lyme arthritis, Chikungunya virus arthritis sequellae occur by a mechanism of molecular mimicry between LFA-1 α and a viral protein, here Chikungunya virus gp1, hence inducing a deleterious auto-immune immune response. In Lyme arthritis, Parvovirus B19 arthritis and Sjögren’s syndrome, such event occurs only in susceptible patients, those having the HLA-DR4 rheumatologic marker of rheumatoid arthritis. Thus, it could be interesting to search HLA-DR4 in Chikungunya infected patients to predict if they will suffer of future arthralgia sequellae.
For Chik, no official clinical specific anti-viral treatment is known, except those discovered in cell cultures [interferon-alpha, ribavirine (Briolant S., 2004) glycyrrhizine (Crance J.M., 2003)]. In 2006, chloroquine (Nivaquine®) was found to inhibit the virus in vitro (de Lamballerie X., unpublished), confirming an old – forgotten - clinical trial with chloroquine phosphate in 1984 (Brighton S.W.), which was successful in 5/10 patients (more : 7/10, if the criteria were those of the patients themselves). Billi M. (2006) advocated a miraculous effect of (unconventional) magnesium chloride (or Nigari, a Japanese algal extract containing 87% magnesium) : This may not be coincidental, because interestingly LFA-1α binds Mg++ : The solution structure of the Mg++ complex of the I-domain of LFA-1 α has been determined by nuclear magnetic resonance (Legge G.B., 2000).

The second lesson is to treat very precociously the patient (like in Lyme disease) with available drugs whether conventional (pegylated interferon α, ribavirine excepted in pregnancy, chloroquine) or unconventional (glycyrrhizine, magnesium chloride, Nigari), before the irreversible phase II, when the arthralgia sequellae are perhaps definitively installed (however, a late treatment can be tried).

Concerning Chik auto-immune thrombocytopenia, corticosteroids are classical ; or as in Parvovirus B19 thrombocytopenia, polyvalent Immunoglobulins. Other measures may be chelation with high affinity column chromatography loaded with platelet gpIb and gp IIIa IIb, treatment with a haptenic form of Chik (epitope-based lead sequence SYLEGTR). This Chik epitope can serve as a protective hapten in a therapeutic purpose.
An anti-Chik vaccine was started in 1980 by the U.S. Army, but stopped. A vaccine developed for Lyme arthritis has been designed which mutated the auto-immune epitope while conserving the protective sequence (Willett T.A., 2004) : This could be also a good model for designing a vaccine against Chikungunya arthritis.
LFA-1 (CD18/CD11a) is the most important beta2 integrin complex for monocyte recruitment, neutrophil activity, and cytotoxic T lymphocyte function. LFA-1 antagonists [statin precursor Mevinolin (Novartis), anti-LFA-1 hydantoïn (Kerry T., 2003) (Boehringer), humanized anti-LFA-1 monoclonal antibodies (Efalizumab) in organ transplantation (Nicolls M.R., 2006)] may be screened as possible useful anti-rheumatic or anti-Chik agents.

For Chik hepatitis, the molecular homology pointed to the interest of Silymarin (Legalon® per os) (documentation of Madaus Laboratory), an emergency intraveinous treatment of Amanites Phalloides intoxication, as a hepatoprotective, non toxic, well tolerated treatment of Chik hepatitis). A more available form Silibinin is under trial in Hepatitis C since 5 years (Poynard T., 2006, trial not yet ended).

Discoveries in Chikungunya virus research represent also progress in comprehension, management and treatment of other rheumatologic alphaviruses (such as Ross River virus in Australia), of Lyme arthritis, Sjögren’s syndrome or Parvovirus B19 polyarthritis ; or for hepatitis from West Nile virus in U.S.A.

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