Non-HIV vaccine antigen from the vaginal microbiota capable of inducing a mucosal neutralizing protective antibody response against HIV infection
10273292 · 2019-04-30
Assignee
Inventors
- Raphaëlle Claude El Habib (Chaponost, FR)
- Régis Sodoyer (Saint Genis les Ollieres, FR)
- Armelle Cuvillier (Saint-Jouvent, FR)
- Christiane Moog (Ostwald, FR)
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
C07K16/1063
CHEMISTRY; METALLURGY
A61K2039/58
HUMAN NECESSITIES
C07K2317/33
CHEMISTRY; METALLURGY
C07K16/1253
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C12N2740/16134
CHEMISTRY; METALLURGY
International classification
A61K39/00
HUMAN NECESSITIES
C12N7/00
CHEMISTRY; METALLURGY
Abstract
A new non-HIV vaccine antigen from Mycoplasma sp. permease capable of inducing a mucosal neutralizing protective antibody response against HIV infection, a neutralizing antibody directed to said antigen, and a method for the identification of new antigens from the mucosal microbiota for the development of vaccines against pathogens.
Claims
1. A method of inducing expression of a HIV-1 cross-reactive and HIV-1 neutralizing antibody in a human subject, comprising administering to the human subject an immunogenic composition comprising: (i) A Mycoplasma genitalium permease antigen comprising an amino acid sequence which is at least 85% identical to SEQ ID NO: 4 or a peptide of at least 5 consecutive amino acids thereof, or (ii) a polynucleotide encoding said antigen in expressible form, in an amount effective to induce expression of an HIV-1 cross-reactive, HIV-1 neutralizing, and anti-Mycoplasma genitalium permease antibody in the human subject, and a pharmaceutically acceptable adjuvant.
2. The method according to claim 1, wherein said permease antigen comprises the protein of SEQ ID NO: 4.
3. The method according to claim 1, wherein said permease antigen comprises the protein of SEQ ID NO: 7.
4. The method according to claim 1, wherein said polynucleotide comprises SEQ ID NO: 5.
5. The method according to claim 1, wherein said polynucleotide comprises SEQ ID NO: 6.
6. The method according to claim 1, wherein the HIV-1 cross-reactive and HIV-1 neutralizing antibody binds to HIV-1 gp41.
7. A method of inducing expression of an HIV-1 cross-reactive and HIV-1 neutralizing antibody in a human subject, comprising administering to the human subject a vaccine composition comprising: (i) a Mycoplasma genitalium permease antigen comprising an amino acid sequence which is at least 85% identical to SEQ ID NO: 4 or a peptide of at least 5 consecutive amino acids thereof, or (ii) a polynucleotide encoding said antigen in expressible form, in an amount effective to induce expression of an HIV-1 cross-reactive, HIV-1 neutralizing, and anti-Mycoplasma genitalium permease antibody in the human subject, and a pharmaceutically acceptable adjuvant.
8. The method according to claim 7, wherein said permease antigen comprises a permease epitope cross-reacting with an HIV-1 gp41 ectodomain or C-terminal tail epitope.
9. The method according to claim 8, wherein said permease epitope cross-reacts with an HIV-gp41 ectodomain epitope from SEQ ID NO: 2.
10. The method according to claim 7, wherein said permease antigen comprises the protein of SEQ ID NO: 4.
11. The method according to claim 7, wherein said permease antigen comprises the protein of SEQ ID NO: 7.
12. The method according to claim 7, wherein said polynucleotide comprises SEQ ID NO: 5.
13. The method according to claim 7, wherein said polynucleotide comprises SEQ ID NO: 6.
14. The method according to claim 7, wherein the HIV-1 cross-reactive and HIV-1 neutralizing antibody binds to HIV-1 gp41.
Description
(1) The practice of the present invention will employ, unless otherwise indicated, conventional techniques which are within the skill of the art. Such techniques are explained fully in the literature. In addition to the above arrangements, the invention also comprises other arrangements, which will emerge from the description which follows, which refers to exemplary embodiments of the subject of the present invention, with reference to the attached drawings in which:
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EXAMPLE 1: IDENTIFICATION OF THE VAGINAL IMMUNE RESPONSE IN UNINFECTED WOMEN HIGHLY EXPOSED TO HIV
(8) 1. Material and Methods
(9) 1.1 Cohorts
(10) A cohort of 32 HIV highly-exposed uninfected women (HEUW) from sero-discordant couples was investigated in collaboration with the GHESKIO Centers (Haitian Group for the Study of Kaposi's Sarcoma and Opportunistic Infections) in Haiti. The control cohort included 15 HIV non-exposed uninfected subjects. None of the subjects had CCR5-Delta 32 homozygous genotype and two were heterozygous, one in HEUW group, the other in control group. The study was approved by ethical committee and informed consent was obtained from all women prior sampling.
