WO2024173143A2 - Vaccines incorporating hiv th/ctl epitope peptides to prevent and treat patients with hiv infection and aids - Google Patents

Abstract

An HIV Th/CTL vaccine composition is provided. The HIV Th/CTL vaccine composition includes a Th/CTL peptide. A method for treating HIV infection and AIDS in a subject is also provided. The method includes administering a pharmaceutically effective amount of the HIV Th/CTL vaccine composition to the subject. An HIV vaccine composition, a Global HIV T vaccine composition, a method for preventing and treating HIV infection in a subject are also provided.

Description

VACCINES INCORPORATING HIV TH/CTL EPITOPE PEPTIDES TO PREVENT

ANO TREAT PATIENTS WITH HIV INFECTION ANO AIDS

The present application is a PCT International Application that claims the benefit of

U.S. Provisional Application Serial No. 63/484,580, filed February 13, 2023, which is incorporated herein by reference in its entirely.

FIELD OF THE INVENTION

The present disclosure relates to vaccines incorporating HIV Th/CTL epitope peptides to prevent and treat patients with HIV infection and AIDS.

BACKGROUND OF THE INVENTION

AIDS is a chronic, potentially life-threatening condition caused by Human Immunodeficiency Virus (HIV). HIV atacks the human immune system. Without treatment, HIV infection will progress In stages with the CD4+ T cell population declining slowly as disease progresses, eventually leading to its late-stage condition, with depletion of the CD4 cells, known as Acquired Immunodeficiency Syndrome (AIDS).

Since the introduction of antiretroviral drugs for treatment of AIDS over the past two decades, HIV related morbidity and mortality has been substantially reduced, and the long term outcome for those with HIV has improved. These drugs have also played a key role in HIV prevention. Antiretroviral treatment (ART) effectively suppresses viremia below the clinically detectable level, increases survival, improves quality of life, and decreases onward transmission.

However, ART is not curative and periodically emerging HIV- 1 from latent reservoir (LR) has to be drug-controlled for the rest of the patients life. Life-long provision of ART poses not only significant economic and logistical challenges, but heightens concerns for social stigma, unwanted toxicity, and emergence of viral drug resistance particularly if the use of ART is suboptimal. In addition, the use of ART for pre-exposttre prophylaxis (PrEP) will further stretch its supply. ¹¹⁵

Vac-cme approach has the potential to prevent HIV infection and cure AIDS in humans. However, despite near 40 years of extensive global research effort, there is currently no vaccine available to effectively prevent HIV infection, treat HIV infection or AIDS. HIV vaccine researchers have long been focused on (1) elicitation of broadly neutralizing antibodies (bNAbs.) by targeting HIV gpl60 Env to directly block viral entry, thus preventing infection by interfering with engagement of host cell receptor (CD4) or coreceptors (CCR5 or CXCR4); or (2) passive administration of bNAbs to protect against heterologous HIV infection. HIV bNAb induction, however, is particularly challenging.' bNAbs take years to develop in natural HIV infections as they often have high levels of somatic mutation, insertions and deletions, with unusually long CDRH3 regions. Thus, even if vaccine elicitation of bNAbs is achieved, response to any one bNAb epitope is unlikely to provide universal sterilizing protection against circulating HIV-l .

Several tines of evidence, including genome-wide association studies in humans and experimental studies with Simian Immunodeficiency Virus (SIV) in macaques, support the notion that CD8* T cells play an essential role in mediating viral control in elite controllers. Mme specifically, the emergence of HJ.V-specifi.c CD8+ T cell responses has consistently been associated with reduction in peak virus replication during primary infection; rapidescape from. HIV-specific CDtH T cell responses has been observed in acute infection, indicating antiviral function for at least a subset of these cells; and in an animal model of AIDS, elimination of CD8+ lymphocytes in SIV infected rhesus monkeys during chronic SIV infection resulted in a rapid and marked increase in viremia that was again suppressed coincidently with the reappearance of SI V-speci fic CD8+ T cells. These results confirm the importance of cell-mediated immunity in controlling HIV-1 infection and support the exploration of HIV T cell vaccination approaches to elicit the effector cell-mediated immunity

