Patent Application
20190298820
The present disclosure relates to certain epitopes in the C-terminal domain of the HIV-1 gp41 protein, and more specifically to compositions and methods for using the epitopes to develop vaccine protection against HIV.
The envelope protein of human immunodeficiency virus (HIV) is synthesized as a gp160 polypeptide, and is cleaved by a cellular protease to yield a non-covalently linked heterodimer—an external gp120 domain and a transmembrane, anchoring gp41 domain. The gp41 protein can be structurally sub-divided into three major domains: the extracellular domain (or ectodomain), the membrane-spanning domain, and the C-terminal tail (CTT). The CTT of gp41 protein has not received much attention as the target for the development of HIV vaccine. Efforts to design immunogens capable of presenting neutralizing epitopes targeting the CTT of gp41 are still largely unsuccessful.
There is a need for broadly neutralizing monoclonal antibodies (mAbs) that target the CTT and can provide protection from the HIV virus. Disclosed herein are compositions and methods that address the shortcomings of the art, and may provide any number of additional or alternative advantages.
The present disclosure relates to anti-HIV antibodies that have been linked to vaccine-induced protection and that interact with conformational epitopes derived from the CTT of gp41 of HIV-1. Embodiments of the disclosure include recombinant nucleic acid compositions containing sequences having 80% or more identity to nucleic acid sequence of the Kennedy Loop, which is a segment of peptides in the CTT. Embodiments of the disclosure include recombinant nucleic acid compositions containing sequences having 90% or more identity to nucleic acid sequence of the Kennedy Loop of gp41. Embodiments of the disclosure include polypeptide compositions containing amino acid sequences having 80% or more identity to amino acid sequence corresponding to the Kennedy Loop of gp41 and are conformationally constrained by the addition of cysteine sequences. Embodiments of the disclosure include polypeptide compositions containing amino acid sequences having 90% or more identity to amino acid sequence corresponding to the Kennedy Loop of gp41 and are conformationally constrained by the addition of cysteine sequences.
Embodiments include vaccine compositions effective against a Human Immunodeficiency Virus-1 (HIV-1) infection and containing a recombinant peptide sequence that has at least 90% or more sequence identity to C-terminal domain of Kennedy loop of a HIV-1 gp41 protein and is conformationally constrained by a two or more cysteine residues. Embodiments include vaccine compositions effective against a Human Immunodeficiency Virus-1 (HIV-1) infection and containing a recombinant peptide sequence that has at least 90% or more sequence identity to N-terminal domain of Kennedy loop of a HIV-1 gp41 protein and is conformationally constrained by a two or more cysteine residues. In certain embodiments, the cysteine residues are the terminal residues of the recombinant peptide sequence.
Embodiments of the disclosure include isolated HIV monoclonal antibodies comprising an antibody binding portion that interacts with a polypeptide with an amino acid sequence 90% or more identical to an amino acid sequence corresponding to the Kennedy Loop of gp41 and is conformationally constrained by the addition of cysteine sequences. Embodiments include monoclonal antibody compositions capable of specifically binding to a recombinant peptide sequence that has at least 90% or more sequence identity to C-terminal domain of Kennedy loop of a HIV-1 gp41 protein and is conformationally constrained by a two or more cysteine residues. Embodiments include monoclonal antibody compositions capable of specifically binding to a recombinant peptide sequence that has at least 90% or more sequence identity to N-terminal domain of Kennedy loop of a HIV-1 gp41 protein and is conformationally constrained by a two or more cysteine residues. Embodiments include monoclonal antibody compositions capable of specifically binding to C-terminal domain of the HIV-1 gp41 protein and containing a heavy chain with a variable region having at least 80% or more sequence identity to the 61p1B2 variable heavy sequence and a light chain with a variable region having at least 80% or more sequence identity to the 61p1B2 variable light chain sequence.
Numerous other aspects, features and benefits of the present disclosure may be made apparent from the following detailed description taken together with the drawing figures. The pharmaceutical compositions can include compositions described herein, other components, or ingredients depending on desired prevention and treatment goals. It should be further understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the disclosure as claimed.
The present disclosure can be better understood by referring to the following figures. The components in the figures are not necessarily to scale. The emphasis is instead placed upon illustrating the principles of the disclosure. In the figures, reference numerals designate corresponding parts throughout the different views.
