Patent Application
20220402978
The present invention relates to a novel class of polypeptides that are suitable as antigens mimicking epitope of spike HIV-1 gp120 glycoprotein recognized by broadly neutralizing antibody VRC01, and thus are suitable as immunogens for stimulation of production of HIV-1-neutralizing antibodies and for development of vaccine preventing HIV infection.
HIV-1 infection and Acquired Immunodeficiency Syndrome (AIDS) represent global pandemic resulting in an estimated 35 million deaths worldwide. Irrespective of intensive research, no commercial vaccine is available. The most important obstacles are an enormous HIV-1 antigenic variability and unique biochemical, biological, and immunological properties of the most promising vaccine candidate, HIV-1 envelope (Env) glycoprotein which is responsible for HIV-1 attachment to and the entry into the host cell (Robinson H L HIV/AIDS Vaccines: 2018. Clin Pharmacol Ther 2018, 104: 1062-73; Moore P L The Neutralizing Antibody Response to the HIV-1 Env Protein. Curr HIV Res 2018, 16: 21-8). Env is a trimer of gp160 proteins cleaved into two functional subunits: the gp41 trans-membrane glycoprotein and virus surface-exposed gp120 glycoprotein. Majority of identified and cloned human antibodies, able to neutralize broad range of HIV-1 Env variants (bn-mAb) including VRC01, recognize gp120 subunits.
Novel strategies in HIV-1 vaccine development were encouraged after identification of several of such bn-mAbs, because they could limit viraemia, as shown for elite neutralizers, group of individuals with broad and potent neutralizing activity. The generation of HIV-1-specific bn-mAbs under natural conditions is a long-term process lasting for years and difficult to be elicited by conventional vaccination. The majority of identified bn-mAbs exhibit unique properties including a long HCDR3, extraordinary frequencies of V(D)J mutations and poly- or autoreactivity with human lipids and proteins, which seem to be a crucial obstacle for the development of a successful vaccination strategy.
Despite increasing knowledge of molecular structure of Env glycoprotein of HIV-1, its interaction with neutralizing antibodies and mechanism of immune response development, current vaccines induce immune response with low efficiency and insufficient breadth of HIV-1 variants. (T. Q. Zhou, I. Georgiev, X. L. Wu, Z. Y. Yang, K. F. Dai, A. Finzi, Y. D. Kwon, J. F. Scheid, W. Shi, L. Xu, Y. P. Yang, J. A. Zhu, M. C. Nussenzweig, J. Sodroski, L. Shapiro, G. J. Nabel, J. R. Mascola, P. D. Kwong, Structural Basis for Broad and Potent Neutralization of HIV-1 by Antibody VRC01. Science 329, 811-817 (2010); K. J. Bar, M. C. Sneller, L. J. Harrison, J. S. Justement, E. T. Overton, M. E. Petrone, D. B. Salantes, C. A. Seamon, B. Scheinfeld, R. W. Kwan, G. H. Learn, M. A. Proschan, E. F. Kreider, J. Blazkova, M. Bardsley, E. W. Refsland, M. Messer, K. E. Clarridge, N. B. Tustin, P. J. Madden, K. S. Oden, S. J. O'Dell, B. Jarocki, A. R. Shiakolas, R. L. Tressler, N. A. Doria-Rose, R. T. Bailer, J. E. Ledgerwood, E. V. Capparelli, R. M. Lynch, B. S. Graham, S. Moir, R. A. Koup, J. R. Mascola, J. A. Hoxie, A. S. Fauci, P. Tebas, T. W. Chun, Effect of HIV Antibody VRC01 on Viral Rebound after Treatment Interruption. New England Journal of Medicine 375, 2037-2050 (2016)).
One of innovative solutions to overcome current problems with development of efficient vaccination strategy against infection by HIV is to stimulate production of neutralizing antibodies targeting Env glycoprotein of HIV-1 virus by method of directed evolution of proteins that can represent protein replicas of epitopes recognized by well-characterized neutralizing antibodies (bn-mAb). These small binding proteins can be then used as recombinant antigens for the construction of a vaccine, which will stimulate immune system of the host to produce serum antibodies of required specificity and neutralizing breadth, analogical to the originally used neutralizing Env-specific monoclonal antibody (bn-mAb).