HIV controllers can suppress viral replication without antiretroviral medications. HIV- specific CD8+ T cells from controllers, compared with those from progressors, have greater capacity to proliferate and develop cytolytic potential upon in vitro antigenic stimulation. By contrast, CD8+ T cells from progressors often exhibit strong ex vivo activation but fail to proliferate or acquire cytolytic capacity due to exhaustion and necroptotic cell death, and these deficiencies are not restored despite prolonged ART. In comparison with progressors, CD8+ T ceil responses in HIV controllers show an overall increased ability to maintain long- term memory' and effector potential, with the ability to kill infected cells before progeny virions are produced, providing a clear rationale for exploiting these features for treatment, prevention and cure. ^{(4 )}

Since specific CD 8+ T ceils are capable of limiting HIV- 1 replication in acute infection or in untreated controllers, an effective HIV vaccine should therefore harness CD8+ T ceils against HIV infection. In light of the extensive genetic diversity of HIV, antigenic diversification is therefore essential in the design of an effective T ceil vaccine to prevent escape from immune selection and therapy, a major obstacle in currem global HIV vaccine development strategy.

Thirty-seven million people are currently living with HIV and AIDS. This situation continues to have devasting healthcare and economic impact throughout the glebe. There remains an urgent need to develop an effective HIV vaccine to effectively prevent HIV infection or treat HIV infection or AIDS.

The present disclosure is directed to a vaccine system against HIV to treat those with HIV infection and AIDS. More specifically, the vaccine system employs as its T cell immunogen ten site-directed HIV Th/CTl. epitope peptides that are conserved in their sequences across all HIV isolates and promiscuous in their respective HLA bindings. They are designed with high precision, optimized for manufacture, and amenable for incorporation in the vaccine formulations to provide optimal T cell immunity to the vaccinees.

REFERENCES

Each patent, pubheation, and non-patent literature cited in the application is hereby incorporated by reference in its entirety as if each was incorporated by reference individually.

(.1 ) Tomas Hanke, Aiming for protective T-cell responses: a focus on the first-generation conserved region HIVconsv vaccines in preventive and therapeutic clinical trials. Expert review of vaccine 2019, Vol. 18, No. 10, 1029-1041 .

(2) Bette Korber et al., T cell-based strategies for HIV-1 vaccines. Vaccines Immunotlterapeufics 2020, Vol, 16 No, 3, 713-722,

(3) lorn E. Schmitz et al , Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes. Science 1999, Vol 283, is. 5403 p. 857-860,

(4) David R. Collins et al., CD8+ T cells in HIV control, cure and prevention. Nature

Reviews Immunology 2020, Vol. 20, 471 .

(5) Chang Yi Wang et al., A multitope SARS-CoV-2 vaccine provides long-lasting B cell and T cell immunity against Delta and Omicron variants J Clin Invest 2022, 132(10):e 157707

(6) Otto O. Yang et al.. Short Conserved Sequences of HI V- 1 Are Highly Immunogenic and

Shift immunodominance J, of Virology 2015 Vol. 89, Is 2 , P. 1195-1204

(7) Hayato Murakoshi l et al., CD8+ T cells specific for conserved, cross-reactive Gag epitopes with strong ability to suppress HIV-1 replication, Retrovirology 2018 15:46

(8) Chengcheng Zou el at, Effec-tive Suppression, of HIV- I Replication by Cytotoxic T Lymphocytes Specific for Pol Epitopes in Conserved Mosaic Vaccine Immunogens. .1. Virology 2019 Vol. 93 Is. 7

SUMMARY OF THE INVENTION

In accordance with some embodiments of the present disclosure, an HIV Th/CTL vaccine composition is provided. The HIV Th/CTL vaccine composition includes a Th/CTL peptide.

In accordance with some embodiments of the present disclosure, a method for treating HIV infection and AIDS in a subject is provided. The method includes administering a pharmaceutically effective amount of the HIV Th/CTL vaccine composition io the subject.

In accordance with some embodiments of the present disclosure, an HIV vaccine composition is provided. The HIV vaccine composition includes the components in the amounts shown in Table 2.