(11) 1.2 ELISA on Vaginal Secretions
(12) Vaginal secretions of HEUW and controls were assayed for HIV gp41-specific IgG1 and IgA antibody responses by ELISA, as previously described (Hocini et al., AIDS Res. Hum. Retroviruses, 1997, 13, 1179-).
(13) 1.3 Western-Blot on Vaginal Secretions
(14) Vaginal secretions of HEUW and controls were assayed for HIV gp160, gp110, gp41, p68, p55, p53, p40, p34 and p25 specific IgG1 and IgA antibodies by Western-Blot as previously described (Hocini et al., AIDS Res. Hum. Retroviruses, 1997, 13, 1179-).
(15) 1.4 Inhibition of Transcytosis Assay
(16) Inhibition of transcytosis of assay was performed as previously described (Blec, L. et al. The Journal of infectious diseases, 2001, 184, 1412-1422).
(17) 1.5 Cloning of Mucosal Cells IgG1 and IgA Variable Regions into Phagemid Vector
(18) Separate libraries of IgA and IgG1 Fab fragments were constructed. Briefly, messenger RNAs extracted from mucosal cells of two selected subjects were used for separate amplification of IgG1 and IgA variable regions. PCR primers corresponding to the 5 end of all VH and VL variable domain families were combined individually with a primer derived from IgG1 or IgA first constant domain (Persson et al., Proc. Natl. Acad. Sci. USA., 1991, 88, 2432-6). The amplified DNA fragments corresponding to heavy and light chains were then cloned into a phagemid vector (Persson et al., Proc. Natl. Acad. Sci. USA., 1991, 88, 2432-6), allowing the surface display of Fab fragment (Barbas et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 7978-82).
(19) 1.6 Sequencing of Recombinant IgG and IgA Fab
(20) Individual clones, from both IgG1 and IgA libraries were sequenced using the standard Sanger dideoxy technique.
(21) 1.7 Expression of Recombinant IgG1 and IgA Fab Fragments
(22) Recombinant Fab fragments were expressed using both E. coli (Studier and Moffat, J. Mol. Biol., 1986, 189, 113-130) and Cell-free translation (Ryabova et al., Methods Mol. Biol. 1998, 77, 179-93).
(23) 1.8 Analysis of VH Mutations
(24) VH mutations of recombinant IgG1 and IgA were analysed as described in Wang et al., BMC bioinformatics, 2008, 9 Suppl 12, S20.
(25) 1.9 Identification of Antibody Epitope by Phage Displayed Random Peptide Libraries Screening
(26) In order to identify the epitope recognized by the antibody, the recombinant antibody Fab fragment was screened against commercial phage displayed random peptide libraries (Ph.D.-12 Phage Display Peptide Library Kit, NEW ENGLAND BIOLABS), according to the manufacturer's instructions. Typically, 4 to 5 rounds of selection (bio-panning) were applied.
(27) 1.10 Statistical Analysis
(28) A Yates' chi-squared test was used to analyze the samples from Table I. The difference between control group and HIV-highly exposed group is statistically significant as long asp value<0.05.
(29) 2. Results
(30) The immune response in the genital tract of a cohort of HIV highly exposed uninfected women (HEUW) from sero-discordant couples was investigated in collaboration with the GHESKIO Centers (Haitian Group for the Study of Kaposi's Sarcoma and Opportunistic Infections) in Haiti. Vaginal fluids from 32 individuals were analyzed by ELISA and Western Blot, and compared with 15 control HIV non-exposed uninfected subjects. None of the subjects had CCR5-Delta 32 homozygous genotype and two were heterozygous, one in HEUW group, the other in control group. Vaginal secretions were tested for specific IgG1 and IgA antibody responses against gp41 in ELISA. ELISA results were confirmed by western blot. Only samples showing a strong positivity with both techniques were retained for further investigation and antibody library construction.