The present invention provides polypeptides mimicking the epitope of glycoprotein gp120 HIV-1 virus, recognized by monoclonal antibody VRC01, developed from artificial binding proteins identified by selection from highly complex combinatorial library of protein variants (
The present invention provides polypeptides mimicking the epitope of glycoprotein gp120 HIV-1 virus, which is recognized by broadly neutralizing antibody VRC01 (i.e., polypeptide antigens). These polypeptides contain an amino acid sequence: X1YKNX2INX3AX4X5VX6X7VKRX8IDX9ILAX10LP (SEQ ID NO. 1), with N-terminally or C-terminally linked alpha-helical structure, said alpha-helical structure is preferably sequence LAEAKVLANRELDKYGVSD (SEQ ID NO. 2). The alpha-helical structure is directly attached to SEQ ID NO. 1.
Preferably, the present invention provides polypeptides containing an amino acid sequence selected from the group comprising:
with N-terminally or C-terminally attached alpha-helical structure.
The present invention preferably provides polypeptides containing sequence selected from the group comprising:
Within the framework of the present invention it has been found that polypeptides of this invention bind to the entire IgG monoclonal antibody VRC01 as well as to the Fab fragment of the VRC01. The polypeptide can be arbitrarily extended at both sides, for instance to contain up to 100 amino acid residues, preferably up to 80 or up to 70 or up to 60 or up to 50 amino acids. The immune response elicited by the polypeptides can be affected by a combination of an initial immunization dose and further booster doses, in which a truncated version of the particular protein sequence can narrow the immune response and production of antibodies to a part of the cognate protein corresponding to SEQ ID NO. 1.
The present invention further provides a DNA sequence selected from the group comprising complementary DNA coding for the amino acid sequence of the polypeptides of the present invention, and DNA hybridizing with said complementary DNA under conditions of high stringency. Conditions of high stringency refer to the following conditions and solution for washing-off the labeled DNA probe: washing solution containing 0.5×SSC+0.1% SDS, temperature of 60° C.
The present invention further includes the use of said DNA sequence for the preparation of polypeptides or recombinant proteins produced in bacterial, yeast, insect, mammal or human host cells, and also these host cells, containing at least one DNA sequence of the present invention.
The present invention further includes the use of said DNA sequence as an active ingredient of DNA vaccine for prevention of HIV-1 virus infection. DNA vaccines contain DNA to be introduced into cells, so the host cells directly produce antigen stimulating preventative immune response. Immune cells recognize such antigen as heterogeneous structure, mature and are responsible for development of antigen-specific immune response. DNA can be introduced into the host organism freely or encapsulated in a protein to simplify host cell entry.
Polypeptides according to the present invention are suitable for use in pharmaceutical technology, especially as mimicking recombinant protein ligands for development of more efficient vaccine preventing HIV-1 virus infection. To this end, it is particularly preferred to attach further auxiliary proteins to the polypeptides of the present invention, said auxiliary proteins being suitable for stimulation of antibody production. Examples of auxiliary proteins include serum albumin, heat shock protein hsp70 or helical spacer protein TolA or its truncated version TolS. These auxiliary proteins can be covalently linked to polypeptides, thus forming a chimeric protein. Furthermore, the polypeptides of the present invention may be modified by an attachment of auxiliary N- or C-terminal sequences (tags), which allow their specific detection or their oriented immobilization to surface of carriers such as nanoliposomes, resulting in enhancement of immunization efficacy. Such tags include, for instance, affinity or detection tags as poly(His), FLAG, AviTag, HA, Myc, S-tag or V5-tag.
