Highly pathogenic Influenza virus A H5N1 Indonesia & Vietnam Hemagglutinin (HA1) contains an urokinase-plasminogen activator explaining the fibrinolysis

Présentation Poster au 8TH EUROPEAN MEETING ON VIRAL ZOONOSES( Saint Raphaël, France, Octobre 2017)

Guy Mong Ky Tran(1) & Adrien Caprani(2)

(1) Retired since 2014 from University of Auvergne Rhone-Alpes, Health Regional Agency, Public Health Department, Hospital Hotel-Dieu, Clermont-Ferrand ; 31 Av du Bois - Chatenay Malabry, France. email: Cette adresse email est protégée contre les robots des spammeurs, vous devez activer Javascript pour la voir.

(2) Association "Positifs", 147 chemin de la futaie, 83550 Vidauban, email: Cette adresse email est protégée contre les robots des spammeurs, vous devez activer Javascript pour la voir.

Avian Influenza is compared to a chicken lethal Ebola for its internal hemorrhagic symptoms. Oseltamivir is inefficient and not recommanded in hemorragic influenza. In 1996, Zhilinskaia IN (Vopr Virusol) found by computer analysis a similarity between Influenza virus hemagglutinin HA and plasminogen activator (PA), explaining the fibrinolysis. We analysed further these results in Influenza virus A H5N1 Indonesia (83% mortality) and Vietnam HA1. We compared to urokinase (U-PA), tissue PA, TSV-PA, Batroxobin. Results : All the 3 active site residues of serine protease (His, Asp, Ser) were found and the best match was with U-PA :

H5N1 Indonesia/Vietnam             IPKSS-W--S-SH

U-PA/t-PA active site His               ILISScWviSaTH

H5N1 Indonesia/Vietnam                  G I HHpNDAAEQTK

U-PA/t-PA active site Asp      (A,L)HH NDI ALQ IR

H5N1 Vietnam                                KGDS- - TIMKS(L,E)EYGNCNTKCQTPMGA I

U-PA/t-PA active site Ser               QGDSggPLVcS L Q    WIRSHTKGEE- NG(A,L)

H5N1 Indonesia/Vietnam 141-YLGKSSF(R,F)N-150

U-PA                                                       YLGR-S(L,R)L N

Conversely, Influenza H7 China HA1 has His replaced by Arg, Asp replaced by Ser. The motif His-Phe (HF), in the pocket binding Plasminogen clivage site Arg-Ala-Arg, was present in Influenza H5N1 and U-PA (319-HF-320); but not in Influenza virus H7 China, replaced by Gly-Gly (inactive). Conclusion : The   plasminogen activator in avian Influenza virus H5N1 hemagglutinin HA1 ( Zhilinskaia ) was confirmed and may explain fibrinolysis in hemorrhagic Influenza. It seems rationale to use plasminogen inhibitors to block this fibrinolysis. A peptidomimetic drug  of the plasminogen Arg-Ala-Arg (RAR) at the cleavage site  Arg / Ala can  be designed by docking. This strategy was successful in HIV-1 tritherapy, with FP peptidomimetics.

Highly pathogenic Influenza virus A H5N1 Vietnam Hemagglutinin (HA2) contains a scorpion alpha-toxin. Sodium channel inhibitors as therapy.

Présentation Poster au 8TH EUROPEAN MEETING ON VIRAL ZOONOSES( Saint Raphaël, France, Octobre 2017)

Guy Mong Ky Tran(1) & Adrien Caprani(2)

(1) Retired since 2014 from University of Auvergne Rhone-Alpes, Health Regional Agency, Public Health Department, Hospital Hotel-Dieu, Clermont-Ferrand ; 31 Av du Bois - Chatenay Malabry, France. email: Cette adresse email est protégée contre les robots des spammeurs, vous devez activer Javascript pour la voir.

(2) Association "Positifs", 147 chemin de la futaie, 83550 Vidauban, email: Cette adresse email est protégée contre les robots des spammeurs, vous devez activer Javascript pour la voir.