In accordance with some embodiments of the present disclosure, an HIV Th/CTL vaccine composition is provided. The HIV Th/CTL vaccine composition includes a) a gp160-region based protein, b.) a mixture of HIV Th/CTL peptides comprising: SEQ ID NOs: 1-10: and c) a UBIThibla peptide (SEQ ID NO: 11). In accordance with some embodiments of the present disclosure, an HIV Th/CTL vaccine composition is provided. The HIV Th/CTL- vaccine composition includes the aforementioned protein and a pharmaceutically acceptable carrier and/or adjuvant.

In accordance with some embodiments of the present disclosure, a Global HIV T vaccine composition is provided. The Global HIV T vaccine composition includes a Th/CTL peptide.

In accordance with some embodiments of the present disclosure, a Global HIV T vaccine composition is provided. The Global HIV T vaccine composition includes a) a Th/CTL peptide selected from the group consisting of SEQ ID NOs.T-1 1 and any combination thereof; and b) a pharmaceutically acceptable excipient.

In accordance with some embodiments of the present disclosure, a method for preventing and treating HIV infection in a subject is provided. The method includes administeriiig a pharmaceutically effective amount of the aforementioned vaccine composition to the subject.

In accordance with some embodiments of the present disclosure, an HIV Th/CTL vaccine composition is provided. The HIV Th/CTL vaccine composition includes the components in the amounts shown in Table 2.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention can be more fully understood by reading the subsequent detailed description and examples with references made to the accompanying drawings, wherein:

Figures 1A-1J: H'PLC profiles for purified HIV Th/CTL peptides with SEQ ID NOs: 1 to 10. Designer peptides were synthesized, purified as described in EXAMPLE 1 , Only designer pepiides that can be purified by preparative HPI..C to high purity with reasonable yield will be selected for use as peptide immunogens.

'Figures 2A and 2B illustrate the components and manufacturing compounding processes of the protein/peptide HIV T ceil vaccine. More specifically, Figure 2A illustrates the components of the HIV Th. CTL vaccine, with optional gp!60-region based protein as the B immunogen. The HIV T cell vaccine composition contains ten synthetic Th/CTL pepiides (SEQ ID NOs: 1-10) for class 1 and II MHC molecules derived from HIV Th/CTL Gag/Pol/VIF proteins, and the UBITh®ia peptide (SEQ ID NO: 1 1 ) as a catalyst for T cell activation. These components are mixed with CpG J (SEQ ID NO: 12) which binds to the positively (designed) charged peptides by dipolar interactions and also serves as an adjuvant, which is then bound to Alum adjuvant to constitute the vaccine compositions. Figure 2B. illustrates the compounding processes for the manufacturing of HIV Th/CTL Vaccines against HIV isolates with optional inclusion of Gpl6()~egio.n based protein as B immunogen. To produce the vaccine composition, sequential addition of peptides, CpGI, ahim adjuvant and finally the protein component is carried out. Specifically, the designer Th/CTL peptides are added to WFI, followed by the addition of CpGl in the mixture to form the peptides /CpGI complex. Thereafter, the protein buffer, Alum and NaCI are added to the solution which now contains peptides/CpGl/Alum/NaCI. Finally, the gp 160-region based protein solution is added io the solution mixture io arrive at the final vaccine product.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is directed to a vaccine system against HIV to prevent infection and treat those with HIV infection and AIDS. More specifically, the vaccine system employs as its T cell immunogen a set of 10 peptides that are conserved in their sequences across all HIV isolates and promiscuous in their respective HLA bindings. They are designed with high precision, optimized for manufacture, and amenable for incorporation in the vaccine formulations io provide optimal T cell immunity to the vaccinees.

In summary, the disclosed vaccine system utilizes bioinformaties including amino acid sequences from HI V Gag, Pol and VIF proteins for the design and manufacture of optimal HI V Gag, Pol and VIF derived antigenic Th/CTL epitope peptides (SEQ ID NOs: 1-10), and formulations thereof, as HI V T cell vaccines for the treatment of HIV infection and AIDS.