(31) Although all subjects were seronegative for HIV infection, 53% of the HEUW were positive in vaginal secretions for anti-gp41 IgA versus only 13% in the control group (p<0.02) whereas no significant difference was observed for anti-gp41 IgG responses (Table I).
(32) TABLE-US-00001 TABLE I HIV-1 specific immune response in vaginal secretions of HEUW by WB and ELISA Positives in Positives in Control HIV highly exposed P Antibody group (n = 15) group (n = 32) value IgA WB/gp160 0 (0%) 3 (10%) NS IgA WB/gp110 0 (0%) 1 (3%) NS IgA WB/p68 0 (0%) 1 (3%) NS IgA WB/p55 0 (0%) 2 (6%) NS IgA WB/p53 0 (0%) 1 (3%) NS IgA WB/gp41 2 (13%) 17 (53%) 0.02 IgA WB/p40 0 (0%) 1 (3%) NS IgA WB/p34 0 (0%) 4 (12%) NS IgA WBP/p25 1 (6%) 2 (6%) NS IgA ELISA/gp41 3 (20%) 9 (28%) NS IgG1 WB/gp160 0 5 (16%) 0.16 IgG1 WB/gp110 0 2 (6%) NS IgG1 WB/p68 0 1 (3%) NS IgG1 WB/p55 2 (13%) 3 (10%) NS IgG1 WB/p53 0 4 (12%) NS IgG1 WB/gp41 2 8 (25%) NS IgG1 WB/p40 0 2 (6%) NS IgG1 WB/p34 0 1 (3%) NS IgG1 WB/p25 0 4 (12%) NS IgG1 ELISA gp41 2 (13%) 7 (22%) NS WB: Western blot; NS: non-significant
(33) The functional activity of the secretions was tested by inhibition of transcytosis of HIV.sub.JRCSF. None of the secretions were positive in transcytosis functional assays, compared with the broad HIV-1 neutralizing monoclonal antibody 2F5 used as positive control. Therefore the transcytosis test was not further used for screening of samples.
(34) To characterize the antibody response, mucosal cells were collected with a cytobrush from two CCR5 wild type subjects selected on the basis of their strong response against gp41 in ELISA and Western Blot and of long term unprotected sexual relationship with at least one HIV-1 seropositive partner. In order to avoid amplifying genes from the partner, all samples positive for the Prostate Specific Antigen (>50 ng/mL PSA) were discarded.
(35) After specific amplification, IgG and IgA variable regions were cloned into a phagemid vector allowing the expression of Fab fragments at the surface of filamentous phages. In a first strategy, phages displaying a Fab binding to a recombinant gp41 antigen would have been selected. This screening step was found to be unnecessary due to the observed strongly oligoclonal profile of the cloned repertoire. In a standard situation, the analysis by sequencing of clones randomly picked from the library should give a whole set of different sequences. However, in the case of the selected subjects, the vast majority of clones was identical or very closely similar and represented a large percentage of the total population (up to 75% for VH chains). This was not due to a bias in amplification or cloning since the same result was obtained when independent libraries were constructed. The same was true for both the IgG1 and IgA libraries. To further characterize this oligoclonal mucosal repertoire, Fab fragments corresponding to the most represented antibodies were expressed as recombinant proteins.
(36) IgG1 (called Toussaint) in association with 2 different light chains, IgA (called Makandal) in association with 2 different light chains, IgG1 (Jacmel) and IgA (Mangue). The most represented light chains were from 1 and 4 families. These two light chains were systematically associated with all the heavy chains since the original VH/VL chain pairing was not possible to determine. Analysis of the VH mutations showed that Makandal had a germinal un-mutated profile. In order to identify the recognized epitope, this antibody was screened against commercial phage displayed random peptide libraries. In two independent screenings, two overlapping peptides allowed for the identification of an epitope corresponding to a region in the C-terminal part of gp41: LVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGP (SEQ ID NO: 3).
(37) It was hypothesized that the mucosal oligoclonal immune response of these women might be responsible for their resistance to HIV infection and that these antibodies were induced in response to a particular microbiota and by cross-reaction with the gp41 domain of HIV envelope, could prevent the viral fusion. In order to see if the sequence of amino acids recognized by Makandal Fab had any homologies with other proteins of the environment, this sequence was compared with a protein sequence databases excluding HIV proteins. Data showed very weak matches, but interestingly, there were consistent matches with bacterial protein sequences. Among these, a permease from Mycoplasma genitalium (GenBank accession number AAC72488.1 or SEQ ID NO: 1) was selected.