During the Influenza H1N1 pandemic, we found in Influenza virus A H1N1 Japan Hemagglutinin (HA1) 246-YFWKLV-251 the active site loop 39-YFWKLA-44 of the scorpion toxin AaHIT4 (Tran GMK, ISHEID Conf, Toulon, France, 2010: 0291 . www.Positifs.org), with the surprising discovery of a RETRO-INVERSO lecture (from COOH- to NH2-terminus) of Influenza virus HA2 466-VKEYL-462 (= HA2 122-118) matching with the crucial NH2-terminus active site of scorpion 1-VKEgYL-6 which induces broadly cross-reactive neutralising antibodies (Devaux C, 1996). We developped further this short retro-inverso lecture and found a complete scorpion alpha-toxin in the highly pathogenic Influenza virus H5N1 Vietnam 1203/2004 HA2 : Influenzavirus(retro-inverso)126-LQLRVKD-Y-LNKVNSD- -HFD-LT  R- ENEMLV-100

Scorpion  Bot9/AaH2/CsE1   -4 -AEIKVKDgYIVNKVNSDgcKYDcL(L,K)gENEFCL- 26

Influenza virus A H5N1                                78-ENLNK K M ED-GFLDV W TY- NA-96

Scorpion  Bot3/AaHP985                               24-EECNK(K,L)gDsGYCD(I,W)TYgDA-44

Influenza virus A                  45-IDgVPNKVNSI-55                     62-QFE(V,A)GR-EF-70

Scorpion  Bot2/AaH2/LqqV  49-ID-LPDKVRTI-58   Bot2/BotXI  58-RI EV AGRcHF-65

All the scorpion active site residues K2,     Y5,     V10,   Y14,  W38 & G61-R62 (AaH2

numbering) matched with Influenza K121, Y119, V115, F110, W92 & G67-R68.

This was very surprising, because Influenza HA2 (45-126) has no cysteine. Many scorpion cysteines (C16, C26, C36) were matched with Influenza Leucines (L108, L80, L89) or gapped (C12, C63). In conclusion, avian Influenza virus A H5N1 HA2 contains a complete scorpion alpha-toxin, but devoid of any cysteine core structure; this means that its receptor is a sodium Na+ voltage-gated channel and consequently Influenza virus can be inhibited by Na+ channel modifiers (vitamin B1, vegetal fatty acid omega-3, antiarythmics, local anaesthetics (procaïne), eugenol, antimalarials (quinine), antiepileptics). Conversely, fatty acid omega-6 are deleterious. For Influenza vaccine, the epitope mimicking the scorpion NH2-terminus seems crucial. This data points to the importance of RETRO-INVERSO lecture (for instance, the RGD adhesion motif) in deciphering protein function. Three homologies of H5N1 with platelet Integrin ITGB3, disintegrin ITGA2b and plasminogen activator explain the hemorrhagic character of avian Influenza.

Avian Influenza virus A H5N1 polymerases PA, PB1 and hemagglutinin contain a platelet integrin ITGB3 inducing auto-immune anti-platelet antibodies and thrombocytopenia.

Présentation Poster au 8TH EUROPEAN MEETING ON VIRAL ZOONOSES( Saint Raphaël, France, Octobre 2017)

Guy Mong Ky Tran(1) & Adrien Caprani(2)

(1) Retired since 2014 from University of Auvergne Rhone-Alpes, Health Regional Agency, Public Health Department, Hospital Hotel-Dieu, Clermont-Ferrand ; 31 Av du Bois - Chatenay Malabry, France. email: Cette adresse email est protégée contre les robots des spammeurs, vous devez activer Javascript pour la voir.

(2) Association "Positifs", 147 chemin de la futaie, 83550 Vidauban, email: Cette adresse email est protégée contre les robots des spammeurs, vous devez activer Javascript pour la voir.