Polypeptides of the present invention, defined by the amino acid sequence shown above, stimulate production of serum antibodies after being used for immunization of experimental animals. Hyperimmune sera of the immunized animals suppressed infection of reporter cells by tested Env-pseudotyped viruses in the model system and this represents one of the key mechanisms of HIV-1 infection control and one of the targets for development of a preventative vaccine which is still not available at the market. The polypeptides were identified from ABD-derived library of randomized peptides as peptides with the highest specific binding to monoclonal antibody VRC01, which was identified in an individual infected by HIV-1 virus as one of the crucial factors maintaining long-lasting low level of HIV-1 in his serum and contributing to resistance against AIDS development even without the use of antiretroviral agents. VRC01 is known by its ability to neutralize a broad spectrum of HIV-1 variant identified in different regions of the world. The developed polypeptides mimic the structure recognized by VRC01 antibody (antibody-recognized epitope). The advantage of these polypeptides, in comparison to the vaccines currently being tested, is their easy preparation, stability and absence of posttranslational modifications, and this enables their easy biotechnological production in prokaryotic host cells Escherichia coli and their further utilization as vaccine antigens.
a-c. Sera from mice immunized with VRA017, VRA177, VRA019, and VRA017S specifically recognize HIV-1 Env.
Multimeric recombinant protein gp120 of “Clade B” with N-terminal His-tag and C-terminal V5-tag was prepared using a protocol described previously (Raska M, Takahashi K, Czernekova L, et al Glycosylation patterns of HIV-1 gp120 depend on the type of expressing cells and affect antibody recognition. The Journal of biological chemistry 2010, 285: 20860-9; Raska M, Moldoveanu Z, Novak J, et al Delivery of DNA HIV-1 vaccine to the liver induces high and long-lasting humoral immune responses. Vaccine 2008, 26: 1541-51; Raska M, Czernekova L, Moldoveanu Z, et al Differential glycosylation of envelope gp120 is associated with differential recognition of HIV-1 by virus-specific antibodies and cell infection. AIDS Res Ther 2014, 11: 23) in concentration 1.2 mg/mL. This protein was used for competition assays with VRA proteins.
Broadly neutralizing human anti-HIV-1 gp120 monoclonal antibody VRC01 (RRID: AB_2491019) was obtained from Dr. John Mascola (cat #12033) (Wu X, Yang Z-Y, Li Y, et al Rational Design of Envelope Identifies Broadly Neutralizing Human Monoclonal Antibodies to HIV-1. Science 2010, 329: 856-61) through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH. Fab fragment of VRC01 was prepared using Fab Micro Preparation kit (Pierce, ThermoFisher Scientific, Waltham, Mass.) according to the manufacturer's instructions. 125 μL of VRC01 antibody (100 μg) was applied onto a column containing equilibrated immobilized papain and incubated for 5 hours at 37° C. The digested antibody was eluted by centrifugation and added onto the column with equilibrated immobilized Protein A. The column was centrifuged to collect Fab fragment. The concentration of purified Fab fragment was measured. VRC01 IgG protein, as well as its Fab, were tested for its activity to bind gp120 when immobilized on Polysorp plate (NUNC, Roskilde, Denmark) in ELISA. Human IgG kappa 1 mg/mL (purified myeloma protein, Sigma-Aldrich, St. Luis, Mo.) was used as a negative control in ELISA as well as an isotype control for preselection in ribosome display, stored as 1 mg/mL source stock at −20° C. VRC01 mAb was used for ribosome display as a target protein, stored as 3 mg/mL source stock in PBS (pH 7.2) at −80° C.