EXAMPLE 2: THE PERMEASE ANTIGEN OF M. GENITALIUM IS CAPABLE OF INDUCING A SYSTEMIC AND A MUCOSAL NEUTRALIZING PROTECTIVE ANTIBODY RESPONSE AGAINST HIV INFECTION IN HAMIGA MICE
(38) 1. Material and Methods
(39) 1.1 Antigens
(40) Recombinant Truncated Permease from M. Genitalium (rPermease or PERM)
(41) Recombinant truncated permease cDNA (SEQ ID NO: 6) prepared by gene synthesis (GeneART) was inserted in a procaryotic expression vector (pSP401, derived from pET28 cer (NOVAGEN); Peubez et al., Microbial Cell factories, 2010, 9, 65-) and expressed in E. coli BL21 strain. Tagged by a Histidine tail, the antigen (SEQ ID NO: 7) was purified by affinity chromatography on His-IMAC (BIORAD) column. The purified recombinant permease protein fragment ([rPermease]=1 mg/mL in Tris 25 mM, Arginine 0.5 M, Sucrose 5% buffer, pH 8) had a purity of 82%.
(42) HIV Gp41 Ectodomain Peptide (GP)
(43) Recombinant HIV-gp41 ectodomain peptide (GP) preparation is disclosed in Krell et al., European Journal of Biochemistry, 2004, 271, 1566-1579. Briefly, a 0.6 kb DNA fragment containing the sequence encoding the ectodomain of gp41 of HIV isolate LAI (amino acid sequence SEQ ID NO: 2, corresponding to amino acid residues 540 to 673 of gp160 (SEQ ID NO: 14)), was obtained by PCR amplification using, as template, a plasmid containing the gp120 sequence and the gp41 sequence of the LAI isolate. The start codon in the forward primer is a naturally occurring methionine residue. A stop codon and the DNA sequence encoding the extension GGGGSHHHHHH (SEQ ID NO: 15) were added to the reverse primer. Platinum HF polymerase (GIBCO INVITROGEN) was used, according to the manufacturer's instructions, for PCR amplification. The PCR amplified fragment was cloned directly into the vector pM1800 using the restriction sites NcoI and XhoI. The expression vector pM1800 is a derivate of pET28c (NOVAGEN, in which the F1 origin of replication has been deleted and replaced with the Cer fragment, allowing for multimer resolution. For protein expression, E. coli BL21(DE3) was transformed with the corresponding plasmids. Tagged by an Histidine tail, the ectodomain antigen (SEQ ID NO: 16) was purified by affinity chromatography on onto a 5 ml Hi-Trap Chelating column (AMERSHAM PHARMACIA BIOTECH). Protein elution was achieved using a buffer comprising 50 mMTris/HCl, 8 M urea, 500 mM NaCl and 500 mM imidazole, pH 8.0. Protein refolding was achieved by dialysis with 50 mM formate, pH 2.8. Protein was then sterile filtered and stored at 45 C.
(44) 1.2 Immunization
(45) Immunization was performed in HAMIGA transgenic mice (EP Patent 1 680 449), a transgenic mouse strain producing human/mouse chimeric IgAs with human IgA heavy chain constant regions and mouse light chain constant regions and (heavy and light chain) variable regions. Estrous cycles of HAMIGA transgenic mice were synchronized by treatment with Depoprovera (5 days before the first immunization, 8 days- and 16 days post-immunization-2 mg/mouse/injection by subcutaneous route). HAMIGA transgenic mice (10 mice per group) were immunized by intradermal (Group ID in ear) or intravaginal (Group VAG) routes twice at two weeks of interval with the recombinant truncated permease of M. genitalium (PERM) in polyI:C adjuvant (10 g/mouse/injection, ratio 1:2.5 PolyI:C (25 g/mouse/injection, INVIVOGEN)). For comparison, ten HAMIGA mice were immunized with gp41-ectodomain polypeptide via the ID route (GP/ID, 10 g/mouse/injection, in polyI:C adjuvant (10 g/mouse/administration, ratio 1:2.5 PolyIC (25 g/mouse/injection, INVIVOGEN)) and via the vaginal route (GP/VAG, 10 g/mouse/administration, in polyI:C adjuvant (10 g/mouse/administration, ratio 1:2.5 PolyIC (25 g/mouse/administration, INVIVOGEN)). As a negative control, five HAMIGA mice were immunized with Bovine Serum Albumin (BSA) antigen by ID route.