General

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All references or portions of references cited in this application are expressly incorporated by reference herein in their entirety for any purpose.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill hi the art to which this invention belongs. The singular terms “a.” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Hence, the phrase “comprising A or B” means including A, or B, or A and B. It is further to be understood that al! amino acid sizes, and all molecular weight or molecular mass values, given for polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed method, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

!. Peptide processing of the target protein and three-wav recognition are required for a Th cell response.

Immune responses require the cooperative interaction between antigen-presenting cells and T helper (Th) cells. The elicitation of an effective antibody response or effector T cell response requires that antigen-presenting cells recognize the target antigenic site of a subject immunogen and that the T helper cells recognize a T helper ceil epitope. Generally, the T helper epitope on a subject immunogen is different from its B cell epitope(s) or related effector T cell (e.g., cytotoxic T lymphocyte or CTL) epitope(s). The B cell and related effector T cell (CTL) epitopes are sites on a desired target immunogen that is recognized by B cells and related effector cells, which results in the production of antibodies or cytokines against the desired target site. The natural conformation of the target determines (he site to which the antibody or related effector T cell directly binds. Evocation of a Th cell response requires a Th cell receptor to recognize a complex on the membrane of an antigen-presenting cell that is formed between a processed peptide fragment of a target protein and an associated class II major histocompatibility complex (MHC). Thus, peptide processing of the target protein and three-way recognition are required for a Th cell response. The three part complex is difficult to define because 1 ) the critical MHC class II contact residues are variably positioned within different MHC binding peptides (Th epitopes); 2) the different MHC binding peptides have variable lengths and different amino acid sequences; and 3) MHC class II molecules can be highly diverse depending on the genetic make-up of the host. The immune responsiveness to a particular Th epitope is, in part, determined by the MHC genes of the host, and the reactivity of Th epitopes differ among individuals of a population. Th epitopes that are reactive across species and individuals (i.e., promiscuous Th epitopes) within a single species are difficult to identify.

Multiple factors are required for each component step of T cell recognition, such as appropriate peptide processing by the antigen-processing cell. presentation of the peptide by a genetically determined class II MHO molecule, and recognition of an MHC molecule and peptide complex by the receptor on Th cells. The requirements for promiscuous Th epitope recognition for providing broad responsiveness can be difficult to determine.

It is clear that for the induction of antibodies and related cytokines against immune responses, the immunogen must comprise both the B cell epitope/effeclor CTL T cell epitope and Th cell determinant(s).

The present disclosure is directed to a vaccine system against HIV isolates to treat those with HIV infection. More specifically, the vaccine system employs as its T cell immunogen site-directed HIV Th/CTL epitope peptides also designed with high-precision, and synthesized for incorporation in the vaccine formulations to provide optimal T cell immunity io the HIV vaccinees.

The disclosed vaccine system utilizes bioinformaiics including amino acid sequences for the optimal design and manufacture of HIV Gag-PoL VIF protein derived Th/CTL epitope peptides (SEQ ID NOs: 1-10) and T helper cell (Th) epitope derived from a pathogen protein (e.g., SEQ ID NO; 1 1 ), and formulations thereof as vaccines against HIV. Each aspect of the disclosed invention is discussed in further details

2, HIV Th/CTi. Peptides

A long-lasting cellular response could augment the initial neutralizing response (through memory B ceil activation) and provide much greater duration of immunity as antibody titers wane.

To provide immunogens to elicit T ceil responses, Th/CTL epitopes from highly- conserved sequences derived from Gag, Pol, and VIF proteins of HIV were identified after extensive literature search and data review. Several peptides within these regions were selected and subject to further designs. Each selected peptide contains Th or CTL epitopes with prior validation of MHC I or II binding and exhibits good manufacturability characteristics (optimal length and amenability for high quality synthesis). These rationally designed Th/CTL peptides were further modified by addition of a Lys-Iys-Lys sequence to the N terminus of the respective peptides to increase their respective positive charges thus facilitating each peptide's subsequent binding to the highly negatively charged CpG oligonucleotide molecule to form immunostimulatory complexes through “charge neutralization”. The designs and sequences of the ten final peptides and their respective HPLC profiles are shown in Table 1 and Figures 1A-LI.

