12ème Symposium International sur le VIH
et les maladies infectieuses émergentes
Toulon juin 2002

1.c. AIDS VACCINE: the scorpion venom model.

TRAN M.K.G.

1.c. AIDS VACCINE : THE SCORPION VENOM MODEL.
THE ENVELOPE GP41 PROTECTIVE SEQUENCE ELDKWA
IS A SCORPION VENOM LOOP ACTIVE SITE.

EXTENDED VERSION /VERSION LONGUE

TRAN M.K.G.*¹, KIRKIACHARIAN S.¹, MAURISSON G.²*, CAPRANI A.*

* Association Positifs, BP 230, 75865 Paris 18, France. E-mail : caprani@ccr.jussieu.fr, positifs@positifs.org.
¹ University Paris-Sud, Faculty of Pharmacy, Therapeutic Chemistry, 92290 Chatenay Malabry, France.
Correspondence : 31, Av du Bois 92290 Chatenay Malabry, France. E-mail : mkg_tran@yahoo.fr.
² Centre Médical Europe, 44, rue d'Amsterdam, 75009 Paris, France.

HIV-1 VACCINE CHALLENGE.

HIV-1 vaccine remains a very difficult challenge (review in : Letvin N.L., 2002), owing to the fantastic variability of the virus. A single patient contains a flurry of pseudotypes in his blood, and the number of different strains can reach the hudge number of a million of different sequences. Such a challenge is impossible to reach, if pharmaceutical companies intend to design 1 million of vaccines for only one illness. We focused primarily on this major difficulty, namely to look for conserved or highly conserved sequences in HIV-1 genome, with the hope that such an approach would limit the infectivity of HIV-1. The second help is provided by naturally observed resistance to HIV-1, whether with exposed uninfected seronegative prostitutes or long-term non progressors. HIV-1 can escape the immune response of the patient by many of the following mechanisms:

The absence of anti-CD4 binding site (Cordonnier A., 1989) antibodies, or their presence at a later stage (too late to avoid intra-cellular infection) at too low levels or affinity [the epitope looks like a " canyon " (Wang J.H., 1990), similar to rhinovirus receptor site, instead of a protruding spike easily recognized by the immune system ; only camel antibodies recognized " canyon " epitopes (Desmyter A., 1996), whereas human antibodies could not].
The absence of neutralizing antibodies against the V3 loop of the actual infectious, already an escape mutant virus (Schreiber M., 1997) (instead, the anti-V3 loop antibodies are directed to the preceeding strain, so the immune system is always one war late).
The enhancing properties of facilitating anti-V3 loop (Levy J.A., 2000) and anti-gp41 (Robinson W.E. Jr, 1991) antibodies render deleterious any approach using these epitopes, whether or not included in an HIV-1 larger complete protein.

For cytotoxic T lymphocytes (CTLs), the down-regulation of major histocompatibility (MHC) class I (except HLA-C ; the presence of HLA-C allows HIV-1 to escape also to the natural killer NK cells) from the cell membrane by HIV-1 Nef (Piguet V., 1999) renders the objective of CTL efficiency dramatically problematic. If Nef is continuously active [Nef is by far the most abundant transcript in infected cells (H9 cells, lymphocytes, macrophages) making 80% amount of mRNA, versus 18% for Rev and only 2% for Tat (Smythe J.A., 1992)], all efforts to obtain CTL and NK killer cell responses are condemned per advance to fail in pure perte. Thus an absolute requirement is to simultaneously neutralize Nef to avoid HLA down-regulation and CTL inefficiency.

The preliminary hopes are the sommation of a few epitopes in a coalition of, for example, 5 or 6 or more if possible short sequences, all of them being highly conserved, to avoid HIV-1 mutation escape, and create a simultaneous attack at many fronts against the virus. If an additive effect is obtained (i.e. ~ 5%+30%+20%+15+É ~ optimistically 70%), the result will attain a level high enough to be considered satisfying (~70% protection), although not able to cure completely the patient (sterilizing vaccine). The advantage of small (~6 residues long) selected epitopes, instead of a big (~hundreds of residues) protein, is the absence of nocivity (no enhancement by facilitating antibodies).

However, the haptenic character of these epitopes makes them non immunogenic, if not covalently linked to a carrier. An interesting possibility is to vaccinate with epitopes associated to a chaperone, like the proteins of the heat shock protein Hsp family, as vehicle for interaction with the antigen presenting cells (dendritic cells): This very old fundamental work (Srivastava P., 1986) finally succeeded remarkably in a recent clinical approach by inducing in 3 patients the regression of liver and spleen melanoma metastatis (Srivastava P., 2002a). In viral infections, this strategy was tried for simian virus SV 40 (found in mesothelioma), herpes virus, hepatitis B virus, lymphocytic choriomeningitis virus, influenza virus, vesicular stomatitis virus (review in : Srivastava P., 2002b).

Our objective is to determine precisely what is the functional role of each sequence (for example, gp41 is an interferon chimera) (Tran M.K.G., 1999), and its implication in the pathology observed (Gag p24 and aphthous ulcer) (Tran M.K.G., 2002). The following sequences are selected for a preliminary partial vaccine :

Sequence 1: Lopalco's gp41 sequence, in exposed uninfected seronegative prostitutes (mucosal IgA) (Pastori C., 2000 ; Mazzoli. S., 1999), that we demonstrated to be an interferon chimera (Tran M.K.G., 1999).
Sequence 2: Lopalco's cellular CCR5 epitope, that we found mimetic of a viral post-V3 loop (Tran M.K.G., 2001)
Sequence 3: Harrer's sequences, including gag p24 in a long-term non progressor (CTL) (Wagner R., 1999).
Sequences 4 : Rowland-Jones' sequences, in exposed uninfected prostitutes (CTL).
Sequence 5 : Chermann's beta 2-microglobulin-like (modified) sequence, in non-progressors (IgG).
Sequence 6 : We focused here particularly on the Belec's sequence,. in exposed unifected prostitutes (mucosal IgA).

BACKGROUND OF STUDY.

One of the possible solutions is to use an HIV1 highly conserved amino acid sequence, for instance ELDKWA , in envelope (env) gp41 , which induced mucosal protective IgA antibodies in 5% exposed nevertheless uninfected African prostitutes (BELEC L, 2000). This sequence is the target of the neutralizing antibody 2F5 (MUSTER T, 1993 ; XIAO Y, 2000) and is also contained in the peptide Trimeris T-20 which blocked cell fusion, virus entry and reduced the viral load in patients in a clinical trial (KILBY JM, 1998).

OBJECTIVE.

To understand the mechanism of action of ELDKWA.

DESIGN.

Amino acid sequence and tri-dimensional (3D) structure comparison.

RESULTS.

Linear comparison was unsuccessful, pointing to the weakness of computer program algorithms. But 3D structure analysis revealed a striking homology with the active site tryptophan (W38) containing loop of the North African scorpion venom Androctonus Australis Hector (AaH II), namely the most powerful scorpion in the world. Every amino acid could be superimposed on the AaH II venom 3D structure. The matches were (QWASL is the chimpanzee SIV gp41°) (in bold, active site):

CONCLUSION.

HIV1 envelope gp41 conserved sequence ELDKWA mimicks the most dangerous scorpion venom known to date (AaH II) in its tryptophan W38 containing active site, and this almost perfect molecular homology is only tridimensional, not linear. The scorpion venom model of AIDS is confirmed and reinforced further, not only for AIDS vaccine (inclusion of this IgA inducing epitope) but also for treatment (Trimeris T-20, and Na+ voltage-dependant channel modifiers like Tacrine).

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