ABD-derived combinatorial library was assembled by PCR (Ahmad J N, Li J, Biedermannova L, et al Novel high-affinity binders of human interferon gamma derived from albumin-binding domain of protein G. Proteins 2012, 80: 774-89) and used for in vitro translation and further ribosome display selection (Kuchar M, Vankova L, Petrokova H, et al Human interleukin-23 receptor antagonists derived from an albumin-binding domain scaffold inhibit IL-23-dependent ex vivo expansion of IL-17-producing T-cells. Proteins 2014, 82: 975-89). Three- and five-round RD selections were performed, 96-well Polysorp plates (NUNC) were coated by VRC01 IgG diluted in coating 100 mM bicarbonate/carbonate solution (pH 9.6) at a concentration according to the adjusted stringency in each round of ribosome display selection procedure: 1st round—50 μg/mL, 2nd round—25 μg/mL, 3rd round—10 μg/mL, 4th round—5 μg/mL and 5th round—5 μg/mL. Preselection procedure was performed in wells coated with human IgG1 kappa antibody (Sigma-Aldrich) at a constant concentration of 25 μg/mL in each round. Final cDNA after the third and fifth round of the selection was amplified by PCR and introduced into a pET-28b vector carrying cloned tolA-AviTag sequence (Krizova L, Kuchar M, Petrokova H, et al p19-targeted ABD-derived protein variants inhibit IL-23 binding and exert suppressive control over IL-23-stimulated expansion of primary human IL-17+ T-cells. Autoimmunity 2017, 50: 102-13) and introduced into E. coli XL1 blue host cells.
Protein variants were produced in the form of fusion recombinant proteins His6-VRA-TolA-AVI allowing in vivo biotinylation of the binding proteins at C-terminus (Krizova L, Kuchar M, Petrokova H, et al. p19-targeted ABD-derived protein variants inhibit IL-23 binding and exert suppressive control over IL-23-stimulated expansion of primary human IL-17+ T-cells. Autoimmunity 2017, 50: 102-13). Truncated fusion VRA proteins were constructed by replacement of full-length tolA (UniProt accession number: P19934, NCBI Reference Sequence: NC_000913.3) with its C-terminal part called tolS via PCR with primers tolA-C-end 5′-ATTAGGATCCCCGTCAGGGGCCGATATCAATAACTATGC-3′ (SEQ ID NO. 9) and tolA-AVI_rev1 5′-TTTCCGCTCGAGCTATTCGTGCCATTCGATTTTCTGAGCCTCGAAGATGTCGTTCAGG CCCGGTTTGAAGTCCAATGGCGC-3′ (SEQ ID NO. 10). VRA protein binders were produced as in vivo biotinylated proteins in E. coli BL21 (DE3) BirA strain with added 50 μM d-biotin (prepared 5 mM solution in 10 mM bicine buffer, pH 8.3) in LB medium containing kanamycin (60 μg/mL) and chloramphenicol (30 μg/mL). Protein production was induced at 35° C. by 1.5 mM IPTG after the culture reached the density OD600=0.6. Cells were harvested in 4 hours after induction, sonicated in TN buffer (50 mM Tris, 150 mM NaCl, pH 8.0), centrifuged (40 000×g, 20 min, 4° C.) and subsequently, bacterial lysates were analyzed or proteins were purified on Ni-NTA agarose column.
Cell lysates of clones of E. coli BL21 BirA host cells producing biotinylated protein variants were prepared using lysozyme solution (PBS buffer, 0.05% Tween, 1% lysozyme, 25 U/mL benzonase, pH 7.4) or using sonicator (Misonix 3000). Polysorp plate (NUNC, Roskilde, Denmark) was coated with VRC01 IgG1 (5 μg/mL) or IgG1 kappa (5 μg/mL) in coating buffer (100 mM bicarbonate/carbonate buffer, pH 9.6) at 7° C. overnight. Next day, the plate was washed by PBST solution (PBS buffer containing 0.05% Tween, pH 7.4) and wells were blocked by PBSTB (PBS buffer pH 7.4, containing 0.05% Tween and 1% BSA). The lysate samples (diluted 33×), purified protein variants as well as ABDwt negative control diluted in PBSTB were applied and their binding was detected using streptavidin Poly-HRP conjugate (Pierce) diluted in PBSTB 1:10 000. The V5-tagged gp120 recombinant protein was diluted in PBSTB and detected by anti-V5 tag-HRP conjugate in PBSTB (1:10 000). Protein binding was visualized by the enzymatic reaction of HRP with OPD substrate (Sigma-Aldrich, St. Luis, Mo., USA) in citrate buffer (3.31% sodium citrate tribasic dihydrate, phosphoric acid, pH 5.0), or TMB-Complete 2 substrate (TestLine Clinical Diagnostics s.r.o., Brno, Czech Republic) and the reaction was stopped by 2 M sulphuric acid, and the absorbance was measured at 492 or 450 nm, respectively. Using this method, almost 800 lysates of bacterial clones were screened and four variants were found, which preferentially bind to broadly neutralizing antibody VRC01 in comparison to control isotype antibody IgG kappa (