(46) 1.3 Vaginal Washes Harvest
(47) Vaginal cavities of immunized mice were washed each day during a complete estrous cycle (4 days), by gently flushing vaginal cavity with 200 L of physiological media (NaCl 0.9%, URGO).
(48) 1.4 Seric IgA and Secretory IgA Concentration Titration by ELISA
(49) Seric IgA from immunized HAMIGA mice (purified by affinity chromatography) and secretory IgA prepared from vaginal washes were titered by ELISA. Briefly, 96-well plates (Maxisorp, NUNC) were coated with 1 g/mL of goat anti-human IgA (BECKMAN COULTER) in PBS buffer overnight at 4 C. Plates were saturated with a BSA 2%/PBS1 buffer during 30 minutes at 37 C. Incubation of the samples (secretory IgA, diluted 10 times in BSA 0.2%/PBS or purified seric IgA, diluted 100 times in BSA 0.2%/PBS) was performed at 37 C. during 2 h. Human IgA reference range (from [control hIgA]=0.2 mg/ml to 1.56 ng/mL) was incubated following the same protocol and revealed by an Alkaline-Phosphatase (AP) labelled-goat anti-hIgA polyclonal antibody (BECKMAN COULTER; diluted 2000 times in BSA 0.2%/BSA).
(50) 1.5 Preparation of Monoclonal IgA
(51) To isolate monoclonal IgA from permease-immunised mice, immortalization of specific B-cell lymphocytes were performed, according to Klher and Milstein protocol (Khler, G. & Milstein, C. European Journal of Immunology, 1976, 6, 511-9). Briefly, splenocytes from permease-immunized mice were fused with mice myeloma cells (X63 Sp2/0) and subcloned on 96-wells plates. The supernatants of hybridoma clones were harvested after three weeks of culture and tested for their neutralizing activity on in vitro HIV infection inhibition assay. Each clone was cryopreserved in DMSO 10%/SVF 20%/DMEM media in liquid nitrogen.
(52) 1.6 IgA Purcation
(53) IgA were purified by affinity chromatography. The monomeric and polymeric forms of IgA were separated by size exclusion column chromatography.
(54) 1.7 Antigen Specificity Analysis
(55) Permease fragment/gp41-ectodomain specific IgA were assessed by ELISA using Maxisorp 96-wells plates (NUNC) coated with 1 to 5 g/mL of antigens overnight at 4 C. Crude supernatants, unpurified IgA from vaginal washes or purified serum IgA (diluted in PBS/Gelatin 0.2%) were incubated 2 hours at 37 C. Specific IgA binding was revealed with an AP-conjugated goat anti-human IgA antibody (1/2000 diluted, BECKMAN COULTER).
(56) 1.8 Cell Preparation
(57) Blood samples were collected from anonymous healthy donors (Etablissement Franais du Sang, EFS). Peripheral Blood Mononuclear cells were obtained by Ficoll-Hypaque sedimentation of Buffy coats. PBMCs were activated with phytohemagglutinin-A (PHA, 1 mg/mL, SIGMA) in RPMI medium-10% fetal calf serum (FCS) supplemented with antibiotics (Penicillin and Streptomycin, 100 Units/mL and 100 g/mL, respectively) at the concentration of 10.10.sup.6 cells/mL for 30 minutes. Cells were then cultivated in RPMI medium-10% FCS supplemented with antibiotics at 2.10.sup.6 cells/mL. After 3 days, cells were frozen. PBMCs from 5 donors were thawed, pooled and culture for one day before being used in the neutralization assay.
(58) TZM-Bl cell line was obtained through the NIH reagent program. TZM-Bl cells were cultured in RPMI medium-10% FCS supplemented with antibiotics (Penicillin and Streptomycin, 100 Units/mL and 100 g/mL, respectively).