To enhance the immune response, a proprietary peptide UBITWla (SEQ ID NO: 1 1) can be added to the peptide mixture of the vaccine composition. UBlTht^la is a proprietary synthetic peptide with an original framework sequence derived from the measles virus fusion protein (MVF). This sequence was also further modified to exhibit a palindromic profile within the sequence to allow accommodation of multiple MHC class II binding motifs within this short peptide of 19 amino acids.

Such immunostimulaiory complexes have been shown to enhance otherwise weak or moderate response of the companion target immunogen (e.g., WO 2020/132275A1.). CpGl is designed to bring the rationally designed immunogens together through “charge neutralization” to allow generation of balanced B ceils (induction of neutralizing antibodies) and Th/CTL responses in a vaccinated host. In addition, activation of TLR-9 signaling by CpG is known to promote IgA production and favor Thl immune response. liBITh®! peptide is incorporated as one of the Th peptides for its “epitope cluster” nature to further enhance the HIV derived Th and CTL epitope peptides for their antiviral activities. The nucleic acid sequence of CpGl is SEQ ID NO: 12. in view of the above, the HIV multi tope T peptide vaccine composition can contain one or more Th/CTL peptides. The Th/CTL peptides can include: a. peptides derived from the HIV Gag-Pol (e.g., SEQ ID NOs: 1-9); b« peptides derived from the HIV VIF protein (e.g., SEQ ID NO: 10); c. an artificial Th epitope derived from pathogen proteins (e.g., SEQ ID NO: 1 1 ).

The vaccine composition can contain one or more of the Th/CTL peptides. In certain embodiments, the vaccine composition conta ins a mixture o f more than one Th/CTL peptide. When present in a mixture, each Th/CTL peptide can be present in any amount or ratio compared to the other peptide or peptides. For example, the Th/CTL peptides can be mixed in equimolar amounts, equal-weight amounts, or the amount of each peptide in the mixture can be different than the amount of the other peptidefsi in the mixture. If more than two Th/CTL peptides are present in the mixture, the amount of the peptides can be the same as or different from any of the other peptides in the mixture.

The amount of Th/CTL peptide(s) present in the vaccine composition can vary depending on the need or application. The vaccine composition can contain a total of between about 0,1 μg to about 100 μg of the Th/CTL peptide(s). In some embodiments, the vaccine composition contains a total of between about 1 ug to about 50 μg of the Th/CTL peptide(s).

In certain embodiments, the vaccine composition contains a mixture of SEQ ID NOs: I- 10 and 11 , These Th/CTL peptides can be mixed in equimolar amounts, equalweight amounts, or the amount of each peptide in the mixture can be different than the amount of the other peptide(s) in the mixture. In certain embodiments, these Th/CTL peptides are mixed in equal-weight amounts in the vaccine composition.

The presence of Th and CTL epi topes in phannaceuticaVvaccine formulations prime the immune response in treated subjects by initiating antigen specific T cell activation, which correlates to protection from and treatment of HIV infection. Additionally, formulations that include carefully selected endogenous Th epitopes and/or CTL epitopes presented on proteins from HIV can produce broad cell mediated immunity, which also makes the formulations effective in treating and protecting subjects having diverse genetic makeups.

Including one or more endogenous HIV Th/CTL epitope peptides in a pharmaceutical composition allows the epitopes to be seen and processed by antigen presenting B cells, macrophages, dendritic cells, etc. These cells process the antigens and present them to the surface to be in contact with the T cells to trigger further T cell responses to help mediate killing of the virus infected ceils. The endogenous HIV CTL epitope peptides contain a Lys-Lys-Lys (KKK) tail at the N-tenniaus.

The endogenous Th/CTL epitope peptides of SEQ ID NOs: 1-10 are particularly useful when used in a pharmaceutical composition that has been formulated into an immunostimulatory complex with a GpG oligonucleotide (ODN), because the cationic KKK tail is capable of interacting with the CpG ODN through electrostatic association. The use of endogenous HIV Th epitopes in the peptide immunogen construct can enhance the immunogenicity of the HIV B cell epitope peptides to facilitate the production of specific high titer antibodies, upon infection, directed against the optimized B cell epitope peptide screened and selected based on design rationales.