The wells of Polysorp plate (NUNC, Denmark) were coated with VRC01 IgG antibody or Fab fragment, diluted in coating buffer (5 μg/mL). The coted wells were blocked with PBSTB solution and V5-tagged gp120 was applied as a serially diluted competitor in PBSTB solution containing VRA protein variant at constant concentration (5 μg/mL) and binding of the in vivo biotinylated VRA017, VRA019 and VRA177 (as His6-VRA-TolA-AVI fusion protein) was detected by streptavidin-HRP conjugate. Alternatively, VRA017 protein was applied as a serially diluted competitor in PBSTB at the constant concentration of V5-tagged gp120 (1.2 μg/mL) and detection of the bound gp120 was performed using anti-V5 tag-HRP antibody conjugate. Results were visualized by the enzymatic reaction of HRP with OPD substrate (Sigma-Aldrich, MO) in citrate buffer, or TMB-Complete 2 substrate (TestLine Clinical Diagnostics s.r.o., Brno) and the reaction was stopped by 2 M sulfuric acid and absorbance at 492 or 450 nm was measured, respectively. This method was used to demonstrate results in which increasing amount of glycoprotein gp120 inhibits binding of VRA017, VRA019 and VRA177 to immobilized VRC01 IgG as well as to Fab fragment prepared by cleavage of VRC01 IgG (
Plasmid DNA coding for full-length protein variants was sequenced (Core facility—Genomics, Faculty of Science, Charles University, BIOCEV, Vestec). Multiple alignments of amino acid sequences were performed using NCBI BLAST (National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov/). Several dozen selected clones were analyzed and the most important ones are shown in Table 1.
Y Y K N L I N N A K T V E G V K A L I D E I L A A L P
A Y K N A I N R A V T V G L V K R V I D G I L A R L P
N Y K N R I N V A L G G T A V K R I I D A I L A A L P
R Y K N D I N P A S R V G A V K R V I D R I L A G L P
R Y K N D I N P A S R V G A V K R V I D R I L A G L P
All experiments were performed on 6-to 8-weeks old female BALB/c mice purchased from AnLab (Brno, Czech Republic) under standard housing conditions according to ARRIVE guidelines. The vaccination experiments were approved by the Ethics Committee of the Faculty of Medicine and Dentistry (Palacky University Olomouc, Czech Republic), and the Ministry of Education, Youth and Sports, Czech Republic (MSMT-15434/2015-7). Preimmune serum samples (130 μl per animal) were obtained using tail vein blood sample collection approach. Each mouse was immunized three times with the corresponding ABD/VRA variant. Two independent immunization experiments were performed. The first experiment used VRA017, VRA019, and VRA177 variants and ABD wild type (ABDwt) for the evaluation of immunogenicity and specificity of elicited murine serum antibodies. Results are presented in
To determine the reactivity of mice sera with HIV-1 Env, a recombinant trimeric Clade B gp120 MBL lacking detection or purification tags was used in ELISA. Maxisorp plates (NUNC, Roskilde, Denmark) were coated with gp120 MBL (50 ng/well) overnight at 4° C. Plates were washed and blocked with 1% BSA/PBS/Tween20 for 3 hours at room temperature. Sera were serially diluted (starting from dilution 1:100) in blocking buffer (in duplicates) and incubated overnight at 4° C., to identify a single dilution corresponding to the linear proportion of titration curves obtained from the majority of VRA-immunized animals used in the final comparison. Final serum dilution was set to 1:400. To detect the bound antibodies specific to gp120, plates were washed and incubated with rabbit anti-mouse IgG, IgG1, IgG2a, and IgM secondary antibody conjugated with horseradish peroxidase (Sigma-Aldrich, St. Luis, Mo., USA) diluted in blocking buffer for 3 hours at room temperature. Signal was developed with O-phenylenediamine-H2O2 substrate. The reaction was stopped with 1 M sulphuric acid and the absorbance was measured at 492 nm. This methodical procedure was used to obtain results shown in