Among the prognostic factors in complicated lethal Influenza A H5N1 Vietnam, a low platelet count in patients is highly correlated to mortality ; oseltamivir is completely inefficient and not recommanded in these cases. This thrombocytopenia was investigated by analysing amino acid sequences of platelet glycoprotein gpIIb/IIIa (integrin ITGA2b/ITGB3) at the epitope ITGB3 49-CAPESIEFPVSEARVLED-66 of Nardi MA (PNAS, 1997), which induced a  thrombocytopenia in mice by inducing high affinity auto-antibodies. Many pathogens mimic this Phenylalanine-Proline FP dipeptide (Tran GMK, ISHEID Conf 2010), most of them being hemorragic fevers (for instance, Ebola, hemorragic dengue fever type 2, leptospirosis Adaman hemorragic fever, etc ). Avian Influenza H5N1 was compared to a chicken Ebola ; Ebola (Sudan) L polymerase was 747-SVFPL(S,E)S-754. We found this crucial auto-immune epitope in Influenza A H5N1 Duck/Guandong/07/2000 RNA-dependent polymerase subunit PA 522-SMEFPLTDPRLE-533. Other Influenza  were H5N1 Goose/Guandong/1/96(Gs/Gd),A/India /Pun/2015(H1N1),A /canine/NY /dog /2008(H3N8). The longest alignment was between a chimera (Goose /Guandong with other Influenza) and ITGB3 :

ITGB3                                                           CDLKENLLKDNCAPESIEFPVSEARVLE

Influenza                                                     CQLKWALGENMAPESMEFPLTDPR-LE

There is an alignment between gallus ITGB3 and Influenza H5N1 Hemagglutinin :

Gallus ITGB3                      DF(E,I)FPRSS

Influenza H5N1 HA                   128-EKIQIFPRSS-137

Vietnam Homo/2005              EKIQIIPKSS

Polymerase PB1 (Kawaoka Y, 1989) of A/Mallard/Shangai/2013 (H5N8) has SMELP(F,S). Conclusion : Influenza virus A H5N1 contains a platelet integrin ITGB 3 with a central motif FP which induces high affinity auto-antibodies against platelets and destroying them, at 3 places (polymerases PA, PB1 and HA). As anti-proteases (Saquinavir, Ritonavir, etc) used in HIV-1 infection mimick this  FP (because FP is present in HIV-1 GAG, cleaved by the protease), they may constitute haptens blocking  deleterious anti-platelet auto-antibodies. We suggest that antiproteases were used in hemorragic influenza,  hemorragic Dengue fever 2, Ebola, Hantaan, Leptospirosa, etc. This dangerous auto-immune epitope must be deleted from Influenza A  vaccine, as well as Dengue 2 vaccine, to avoid enhancing hemorragic auto-antibodies.

The scorpion toxin model of AIDS : The concept of voltage-dependent sodium channel as a receptor of HIV-1.

Présentation orale au 19° congrès mondial de la Société internationale de Toxinologie de Haikou(Chine)

Ce concept de vaccin reprend le brevet de Biosantech –E.Loret  sur la protéine Tat mutée (http://www.positifs.org/fr/publications/our-publications/242-c104-bis-hiv-therapeutic-vaccine-biosantech-erwann-loret-unjustifiable-attacks.html) pour lequel il explique le mécanisme d’action et identifie dans  la glycoproteine gp41 un site conservé pour toutes les souches de VIH. Un nouveau vaccin comportant la protéine Tat mutée du brevet Biosantech  et  le site  conservé  de la gp41 qui est une toxine de scorpion apportera une vraie réponse prophylactique et thérapeutique.

This vaccine concept is based on Biosantech-E.Loret's patent on mutated Tat protein (http://www.positifs.org/fr/publications/our-publications/242-c104-bis-hiv-therapeutic-vaccine-biosantech-erwann-loret-unjustifiable-attacks.html) for which it explains the mechanism of action and identifies in glycoprotein gp41 a site preserved for all strains of HIV. A new vaccine comprising the Biosantech patent mutant Tat protein and the conserved site of gp41 which is a scorpion toxin will provide a true prophylactic and therapeutic response.