Reference will now be made to the exemplary embodiments illustrated in the drawings, and specific language will be used here to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended. Alterations and further modifications of the inventive features illustrated here, and additional applications of the principles of the disclosures as illustrated here, which would occur to one skilled in the relevant art and having possession of this disclosure, are to be considered within the scope of the disclosure.
The disclosure provides methods, compositions and kits for preventing an HIV infection. For example, HIV envelope-like polypeptides (wild-type HIV polypeptides and mimotopes) may be administered to an individual so as to induce a protective immune response to HIV. Alternatively, antibodies directed to the HIV envelope-like polypeptides may be administered to an individual to treat or prevent an HIV infection and/or one or more symptoms associated with the infection (e.g., AIDS).
As used here, the following terms may have the following definitions:
The term “HIV” is meant to include different form of the Human Immunodeficiency Virus, such as HIV-1 and HIV-2 and also the simian immunodeficiency virus (SIV). HIV is organized into groups and subtypes (clades).
The terms “env polypeptide” or “envelope polypeptide” refer to a molecule derived from an HIV envelope protein. The envelope protein of HIV is a glycoprotein of about 160 kd (gp160). During virus infection of the host cell, gp160 is cleaved by host cell proteases to form gp120 and the integral membrane protein, gp41. The gp41 portion is anchored in (and spans) the membrane bilayer of the virion, while the gp120 segment protrudes into the surrounding environment. env polypeptides can exist as monomers, dimers or multimers.
As used herein, the term “vaccine(s)” or “vaccine composition” means a recombinant product, the administration of which is intended to elicit an immune response(s) that can prevent and/or lessen the severity of one or more infectious diseases.
As used herein, a “mimotope” is a polypeptide, which differs from an envelope polypeptide by one or more amino acids but which mimics the three dimensional structure of a wild-type envelope epitope. A mimotope generally in the context of a larger protein backbone called carrier is able to stimulate a host's immune system to produce an antibody antigen-specific response. The host generates antibodies that specifically bind to the mimotope and the corresponding wild-type envelope epitope.
As used herein, “antigen” refers to a molecule containing one or more epitopes/mimotope (either linear, conformational or both) that will stimulate a host's immune system to make a humoral and/or cellular antigen-specific response. The term is used interchangeably with the term “immunogen.” Normally, a B-cell epitope will include at least about 5 amino acids but can be as small as 3-4 amino acids. A T-cell epitope, such as a CTL epitope, will include at least about 7-9 amino acids, and a helper T-cell epitope at least about 12-20 amino acids. Normally, an epitope will include between about 7 and 15 amino acids, such as, 9, 10, 12 or 15 amino acids. Antigens of the present disclosure include the polypeptides of
The term “specifically binds” or “specifically binding” means a high avidity and/or high affinity binding of an antibody to a specific antigen (e.g., the Kennedy loop or fragments thereof). Antibody binding to its epitope on this specific antigen is stronger than binding of the same antibody to any other epitope, particularly those which may be present in molecules in association with, or in the same sample, as the specific antigen of interest. Antibodies which bind specifically to a polypeptide of interest may be capable of binding other polypeptides at a weak, yet detectable, level (e.g., 10% or less of the binding shown to the polypeptide of interest). Such weak binding, or background binding, is readily discernible from the specific antibody binding to the polypeptide of interest, e.g., by use of appropriate controls.
As used herein, unless otherwise noted, the terms “treating”, “treatment” and the like, shall include the management and care of a subject or patient (preferably mammal, more preferably human) for the purpose of combating a disease, condition, or disorder and includes the administration of a compound of the present disclosure to prevent the onset of the symptoms or complications, alleviate the symptoms or complications, or eliminate the disease, condition, or disorder. The term “therapeutically effective amount” as used herein, means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
Illustratively, an effective amount of the compositions of this disclosure ranges from nanogram/kg to milligram/kg amounts of the compositions alone or in combination with other compounds for young children and adults. Equivalent dosages for lighter or heavier body weights can readily be determined. The dose should be adjusted to suit the individual to whom the composition is administered and will vary with age, weight and metabolism of the individual. The exact amount of the composition required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the particular peptide or polypeptide used, its mode of administration and the like. An appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. One skilled in the art will realize that dosages are best optimized by the practicing physician or veterinarian and methods for determining dose amounts and regimens.