In some embodiments, the pharmaceutical composition contains one or more HI V B epitopes together with one or more separate peptides containing an endogenous HIV Th/CTL epitope peptide (SEQ ID NOs: 1-10, or any combination thereof). 3. Excipients

The vaccine composition can also contain, a pharmaceutically acceptable excipient.

As used herein, the term “excipient” or “excipients” refers to any component in the vaccine composition that is not (a) the gp!6O region-based designer protein or (b) the Th/CTL pepiide(s). Examples of excipients include carriers, adjuvants, antioxidants, binders, buffers, bulking agents, chelating agents, coloring agents, diluents, dislntegrams, emulsifying agents, surfactants, solvents, fillers, gelling agents, pH buffering agents, preservatives, solubilizing agents, stabilizers, and the like. Accordingly, the vaccine composition can contain a pharmaceutically effective amount of an active pharmaceutical ingredient (API), such as the gplfiO region -based designer protein and/or one or more Th/CTL peptides, together with a pharmaceutically acceptable excipient.

The vaccine composition can contain one or more adjuvants that act to accelerate, prolong, or enhance the immune response to the API without having any specific antigenic effect itself. Adjuvants can include oils, oil emulsions, aluminum salts, calcium salts, immune stimulating complexes, bacterial and viral derivatives, virosomes, carbohydrates, cytokines, polymeric microparticles. In certain embodiments, the adjuvant can be selected from a CpG oligonucleotide, alum (potassium aluminum phosphate), aluminum phosphate (e,g. .ADJU-PHOS®), aluminum hydroxide (e.g, ALHYDROGEL®), calcium phosphate, incomplete Freund's adjuvant (IFA), Freund’s complete adjuvant, MF59, adjuvant 65, Lipovant, 1SCOM, liposyn, saponin, squalene, LI 21, EMULSIGEN®, EmulsIL-fin®, monophosphoryl lipid A (MPL), Quil A, QS21 , MONTANIDE® ISA 35, ISA 50V, ISA

50V2, ISA 51, ISA 206, ISA 720, liposomes, phospholipids, peptidoglycan, lipopolysaccahrides (LPS), ASO1, ASO2, ASO3, ASO4, AF03, lipophilic phospholipid (lipid A), gamma inulin, algammalin, glucans, dextrans, glucomannans, galactomanmrns, levans, xylans, dimethykliocAadecylanimonium bromide (DDA), as well as the other adjuvants and emulsifiers.

In some embodiments, the vaccine composition contains ADJ1J-PHOS® (aluminum phosphate), MONTANIDE™ ISA 51 (an oil adjuvant composition comprised of vegetable oil and manmde oleate for production of water-in-oil emulsions), TWEEN® 80 (also known as: Polysorbate SO or Polyoxyethylene (20) sorbitan monooleate), a CpG oligonucleotide, and/or any combination thereof. In other embodiments, the pharmaceutical composition is a water-in-oil-in-waler (i.e., w/o/w) emulsion with EMULSIGEN or EMULSIGEN D as the adjuvant.

In certain embodiments, the multitope protein/peptide vaccine composition contains ADJU-PHOS® (aluminum phosphate) as the adjuvant to improve the immune response. Aluminum phosphate serves as a Th2 oriented adjuvant via the nucleotide binding oligomerization domain (NOD) like receptor protein 3 (NLRP3) inflammasome pathway. Additionally, it has pro-phagocytic and repository effects with a long record of safety and the ability to improve immune responses to target proteins in many vaccine formulations.

The vaccine composition can contain pH adjusters and/or buffering agents, such as hydrochloric acid, phosphoric acid, citric acid, acetic acid, histidine, histidine HOHaO, lactic acid, tromethamine, gluconic acid, aspartic acid, glutamic acid, tartaric acid, succinic acid, malic acid, fumaric acid, a-ketoglularic acid, and arginine HCI,

The vaccine composition can contain surfactants and emulsifiers, such as olyoxyethylene sorbitan fatty acid esters (Polysorbate, TWEEN®), Polyoxyethylene 15 hydroxy stearate (Macrogol 15 hydroxy stearate, SOLUTOL HS15®), Polyoxyethylene castor oil derivatives (CREMOPHOR.® EL, ELP, RH 40), Polyoxyethylene stearates (MYRJOv), Sorttitan fatty acid esters (SPAN®), Polyoxyethylene alkyl ethers (BRIM), and Polyoxyethylene noaylphenol ether (NON OXYN OL®) .