Tat Oyi vaccine cysteine-rich domain: Molecular homology with Conus Consors toxin
CcTx and scorpion Leiurus Quinquestriatus Hebraeus alpha-like toxin Lqh3.
Tat Oyi vaccine, based on spontaneous retro-seroconversion of 23/25 Gaboneses,
obtained significant results: Retro-seroconversion in a macaque and 2 beginnings of retroseroconversion
in 2 patients and 10 indetectable DNA patients after 3 years. Tat Oyi has 2
crucial mutations: C22S and F98E (which creates a 97-PE-98 motif with a cis-bond Pro-Glu).
Protective IgM of innate immunity, possibly explaining natural chimpanzee AIDS
resistance, are directed against 2 epitopes (Rodman TC 1992, 1993, 2001 ; Nicoli F, 2016):
the basic region and the cysteine-rich domain, also targeted by IgG. Protection of Thaï AIDS
vaccine was conferred by a conotoxine-like HIV-1 V2 loop (Tran GMK, 2013). We searched
cysteine-rich toxins (conus, scorpion) binding to voltage-gated sodium (Nav) channel in Tat
by amino acid (AA) sequences and tridimensional (3D) structures comparison.
Results : 1°) Tat Oyi (9-42) is homologous to Conus consors CcTx
1-APWLVPSQIPTTCCGYNPGTMCPSCMCTNTC-32.
2°) Tat Oyi (39-44) is aligned with Lqh3 37-CGFKVGHGLA-46, Tat (35-QCHL/F-32)
with Lqh3/Bom4/Bot1 63-EKCHF-67 ; Tat Oyi 29-RKCYCNNS-22 with Lqh3/Bom4 14-
HHCFPNSYC-22; Tat Oyi 96-DPED-99 with 8-QPEN-11 (OcyTx1 11-DPED-14).
All the Lqh 3 active site AA (P9, E10, H15, F17, F39, L45, H66)
are found in Tat Oyi (P97, E98, K28, L32, F38, L43, H13).
Tat Oyi residues K28, L32, F38, L43, H13 are almost 100% conserved in all Tat. (H13
replaced H 33 in 3D structure).
3°) TAT group O chimera (mainly O.CM.96.CMA102) has the COOH-terminus
YHCSKDSCNCCTRISGQ*YC matching with
AaH2/Boma6d/Bot2 14-YHCGKNSYCNEECTKLGESGYC-35.
Conclusions : Tat Oyi vaccine mechanism of action is a molecular homology with
ligands of Nav channel (Conus CcTx and scorpion Lqh3); five Tat conserved AAs in all HIV-
1 strains are implicated in Tat Oyi protection, by a partial cross-reactivity. This therapeutic
vaccine is promising for a rapid development of prophylactic vaccine, as in rabies vaccine.