The compositions herein are formulated in accordance to the mode of potential administration. Thus, if the composition is intended to be administered intranasally or by inhalation, for example, the composition may be a converted to a powder or aerosol form, as conventional in the art, for such purposes. Other formulations, such as for oral or parenteral delivery, are also used as conventional in the art. Compositions for administration herein may form solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
Optimal dosages to be administered may be readily determined by those skilled in the art, and will vary with the particular compound used, the mode of administration, the strength of the preparation, the mode of administration, the number of consecutive administrations within a limited period of time (e.g. up to 60 minutes) and the advancement of the disease condition. In addition, factors associated with the particular patient being treated, including patient age, weight, diet and time of administration, will result in the need to adjust dosages. The term “subject” as used herein, refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment. Preferably, the subject has experienced and/or exhibited at least one symptom of the disease or disorder to be treated and/or prevented.
The following Examples are set forth to aid in the understanding of the disclosure, and are not intended and should not be construed to limit in any way the disclosure set forth in the claims which follow thereafter.
Six rhesus monkeys (RMs) from a vaccine study were found to be protected from virus challenges after vaccination either completely or partially, the latter being defined as 1 log lower peak viremia compared to the mean viremia of the unvaccinated controls (one rhesus monkey had borderline protection, and five had clear vaccine failure during the first five low-dose challenges with a heterologous simian-human immunodeficiency virus (SHIV), i.e., a virus with an HIV-1 envelope that differed from the HIV-1 envelope given as an immunogen in the form of trimeric gp160 (
The Kennedy Loop region of gp41 was designed and constructed as a novel constrained loop, linked to green fluorescent protein (GFP) and was used as a bait to isolate B cells specific to the Kennedy Loop. The entire Kennedy Loop of 40 amino acids was used to generate cysteine-constrained loops of sub-regions thereof (See
Analysis of the protein sequences of the Kennedy Loop for structural stability and hydrophobicity index showed that the Kennedy Loop is very flexible and may not retain any structure. Keeping this in mind, two cysteine amino acids were synthesized at the ends of all the sequences to create novel constrained and structurally stable epitope loops. DNA sequences were synthesized after codon optimization for recombinant expression in E. coli. The DNA and amino acid sequences, as shown in
The Kennedy Loop-GFP recombinant fusion proteins and scrambled Kennedy Loop-GFP recombinant fusion proteins were tested for their binding specificity to sera of rhesus monkeys # RTr-11 and # RRi-11 (vaccine-protected monkeys) as well as to rhesus monkey # RDo-11 (vaccinated rhesus monkey with vaccine failure; please see black boxed rhesus monkey in
The Kennedy Loop-GFP recombinant fusion proteins and scrambled Kennedy Loop-GFP recombinant fusion proteins were also tested for their specific binding and non-binding to a panel of monoclonal antibodies, as shown in
Four years after the vaccination, the vaccine-protected rhesus monkeys were boosted with the two out of the three original immunogens (trimeric clade C HIV1084i gp160 and HIV-1 Tat) to expand the memory B cell pool. The antigens (trimeric HIV-1 1084i gp160 and HIV-1 Tat) were the exact same immunogens (even same batch) that those used for vaccination. Additionally, Gag-Pol particles had been part of the vaccine regimen but were not included in the boost. FACS sorting for single memory B cells was done 14 days after the vaccine boost in rhesus monkey # RTr-11. K-CTcc fused to GFP was used as a recombinant bait protein for the isolation of cognate memory B cells from PBMC of rhesus monkey # RTr-11. Live cell flow cytometry sorting for single memory B cells was performed 14 days after the vaccine boost in rhesus monkey # RTr-11. Kennedy Loop-specific single memory B cells were sorted into a PCR plate as follows: CD3−, CD19+, CD27+, IgG+ and K-CTcc-GFP+ cells (
The B cells sorted with fluorescently-tagged K-CTcc were used to generate recombinant monoclonal antibodies. Using single-cell cDNA synthesis and nested PCR for IgG-specific gene amplification, we were able to isolate 29 gamma, 12 kappa and 17 lambda genes. After cDNA synthesis, light chain variable (VL) Ig genes and heavy chain variable (VH) Ig genes were amplified by semi-nested PCR with a set of newly developed primers specific to rhesus monkey VL and VH Ig genes. Primers for amplification of rhesus monkey immunoglobulin VH and VL genes were selected as previously described in Sholukh A M, et al. (2012) (Isolation of Monoclonal Antibodies with Predetermined Conformational Epitope Specificity. PLoSONE 7(6): e38943. doi:10.1371/journal.pone.0038943).