The vaccine composition can contain carriers, solvents, or osmotic pressure keepers, such as water, alcohols, and saline solutions (e.g., sodium chloride). The vaccine composition can contain preservatives, such as alkyl/aryl alcohols (e.g., benzyl alcohol, chlorbmanol, 2-ethoxyethanol), amino aryl acid esters (e.g., methyl, ethyl, propyl butyl parabens and combinations), alkyl/aiyl acids (e.g., benzoic acid, sorbic acid), biguanides (e.g., chlorhexidine), aromatic ethers (e.g., phenol, 3-cresol 2-phenoxyethanol), organic mercurials (e.g,, thimerosak pheriylmerctrrate salts).

4. Formulations

The vaccine composition can be formulated as immediate release or for sustained release formulations. Additionally, the vaccine composition can be formulated for induction of systemic, or localized mucosal, immunity through immunogen entrapment and coadministration with microparticles. Such delivery systems are readily determined by one of ordinary skill in the art.

The vaccine composition can be prepared as an injectable, either as a liquid solution or suspension. Liquid vehicles containing the vaccine composition can also be prepared prior to injection. The vaccine composition can be administered by any suitable mode of application, for example, t.d., i.p., i.m., intranasal I y, orally, subcutaneously, etc. and in any suitable delivery device. In certain embodiments, the vaccine composition is formulated for subcutaneous, intradermal, or intramuscular administration. The vaccine composition can also be prepared for other modes of administration, including oral and intranasal applications.

The vaccine composition can also be formulated in a suitable dosage unit form, in some embodiments, the vaccine composition contains from about 1 ug to about 1 ,000 μg of the API (e,g., the gpldO region-based designer protein and/or one or more of the Th/CTL peptides). Effective doses of the vaccine composition can vary depending upon many different factors, including means of administration, target site, physiological state of the subject, whether the subject is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the subject is a human, but nonhuman mammals can also be treated. When delivered in multiple doses, the vaccine composition may be conveniently divided into an appropriate amount per dosage unit form. The administered dosage will depend on the age, weight and general health of the subject as is Well known in the therapeutic arts.

In some embodiments, the vaccine composition contains a gpI60 region-based designer protein or peptide, and one or more Th/CTL peptides in a formulation with additives and/or excipients, tn certain embodiments, the vaccine composition contains a gp.160 region-based designer protein and more than one Th/CTL peptides in a formulation with additives and/or excipients. A vaccine composition containing a mixture of more than one Th/CTL peptides can provide synergistic enhancement of the immunoefficacy of the composition. A vaccine composition containing a gp 16{)-based designer protein and more than one Th/CTL peptides in a formulation with additives and/or excipients can be more effective in a larger genetic population compared to compositions containing only the designer protein or one Th/CTL peptide, due to a broad MHC class 11 coverage, thus providing an improved immune response to vaccine composition.

When the vaccine composition contains a gp160 region-based designer protein and one or more Th/CTL peptides as the API, the relative amounts of the designer protein and the Th/CTL peptides can be present in any amount or ratio to each other. For example, the designer protein and the Th/CTL peptide(s) can be mixed in equimolar amounts, equalweight amounts, or the amount of the designer protein and the Th/CTL peptidefs) can be different. In addition, if more than one Th/CTL peptide is present in {he composition, the amount of the designer protein and each Th/CTL peptide can be the same as or different from each other. In some embodiments, the molar or weight amount of the designer protein is present in the composition in an amount greater than the Th/CTL peptides. In other embodiments, the molar or weight amount of the designer protein is present in the composition in an amount less than the Th/CTL. peptides. The ratio (weightrweight) of the designer protein to Th/CTL pepiide(s) can vary depending on the need or application. In some instances, the ratio (w:w) of the designer peptide to Th/CTL peptide(s ) can be 70:30, 80:20, or 90: W.