(59) 1.9. Virus Preparation
(60) Primary HIV-1 isolates were amplified on human blood leukocytes, as described previously (Holl et al., Blood, 2006, 107, 4466-4474). Virus stocks collected at peak virus production were concentrated 70-fold with a 100-kDa cutoff polyethersulfone filter (Centricon Plus-70 Biomax Filter; MILLIPORE). Primary HIV-1 isolates were obtained from the National Institute for Biological Standards and Control (NIBSC): HIV-1.sub.SF162 isolate (subtype B, R5), HIV-1.sub.QH0 isolate (subtype B, R5 strain, R5), HIV-1.sub.89.6 (sub-type B, X4R5), Viruses HIV-1.sub.DU174 (subtype C, R5), HIV-1.sub.92BR025 (subtype C R5), HIV-1.sub.92UG024 (subtype D, X4), HIV-1.sub.KON (subtype CRF02-AG, X4). HIV-1 BaL (subtype B) was provided by S. Gartner, M. Popovic, and R. Gallo (NIH).
(61) Pseudoviruses HIV-1.sub.SF162 and HIV-1.sub.QH0 (SF162.LS and QH0692.42) were produced by co-transfection of 293T cell line with EnvC3 back bone and Envs from HIV-1.sub.SF162 and HIV-1.sub.QH0 respectively.
(62) 1.10 Neutralization Assays
(63) Neutralization assays were performed on human PBMC and TZM-bl cells, as previously described (Mascola et al., J. Virol., 2005, 79, 10103-10107), using two standard reference strains of clade B as Env-pseudotypes viruses (SF162.LS and QH0692.42; Li et al., J. Virol., 2005, 79, 10108-10125). The 50%, 70%, 80% or 90% inhibitory dose was defined as the sample concentration that caused 50%, 70%, 80% or 90% reduction in relative luminescence units (RLU; Li et al., J. Virol., 2005, 79, 10108-10125) in TZM-bl or percentage of infected cells in PBMC, respectively.
(64) 1.11 Fc-Mediated Inhibition of HIV Replication in MDMs
(65) Fc-mediated inhibition assay was performed on Monocyte derived macrophages (MDMs). MDMs were generated by culture of CD14+ monocytes with GM-CSF for 5 days. Inhibition of cell free HIV-1 SF162 or HIV-1 BaL replication in MDMs was assessed as previously described by Holl et al. (J. Virol., 2006, 80, 6177-6181; J. Immunol., 2004, 173, 6274-6283). Briefly, antibodies and virus were incubated for 1 h before addition to MDMs. Virus replication was measured after 48 h by the intracellular staining of p24 in MDMs by flow cytometry. Percentage of infected cells compared to control infected macrophages without antibodies was determined.
(66) 2. Results
(67) A truncated permease antigen from M. genitalium (SEQ ID NO: 7, corresponding to amino acid sequence 431-875 of M. genitalium permease sequence SEQ ID NO: 1) was expressed in E. coli and used to immunize HAMIGA transgenic mice expressing chimeric human IgA. Groups of ten HAMIGA mice received two administrations of permease fragment in Poly I-C adjuvant via the intradermal (Group PERM/ID) and vaginal routes (Group PERM/VAG). At the same time, groups of ten HAMIGA mice were immunized with gp41-ectodomain polypeptide (SEQ ID NO: 16) (Group GP/ID; Group GP/VAG). As a negative control, five HAMIGA mice were immunized with BSA antigen (Group BSA) by ID route. IgA from vaginal washes and purified serum IgA from immunized and control HAMIGA mice were analysed.
(68) To identify the immunization route and the antigen able to induce neutralizing immune response, neutralizing activity of seric IgA from each immunized mouse was measured against SF162 virus strain, a tier 1 isolate, in a PBMC-based neutralizing assay (
(69) In addition, only vaginal IgA pools from PERM/ID and PERM/VAG immunized group (n=10) exhibited a strong neutralization activity against HIV-1.sub.SF162 (a tier 1 isolate,) with the lowest IC90 (in a range of 1.5 to 2.5 g/mL; Table II). Vaginal IgA pools from PERM/VAG immunized group (n=10) reached an IC50<2.5 g/mL against QHO isolate (a tier 2 isolate) when performed in a TZM-bl cells based infection inhibition assay.