N=A+G+C+T; V=A+C+G; D=A+T+G; B=T+C+G; H=A+T+C; W=A+T; S=C+G; K=T+G; M=A+C; Y=C+T; R=A+G.
Using these primers under the experimental conditions above, about 29 gamma, 12 kappa and 17 lambda genes were isolated. The pairs of VH and VL genes obtained after two rounds of PCR were sequenced to assess productivity and gene rearrangement as well as to obtain sequence information for the beginning of framework region 1 (FR1). After amplification of VH/VL pairs with cloning primers, the PCR fragments were inserted into vectors of the pFUSE2- family that contain constant region sequences of human Ig light (Igk or Ig12) or heavy (Igc1) chains. This cloning strategy yielded chimeric simian-human IgG1 monoclonal antibodies.
About 10 pairs of full-length Ab genes were cloned into commercial vectors in the first round of screening. Antibody genes from those single B cells that were able to provide a pair of VH and VL genes (gamma with kappa or gamma with lambda) by nested PCR were cloned into pFUSE2 vectors (as shown in
Embodiments of the disclosure include isolated HIV monoclonal antibodies, or antigen binding fragment thereof, containing a heavy chain and a light chain. In certain embodiments, the heavy chain includes a heavy chain variable region with an amino acid sequence at least 80% identical to one of the amino acid sequences set forth in
Small scale expression after co-transfection of 293T cells revealed expression of 4 monoclonal antibodies. Full-length IgG1 monoclonal antibodies were produced by transient co-transfection of the paired heavy and light chain pFUSE plasmids into Expi293F cells (Invitrogen) grown in serum-free FreeStyle™ 293Expression Medium (GibcoH Invitrogen) using the TransITPRO™ Transfection Kit (Mirus Bio). Cells were cultivated for 4 days at 37° C./8% CO2 with continuous shaking at 135 rpm. Supernatants were collected, filtered through 0.22 μm filters and supplemented with Halt Protease Inhibitor Cocktail (ThermoFisher) and 1006 penicillin-streptomycin solution (GibcoH Invitrogen). Next, supernatants were tested for binding to HIV-1 env and mimotopes, and positive monoclonal antibodies were affinity-purified using protein A agarose (GE Healthcare) according to manufacturer's instructions. IgG concentrations were determined by measuring absorbance at 280 nm on Nanodrop 1000 (Thermo Scientific) using the IgG default protocol. In previous attempts using linear mimotopes without C-C constraints, most of the monoclonal antibodies generated from single-cell sorted memory B cells did not show the expected specificity for the bait protein(s) and/or HIV-1 env or peptides thereof.
Glycerol stocks of bacterial cultures with mAb plasmids were expanded to make
Attorney Docket No. 0074714.000104 midi and maxi preps; plasmids were used to transfect large cultures of Expi293F cells to produce glycosylated full-length monoclonal antibodies. These were expressed (yield 5 to 10 μg/ml) and purified using protein A affinity chromatography.
At least seven monoclonal antibodies were tested and were analyzed for specific binding to Kennedy Loop-GFP mimotopes, native Kennedy Loop-GPF fusion protein, and gp41 or gp160 of some HIV envelopes. Monoclonal antibodies, which showed specific binding to consensus HIV-1 clade C env peptides representing the Kennedy Loop region, were identified. Six anti-Kennedy Loop monoclonal antibodies were tested for their binding efficacy. Binding of anti-Kennedy Loop monoclonal antibodies to HIV-1 envelope proteins obtained from the NIH AIDS Research and Reference Reagent Program (ARRRP) was analyzed. Five of them showed specific binding to K-CTcc and HIV-1 gp160, as shown in
Neutralization by TZM-bl cell-based assays was not observed, but the monoclonal antibodies did show virus neutralization in human peripheral blood mononuclear cell (PBMC)- based assays. TZM-bl cell based assay utilize a genetically engineered cell line (TZM-bl) that are susceptible to infection by most strains of HIV-1, SIV, and SHIV. This assay as many other assays is based on the same principle, measuring reductions in virus infectivity as described in Curr. Protoc. Immunol. 2005 Jan; Chapter 12: Unit 12.11. doi: 10.1002/0471142735.im1211s64 by D. Montefiori. However, TZM-bl cells display an abnormally high number of CCR5 coreceptor molecules on the cell surface, which yield false positive neutralization data for a number of anti-gp41 neutralizing monoclonal antibodies, especially those with a relatively slow on-rate.
