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

3.c. Thailand and Africa vaccines with HIV-1 gp41 LQAR (Leu Gln Ala Arg):
Thailand strains are the most homologous to interferon-beta 2 (IFN- b2).

TRAN M.K.G.

3.c. Thailand and Africa vaccines with HIV-1 gp41 LQAR (Leu Gln Ala Arg):
Thailand strains are the most homologous to interferon-beta 2 (IFN-b2).

EXTENDED VERSION /VERSION LONGUE

* 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 cellular CCR5 epitope, that we found mimetic of a viral post-V3 loop (Tran M.K.G., 2001)
Sequence 2: Belec's sequence,. in exposed unifected prostitutes (mucosal IgA)
Sequence 3: Harrer's sequences, including gag p24 in a long-term non progressor (CTL) (Wagner R., 1999).
Sequence 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 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).

INTRODUCTION.

Mutation escape is one of the most important problem to solve in the fight against AIDS. An unexplained mutation escape (A --> T 582) was described by Reitz M.S.Jr. (1988) in the HIV-1 HXB2 envelope transmembrane gp41 sequence 579 RILAVERY 586. We tried to elucidate the nature of this sequence RILAVERY and its biological importance (Tran M.K.G., 1999), in view of the existence of interferon (IFN) disturbances in AIDS (acid-labile IFN is a very precocious marker of AIDS progression) (Ruffault A., 1989 ; Voth R., 1989). Thus by protein amino acid sequences and tridimensional (3D) structure analysis, we compared HIV gp41 sequences and IFN sequences. This site is mimicking in 3D a new IFN, which is a chimera between IFN-alpha (IFN-a) and mouse IFN-beta (probably the mysterious "acid-labile" IFN, a precocious marker of AIDS progression).A and K are mutation escapes.

So a new HIV receptor may be a special IFN receptor subtype, common to both IFN-alpha and mouse IFN-beta. Antibodies and vaccine focused on this gp41 epitope seem to be important.

Independently from this work, Lopalco L (2000) found, in 50% exposed but uninfected African prostitutes, vaginal mucosal IgA antibodies against a sequence LQAR of HIV1 envelope gp41. A vaccine based on nasal spray (which induces widespread mucosal IgA in the vagina) may be feasable in Africa, with a possible protection of 50% against HIV-1 sexual transmission. The conservation of this epitope is very interesting, owing to HIV1 hypervariability. 

OBJECTIVE.

We try to elucidate the mechanism of protection conferred by HIV-1 gp41 tetrapeptide LQAR.

METHOD.

We analyzed protein amino acid (AA) sequences, active site residues, DNA nucleotidic sequences and three-dimensional (3D) structure analysis, and compared HIV-1, HIV-2, SIV gp41 sequeKnces of the up-to-date Los Alamos databank (Korber B., 1998 ; Kuiken C., 1999, 2000) and IFN sequences [IFN-alpha (A, B, C, D, E, F, G, H, I), IFN-beta (human, mouse, monkey, bovine), IFN-beta 2 (IL-6), IFN-gamma, IFN-delta, IFN-tau, IFN-omega, Ovine Trophoblast Protein OTP1, pseudo-genes] quoted in the litterature (Goeddel D.V., 1981) (Martal. J.L., 1998). The lentivirus Visna was added, as well as other immunosuppressive retroviruses Moloney MuLV, BLV, HTLV-I and II.

RESULTS.

There is an hexapeptide VERYLK in HIV-1 gp41 almost identical to mouse (but not human) IFN-beta sequence VQRYLK (E = Glutamic acid, is homologous to Q = Glutamine). Furthermore, the ustream sequence ILAV of gp41 is identical to IFN-alpha sequence ILAV. This is highly significant because the tyrosine Y 123, conserved in all known mammalian IFNs-alpha and -beta, is the active site residue (McInnes B., 1989). The 2 IFNs have also the same receptor. HIV-1 can escape immune neutralizing antibodies by a mutation escape (A --> T 582) located in gp41 sequence 579 RILAVERY 586 (Reitz M.S.Jr., 1988). A Lysine K (underlined) to Arginine R or Glutamine Q substitution in the same sequence AVERYLK abolished recognition by Cytotoxic T-Lymphocytes (CTLs) (Dai L.C., 1992; Hammond S.A., 1991).

Downstream, there is a sequence CAW... C common to IFN-delta and SIV AGM gp41. However, this linear alignment, centered on the 2 cysteines, may be more phylogenetic & darwinian, than functionnal & medical.. We obviously prefer... another alignment, in 3D, centered on tryptophan W, with the HIV-1 gp41 W 595 (preceding the cysteine 597 of WGC), matched with the IFN-alpha/beta W 141 (following the cysteine of CAW). This has the advantage to discard the deleterious, enhancing HIV gp41 epitope of Robinson W.E. Jr. (1990) which is included between the 2 cysteines CSGKLIC.