Competition of VRC01 with Hyperimmune Mice Sera for Gp120 Binding Tested by ELISA
Plates were coated as described above for Env-specific serum antibody determination by ELISA. VRC01 serially diluted in blocking buffer (in doublets) was applied with mouse sera diluted 1:400. To detect bound murine antibodies, plates were washed and incubated with rabbit anti-mouse IgG secondary antibody conjugated with horseradish peroxidase diluted in blocking buffer for 3 hours at room temperature. Plates were developed and measured as mentioned above. This experimental procedure was performed to obtain results shown on
Neutralization assay was performed using various pseudoviruses from clade B and clade C produced in HEK293/17 cell line. Cells at 60-90% confluency in 75 cm2 culture flask were co-transfected using transfection reagent FuGene6 (Promega, Madison, Wis.). Before transfection, 8 μg of plasmid pSG3deltaEnv, 4 μg of plasmid encoding Env and 48 μl of FuGene6 were mixed with DMEM culture medium in a total volume of 800 μl and incubated 30 minutes at room temperature. Then, the mixture was added to 12 ml of RPMI-1640 in a flask with cells. After 2 days, culture medium with produced pseudoviruses was harvested, aliquoted and stored at −80° C. until used. Neutralization assay was performed using TZM-bl cell line stably expressing CD4 receptor, CCR5, and CXCR4 co-receptors and containing genes for luciferase and β-galactosidase under the control of HIV-1 long-terminal-repeat promotor (NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH). Serially diluted serum samples in duplicates were incubated with pseudoviruses at approximately 150 000 RLU in 150 μl of DMEM. After 90 minutes' incubation at 37° C., 100 μl of cells at a density of 105 cells/ml was added. The plate was incubated at 37° C. in 5% CO2 atmosphere for 48 hours. Then, 150 μl of culture medium was removed and 100 μL of lysis buffer containing luciferin (Promega) was added. After 2 minutes, 100 μL of lysed cells were transferred into black 96-well plates and luminescence was measured using HP luminometer. This methodical procedure was used for obtaining of a set of neutralization results summarized in Table 2. Our results show that sera of mice immunized with VRA177 and VRA017 proteins exhibit a neutralization activity, which is presented on the set of 13 prepared HIV pseudoviruses. Mice sera of VRA177 variant blocked the binding of 5 types of pseudoviruses to indicator human cells, while sera of mice immunized with VRA017 blocked binding of 2 types of pseudoviruses only. The best neutralizing activity was achieved by a combinatory immunization with VRA177TolA and VRA017S eliciting high titers of serum antibodies blocking the binding of 8 of 13 tested pseudoviruses. Detailed binding curves of hyper immune and naïve mice sera for each type of pseudovirus are shown on
Protein samples (0.2 mg/mL) in PBS and 5× Sypro Orange dye (Sigma-Aldrich) were mixed in a total volume of 25 μL and measured using the real-time PCR Detection System CFX96 Touch (Bio-Rad Laboratories, Hercules, Calif.) as described previously (Kuchar M, Vankova L, Petrokova H, et al Human interleukin-23 receptor antagonists derived from an albumin-binding domain scaffold inhibit IL-23-dependent ex vivo expansion of IL-17-producing T-cells. Proteins 2014, 82: 975-89). Thermal stability of proteins VRA017S, VRA019S and VRA177S is shown in
Differences between groups and statistical significance were determined by analysis of variance (ANOVA), Kruskal-Wallis test and Dunn's post-test. All statistical analyses were performed using SPSS v. 21 statistical packages (IBM Corp., Armonk, N.Y.) or GraphPad Prism 5 Software (GraphPad Software Inc, San Diego, Calif.).
| Number | Date | Country | Kind |
|---|---|---|---|
| PV 2019-585 | Sep 2019 | CZ | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CZ2020/050066 | 9/8/2020 | WO |