(70) TABLE-US-00002 TABLE II HIV-1 specific neutralizing activity of purified vaginal IgA pools from immunized HAMIGA mice Target cells PBMC TZM Percentage of inhibition of HIV-1 infection IC90 IC50 Group of immunized HIV-1 virus HAMIGA (n = 10) SF162 QHO SF162 QHO Vaginal IgA GP/ID 0.010 >0.010 >0.01 >0.01 Vaginal IgA GP/VAG >0.016 >0.016 0.016 0.016 Vaginal IgA PERM/ID 0.0015 >0.0015 >0.0015 >0.0015 Vaginal IgA PERM/VAG 0.0025 >0.0025 0.002 0.0025 Vaginal IgA Pre-immune >0.0035 >0.0035 >0.0035 >0.0035 Vaginal IgA BSA 0.0115 >0.0115 >0.0115 >0.0115 *HIV-Infection Inhibition titers are expressed in mg/mL
(71) The supernatants of hybridoma clones from permease-immunised mice were tested for neutralizing activity in a PBMC assay against the HIV-1.sub.SF162 isolate. Out of 900 hybridoma supernatants, 141 showed 80% neutralisation of the viral infection including 34 that showed 90% neutralization potency. One clone, C3G4, was sub-cloned three times and anti-permease specificities were confirmed by ELISA. One clone named C3-G4.EA5.IH2.JA11 was finally stabilized. The variable segments of the neutralizing immunoglobulin, a human chimeric IgA1,.sub., were sequenced and analysed on IMGT data base (www.imgt.org/vquest).
(72) TABLE-US-00003 TABLEIII SequenceandanalysisofthevariablesegmentsoftheHIV-1 neutralizinghumanchimericmonoclonalIgA1,.sub.K,C3G4 Heavychain IgH-V IgH-D IgH-J mabhIgA1(C3G4clone) 5-12-1*01F 1-1*01 2*01F MousegermlinegeneIdentity 93.06% 82.98% AAIgHVsequence EVQMVESGGGLVKPGGSLKLSCAASGFAFNKYDMSVVVRQTPAK RLEVVVAYISGGGGHTYYRDTLKGRFTVSRDNAKNTLYLQMNSLKS EDTAMYYCTRHGTSWDYWGQGT(SEQIDNO:8) Lightchain IgH-V IgH-J mabhIgA1(C3G4clone) 15-103*01 1*01F MousegermlinegeneIdentity 95.34% 94.74% AAIgLVsequence DIQMNQSPSSLSASLGDTISITCRASQNINFWLSVVYQLKPGNIPKQ LIYKTSNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCLQGQS YPWTFGGGTKLEIK(SEQIDNO:9)
(73) The supernatant of C3G4 contains both a monomeric and polymeric monoclonal IgA1 (
(74) Monomeric IgA and polymeric IgA enriched fractions of purified C3G4 were primarily tested in a PBMC neutralizing assay with two HIV-1 virus isolates, SF162 and QHO (
(75) Monomeric and polymeric forms of C3G4 were also tested in a macrophage-based neutralization assay. The assay shows the Fc-mediated inhibitory activity of the antibodies. As shown in Table IV, both forms presented 80% inhibition potency in PBMC and Macrophages-based assays, in presence of 6 g/mL and 14 g/mL of C3G4 IgA, respectively. Ninety percent inhibition of HIV-1 infection was achieved when both tests were performed with 58 g/mL of C3G4 IgA polymeric form. Polymeric form of C3G4 IgA showed a greater neutralizing potency than monomeric form. The higher avidity of polymeric form may allow the antibody to recognize and capture several viral particles, as opposed to the monomeric form.
(76) TABLE-US-00004 TABLE IV HIV-1 specific neutralizing activity of monomeric or polymeric enriched fractions of C3G4 IgA antibody Human cell type PBMC Macrophages HIV-1 isolate SF162 BaL HIV-1 infection inhibition titer (in mg/mL) IC80 IC90 IC80 IC90 Sam- C3G4-purified IgAl >0.125 >0.125 0.031 0.200 ples (containing two forms mix) C3G4-enriched 0.094 >0.094 0.023 0.094 monomeric IgA1 C3G4-enriched 0.006 0.058 0.014 0.058 polymeric IgA1
(77) Neutralizing activity of monoclonal C3G4 IgA1 was also tested on different primary HIV-1 isolates (
(78) These data demonstrate that Mycoplasma genitalium permease antigen represents a HIV-1 vaccine candidate able to prime a mucosal and systemic broadly-neutralizing protective antibody response against HIV infection in the immunized individuals.
(79) Discussion
(80) In this study, using HAMIGA mice to produce IgA, immunization with M. genitalium permease adjuvanted with polyI:C administered via the ID or vaginal routes elicited specific IgA Abs able to recognize the HIV-Gp41 and to neutralize HIV-1 primary isolates.