Envelope gp41 contains toxins of Androctonus Crassicauda : A cross-reactive vaccine
There is a scorpion toxin, mainly Androctonus Crassicauda (A.Cra, AC), in HIV-1 envelope
gp41. We found in 2002 (13th ISHEID Conf, Toulon, France) scorpion toxin active sites W38,
R2 an Y5: HIV-1/2 /SIV gp41 chimera 662-A/E F/L N/D Q/KWASLW/FGN-672
α-toxinAc2/AaH2/Amm5, 8/Bot3/Lqh2 /Lqq5 A/E46,F/L15,N/D44, 37-QWASPYGN-44
HIV-1 gp41 MSD (Membrane Spanning Domain)…VNR 706-VRQGYSPLSFQT-719
Scorpion α-toxinAc2/Bot9/Css2/Cn2,3 signal peptide…VES 1-VRDGY-PVSLNT-10
The hydrophobic gp41 MSD corresponds exactly to the scorpion toxin signal peptide.
We completed here with α-toxin Y14, the α-helix 21-29, GXC motif and COOH-terminus :
HIV-1 B.US.WEAU160 gp41 791-EALKY-CWNLLQYWSQELKN-809
α-toxinAc4/A.Cra3/CsEV3/Ts5/AahIT1 10-DGCKYgCWNLLEYCTNECKD-29
HIV-1/HIV-2 824-EWTDRvCEIVQGACR-838
Scorpion Lqq5 28-ECTEK-CgELNGYCQ-42
HIV-1 gp41 (in retro-inverso : from 737 to 729) 737-TGEPRDPGR-729
BmKM8 /α-toxinAc1, 4 COOH-terminus 54-S(R,E)IKDPGK-62
Gp41 COOH-terminal tail is exposed to the surface by carbohydrates (750-NGS-753, 816-
NAT-818) and the Kennedy epitope 728-745.
Gp41 SWSNKS « 3S » is a potential therapeutic AIDS vaccine (Vieillard V, 2008):
Scorpion chimera COOH-terminus CKLACY-SVP-WNPTWS- RSNTCGKK
HIV-1/CPZ.cd90.ant gp41 ectodomain 598-C-KLVCYtSVPdWVPSWSNKSQTCAKN-624
It could be interesting to vaccinate with the 5 toxins (α-toxin Ac1, 2, 3, 4 /A.Cra3) of
Androctonus Crassicauda venom, as it is highly cross-reactive with Old World scorpions, (as
α-toxin Ac2 contains all the active site R/K2, Y5, Y14, W38, P41, K58, R/K 62);
corresponding HIV-1 gp41 R/K707, Y710, Y795, W666, L/i669 R733, R729 are
conserved; gp120 V3 loop R/H322, R/Q326 are only highly conserved. HIV-1 gp41 662-
ELDQ/KWASL-669 (entry fusion inhibitor T-20, enfurvitide), the target of 2F5 antibody,
neutralizes primary isolates of African, Asian, American and European HIV-1 strains (L663,
W666, L669 are conserved). In HIV-1 patients, the level of this antibody is very low. In HIV-
1 gp41, α-toxin Ac2 is incomplete, as its COOH-terminus
48-CYKLPDHVRTKGPGR-62 is lacking and mimics HIV-1 B.US.RF gp120 V3
loop 309-CTRPN∗RKSITKGPGR-326 (∗ is glycosylated NNT) (Tran GMK, 1989).
The link between TAT and gp41, 2 scorpion toxins, is Luman, a transcription factor :
gp41 inhibits Luman (an inhibitor of TAT) ; Luman induces human herpes virus reactivation.
There are Androctonus toxins in Nef :
HIV-1 Nef chimera 120-YIPDWQNYTKGPGvR-134 137-YFGFCY-WKLV-146
A.Crassicauda α-TxAc2 49-YKLPDHVRTKGPGR-62, AaHIT4 34-YYGYCYfWKLA-44
α-defensin 1-4, a major natural immunity HIV-1 inhibitor in long term non progressors
(Zhang L, 2003; Wu Z, 2005), is a scorpion venom family member and aligned with Nef
(Tran GMK, 2003).
HIV-1 protease has a Buthus/A.Cra toxin (phylogentically, spider venom contains
protease for preys digestion): BomPI, Bot2 56-VPIRIEGK-62, HIV-1 protease 11-
VPIRIEGR-18, α-toxin Ac1 56-VPIK(P,D)SQ-63.
Until now no research was done on mutations of the Na+ voltage-gated channel in
HIV-1 resistent patients : This could be helpful for HIV-1 cure, as observed in « the patient of
Berlin » (by targeting the CCR5delta32 mutation).
Lethal Australian spider Atrax Robustus toxin active site matches with HIV-2
envelope gp120 V2 loop, in three-dimensional structure:
Spider key residues ( 4-KR-5, M18, K19, Y22, 23-AWY-25, Q27, C31) superimpose
on HIV-2 residues (180-KK-181,M172, K173, Y183,186-AWY-188, Q190, C194).
To summarize, HIV-1 vaccine with the 5 α-toxins of scorpion A. Crassicauda (AC) venom
has a broad spectrum and can protect against mainly gp41, and accessorily Nef, protease and
some envelope gp120 V3 loops. A therapeutic vaccine with scorpion AC 5 attenuated α-
toxins would evaluate this concept of molecular mimicry.

Ligands of Na+ voltage-gated channel are anti-HIV-1 drugs
In conclusion, HIV-1 contains many scorpion toxins, but devoid of any classical complete
cysteine core structure; this means that its receptor is a sodium voltage-dependent channel and
consequently HIV-1 can be inhibited by Na+ channel modifiers (vitamin B1, vegetal fatty
acid omega-3, antiarythmics, local anaesthetics (procaïne), eugenol, antimalarials (quinine,
chloroquine), antiepileptics, lithium, ginsenoside, tetrodotoxin. Tacrine (Fredj G, Maurisson
G) was the first prototype in AIDS treatment, but was limited by its hepatotoxicity : The
hydroxylated metabolite of Tacrine has the structure of grayanotoxin. Vegetal fatty acid
omega-3 has no toxicity. As Na+ channel does not mutate, no chemoresistence occur during
treatment : This is a crucial advantage over drugs directed against HIV-1 proteins (reverse
transcriptase, protease, integrase).