In the IFN tridimensional (3D) structure, there is also another patch between IFN and gp41.This patch is in the amino-terminus of the IFN-alpha 2, in particular residue 33, which is critical for biological activity (Camble R., 1986).

Taken together, the following synopsis is presented between HIV-1 gp41 and IFN-alpha/beta chimera: Underlined residues A 582 and K 586 were HIV-1 gp41 mutation escapes, respectively to antibodies and CTLs. In italics, are the IFN residues conserved in all IFNs: Y123 (activity), R33, Q40 (receptor binding), W141 (hydrophobic core) (Martal J.L., 1998). 

								5	8	2								5	8	8
HIV-1/SIV gp41	5	8	0		I	L	A	V	E	R	Y	L	K		C	A	W	5	9	6
IFN-a/mouse IFN-b chimera	1	1	7		I	L	A	V	Q	R	Y	L	K		C	A	W	1	4	1
	1	1	7			1	2	3		1	2	5						1	4	1
Complement C3d							A	V	E	N	Y	L	Q	K

The general 3D structure of IFNs is also strikingly reminiscent of gp41: Anti-parallel alpha-helices linked by loops (Chan D.C., 1997) (Hunter E., 1997).

At the DNA nucleotidic level of the peptide VQRYLK (Val Gln Arg Tyr Leu Lys), the homology is 83,33% (15/18 bases identity), raising to 85,71% (18/21), if a chimera of Visna-HIV is compared to mouse IFN-beta. Visna an interesting sheep model of human Multiple Sclerosis (MS), and, intrigingly, IFN-beta has been precisely approved in MS treatment. Perron H.(1997) isolated frequently a novel retrovirus in MS

(A = Adenine, C = Cytosine, T = Thymine, G = Guanine)l

This gp41 site is mimicking a new IFN, among the hundred of this superfamily members, which is a chimera between IFN-alpha and mouse IFN-beta (probably the mysterious "acid-labile" IFN). Thus, a new HIV receptor is a special IFN receptor subtype, common to both IFN-alpha and mouse IFN-beta. Interestingly, Delcayre A.X. (1991) found that the major IFN-alpha receptor on Burkitt B lymphoma cells Raji-3 was the Epstein-Barr virus(EBV) gp 350/complement C3d receptor CR2 (= CD21). The homology between EBV and C3d (Nemerow G.R., 1987) includes a doublet LY, which is the inverted sequence of YL present in HIV-1 gp41 & IFN

Complement may play an important role in host defence against HIV-1, as Cameron P.U. (1988) showed 42% HIV-1 infected patients with generalised lymphadenopathy had complement C4B null alleles, compared with 18% of seronegative homosexuals and 15% of controls. An absolute priority is the isolation and cloning of this unknown, natural, HIV-1 gp 41-mimetic IFN, with primers synthetized on the basis of our molecular mimicry. Possibly, the sophisticated design of a special HIV-1-specific IFN chimera reproducing exactly this sequence has some value in AIDS treatment, including perhaps EBV-induced B lymphomas, and may be more efficient than available commercial IFNs, whether -alpha or -beta. Antibodies and vaccine focused strictly on this epitope, excluding the downstream enhancing epitope of Robinson, seem to be important.

LQAR is almost identical to the binding site of bovine IFN-beta 2 LEAR (E = glutamic acid & Q = glutamine). LQAR exists in Thailand strains (AE, TH strains) of the Los Alamos databank. So a LQAR vaccine would be protective also in Thailand. Among HIV-1s, the TH strains are the most homologous to rat IFN-beta after the region LAVERYL, spanning 10 AA, which is highly significant : Assuming that there are 20 residues, the probability of finding 2 identical decapeptides (10 AA) is approximatively 1 chance in 20 power 10 (= 10,240,000,000,000). In the IFN 3D structure, the cystine bridge C-C near LEAR and AW joins in proximity LEAR with the important YLK.

CONCLUSION.

In conclusion, a vaccine against sexual transmission in Africa and Thailand seems possible with the IgA-inducing HIV-1 gp41 epitope LQAR ; an interesting approach may perhaps be to present LQAR to dendritic cells via chaperon Hsp, in order to amplify the immune response. Mucosal vaginal IgA induction is feasable by the nasal route ; another induction of vaginal IgA by a protective (5/6 cases) vaccine was achieved in a female macaque model, by encapsulation of a formaldehyde-inactivated SIV virus with polyester microspheres (Marx P., 1993, Science).

As LEAR is an IFN-beta 2 binding site to IFN receptor, HIV-1 gp41 is a viral IFN-beta 2 homologue. The best homology is with AE THailand strains, and opens a new avenue of HIV-1 treatment: The competitive inhibition by IFN-beta 2. An homologous IFN, ovine IFN-tau, has been demonstrated to potently inhibit HIV-1 replication in cell cultures (lymphocytes and macrophages) (Martal J.L., 1998).

In Thailand, prostitutes may be screened for anti-LQAR IgA.

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