(81) IgA is the most abundant antibody isotype produced weekly and delivered by secretion in mucosal area. Because HIV infection is usually initiated at mucosal site of entrance, the inventors assumed that IgA would better mimic the response in mucosa, including the genital mucosa. Pivotal role of polymeric form of IgA is to prevent pathogen entry by immune exclusion, but also polymeric IgA are assumed to agglutinate the virus and block transcytosis (Chomont, N. et al. Virology, 2008, 370, 246-254) and with more efficacy than their IgG homologue (Hur, E. M. et al. Blood, 2012, 120, 4571-82).
(82) The integrity of the mucosal immune system appears to be critical for the rate of the HIV infection progression, because it is severely compromised during acute HIV infection. Beside the massive depletion of CD4+T cells, the frequency of IgA-producing B-cells in mucosal area drops dramatically, decreasing luminal secretory IgA concentrations involved in pathogens-immune exclusion protective pathway (described in both HIV and SIV infection; Alam et al., J. Viral., 2008, 82, 115-125; Muro-Cacho et al., J. Immunol., 1995, 154, 5555-5566; Douek et al., 2006, Nat. Immunol., 2006, 7, 235-239).
(83) Transgenic mice expressing human chimeric IgA (EP patent 1 680 449) were used to ensure selection of a monoclonal IgA class antibody since a conventional immunization of wild type mice usually conduct to an IgG specific response. This mouse line was modified to express a constant human IgA heavy chain whereas constant light chain and variable genes remain from mouse origin. The C3G4-neutralizing antibody was induced by active immunization where the murine and not human repertoire has been involved in the antigen recognition. The possibility to generate the same response in human can only be evaluated in clinical trials.
(84) Resistance to HIV infection is a rare phenomenon. Although several mechanisms may contribute including genetic factors (CCR5 d32 mutation, MHC allotype.) or immune factors such as specific IgA in vaginal secretions directed against the HIV-1 gp41, other factors may be involved.
(85) The human and murine Ab response to many common pathogens is oligoclonal, with a restricted usage of Ig V-genes (Bos, N. A. et al., Infection and Immunity, 1996, 64, 616-623; Baxendale, H. E. & Goldblatt, D., Infection and Immunity 2006, 74, 1025-1031). In addition, in the mucosa exposed to a multitude of germs, these Abs are polyreactive and establish a natural immune homeostasis (Shimoda et al., Immunology, 1999, 97, 9-17).
(86) Therefore, the inventors propose that the innate response to the genital tract microbiota (commensals or pathogens) could also play a role against HIV (or other pathogens) infection. The innate characteristic of the response is suggested by the oligoclonal profile of IgA found in the HEUW and the germinal feature of the Ab. The isolation of an Ab against M. genitalium cross-reacting with gp41 and neutralizing HIV-1 provides additional evidence. Whereas current thinking is that vaccine should induce hypermutated Ab in order to neutralize HIV-1 (Klein, F. et al., Cell, 2013, 153, 126-38), the inventors propose that in mucosal site, main portal of entry of the virus, non affinity matured Ab elicited by specific commensals or pathogen, could neutralize and protect against HIV infection. HEUW may have a particular unique flora with native immune response that contributes to resistance to HIV-1 infection. Comparison of the microbiota in HEUW and women who later becomes infected will help to clarify this assumption.
(87) The key for an effective vaccine strategy seems to be the identification of the priming antigen which elicits ancestor B lineage selection and proliferation for a future maturation all along its immune history. For some viral infections, the antigen that initially activates certain naive B-cell lineages and that stimulates the memory B-cells may not be identical.
(88) The inventors propose that this population of naive B precursors may be recruited and primed by antigens, not only from past infections but from current natural commensal micro-organisms colonization. Immunization by Mycoplasma genitalium antigens induced cross-reactive anti-HIV antibodies and elicited a systemic and mucosal broadly neutralizing anti-HIV response. IgH variable segment of one of the neutralizing monoclonal antibodies from the present study reveals a highest homology with the murine germline ancestor IgVH5-12 (
(89) The inventors propose M. genitalium permease as a promising candidate to initiate a vaccine protocol mainly at the mucosal level based on its ability to prime naive B-cell clones, able to further recognize native HIV-antigens.