The newly isolated anti-Kennedy Loop monoclonal antibodies had no reactivity to self-antigens, such as ds DNA, SM proteins, RNPs, SS-B/La antigens, cardiolipin, and SS-A/Ro antigens and were negative against scrambled sequences of Kennedy Loop proteins. Auto reactivity was tested with an anti-dsDNA EIA kit, anti-Sm/RNP EIA kit, anti-Sm EIA kit, autoimmune EIA anti-SS-A/Ro Test, autoimmune EIA anti-SS-B/La test, Bio-Rad Kallestad ANA screen (all Bio-Rad) and QUANTA LiteH ACA IgG III (INOVA Diagnostics). Assays were performed on automated PhD System (Bio-Rad) and DSXTM System (Dynex Technologies).
Monoclonal antibodies did not bind to or capture virion as expected. ELISA plates (Nunc) were coated with 5 μg/ml of goat anti-human IgG Fc specific Ab (Jackson Immuno Research) overnight at 4° C. After blocking and washing, monoclonal antibodies were added at 5 μg/ml and incubated for 2 hours. The plates were washed, SHIV-1157ipELp (the SHIV strain that had been used as challenge virus in the vaccinated rhesus monkeys earlier) was added to the monoclonal antibodies and incubated for 20 hours, after which the plates were washed again and incubated with 0.5% Triton X-100 for 1 hour to release p27 from the virus bound to the various monoclonal antibodies. The amount of p27 released was determined using a p27 SIV capture kit (ABL, Inc).
The non-binding of anti-Kennedy Loop monoclonal antibodies to virions was expected, because the Kennedy Loop is not exposed on intact virions. Rather, the Kennedy Loop is transiently exposed at the time of either virus entry into or exit from host cells. During this transient phase, structural changes in the HIV-1 env trimer lead to unfolding of some env domains. The Kennedy Loop is thought to appear transiently on the cell surface thereby giving antibodies a chance to hit otherwise unexposed target epitopes crucial for viral entry into the cells. Virion capture experiments were performed multiple times, even with improved, bead-based techniques and including multiple controls.
Engineered SupT1 cells overexpressing CCR5 receptors (SupT1.R5) were exposed to a high inoculum of HIV-1 (in this case HIV-1 1084i) and incubated for 48 h. Cells were then incubated with anti-Kennedy Loop monoclonal antibodies or isotype controls for 1 hour at 4 ° C. followed by binding to goat anti-human Fc FITC-labeled antibody. Cells were
Attorney Docket No. 0074714.000104 fixed and kept for flow cytometry. Simultaneously, a control experiment was also run on non-infected cells to rule out any background issues. Results of one of the representative assay are shown in
Monoclonal antibodies exhibiting competitive inhibition between Kennedy Loop-GFP recombinant fusion protein and recombinant bacteriophage displaying the Kennedy epitope were isolated from a vaccine-protected rhesus monkey. Competition phage ELISA was performed between K-NTcc recombinant fusion protein and the recombinant phages bacteriophages displaying Kennedy-region mimotopes that had been isolated from vaccine-protected rhesus monkeys. The names of the mimotopes are listed in the right upper corner of the graph in
Strong antibody-dependent phagocytosis (ADCP) was observed in the presence of these monoclonal antibodies. Monoclonal antibodies were incubated with inert fluorescent beads coated with gp160 antigen. After 1 hour, the samples were incubated with THP-1 monocytic cells, and about 12 to 24 hours later, the cells were fixed and analyzed on flow cytometry.
The link of antibodies to the gp41 Kennedy Loop with vaccine-induced protection demonstrates the suitability of this region for vaccine design. Embodiments described here
Attorney Docket No. 0074714.000104 include isolation and characterization of the selective monoclonal antibodies with the use of several Kennedy Loop-based mimotopes. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims. LISTING OF CLAIMS This listing of claims replaces all prior versions, and listings, of claims in the application.
This invention was made with government support under Grant Nos. P01 AI048240 and R01 AI100703 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US17/60342 | 11/7/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62418729 | Nov 2016 | US |