5. Methods

The present disclosure is also directed to methods for making and using the vaccine composition and formulations thereof. a. Methods for Manufactttrmg the gplSO Region-Based Designer Protein and Th/CTL Pentides

The disclosed Th/CTL peptides can be manufactured according to the methods described in Example 1 . h. Methods for Using the Vaccine Composition

In ireaiment applications, the disclosed mullitope protein/peptide vaccine composition can be administered to a subject with HI V infection or AIDS to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease.

The amount of the vaccine composition that is adequate to accomplish prophylactic treatment is defined as a prophylactically-effective dose. The disclosed multitope proiein/peptide vaccine composition can be administered to a subject in one or more doses to produce a sufficient immune response in order to prevent or treat an infection by HIV. Typically, the immune response is monitored, and repeated dosages are given if die immune response starts to wane.

The vaccine composition can be formulated as immediate release or for sustained release formulations. Additionally, the vaccine composition can be formulated for induction of systemic, or focalized mucosal immunity through immunogen entrapment and coadministration with microparticles. Such delivery systems are readily determined by one of ordinary skill in the art.

The vaccine composition can be prepared as an injectable, either as a liquid solution or suspension. Liquid vehicles containing the vaccine composition can also be prepared prior to injection. The vaccine composition can be administered by any suitable mode of application, for example, Id., i.p„ Im., mtranasally, orally; subcutaneously etc. and in any suitable delivery device. In certain embodiments, the vaccine composition is formulated for subcutaneous, intradermal or intramuscular administration. The vaccine composition can also be prepared for other modes of administration, including oral and inlranasa! applications.

The dose of tlie vaccine composition will vary depending upon the subject and the particular mode of administration. The dosage required will vary according to a number of factors known to those skilled in the art, including, but not limited to the species and size of the subject. The dosage may range from I μg to 1 ,000 μg of the combined weight of the designer protein and the Th/CTL peptides. The dosage can be between about 1 μg to about 1 .mg, between about 10 μg to about 500 ug, between about 20 μg to 200 μg of the combined weight of the designer protein and the Th/CTL peptides. The dosage, as measured by the combined weight of the designer protein and the Th/CTL peptides is about. 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about ! 00 μg, about 1 10 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about 160 p.g, about 170 μg, about 180 μg, about 190 μg, about 200 μg, about 250 ug, about 300 μg, about 400 μg, about 500 μg, about 600 μg, about 700 μg, about 800 μg, about 900 μg, about 1 ,000 μg. The ratio (weightrweight) of the designer protein to Th/CTL peptide(s) can vary depending on the need or application. In some instances, the ratio (WAV) of the designer protein to Th/CTL peptide(s) can be 70:30, 80:20, or 90: 10. In specific embodiments, the vaccine composition contains the components shown in Table 2,

The vaccine composition can be administered in a single dose, in multiple doses over a period of time, or by continuous infusion. The vaccine composition can be administered continuously or according to a specific dosage schedule. The effective doses may be extrapolated from dose-response curves obtained from animal models. In some embodiments, the vaccine composition is provided to a subject in a single administration. In other embodiments, the vaccine composition is provided to a subject in multiple administrations (two or more). When provided in multiple administrations, the duration between administrations can vaiy depending on the application or need, In some embodiments, a first dose of the vaccine composition is administered to a subject and a second dose is administered about 1 week to about 12 weeks after the first dose. In certain embodiments, the second dose is administered about 1 week, about 2- weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks after the first administration. In a specific embodiment, the second dose is administered about 4 weeks after the first administration.

A booster dose of the vaccine composition can be administered to a subject following an initial vaccination regimen to increase immunity aaainsi HIV, In some embodiments, a booster dose of the vaccine composition is administered to a subject about 6 months to about 10 years after the initial vaccination regimen. In certain embodiments, the booster dose of die vaccine composition is administered about 6 months, about I year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years after the initial vaccination regimen or after the last booster dose.

SPECIFIC EMBODIMENTS

(1 ) An HIV Th/CTL vaccine composition comprising a Th/CTL peptide. (2) The HIV Th/CTL vaccine composition according to (I), wherein the Th/CTL peptide is derived from the HIV Gag-poi protein of SEQ ID NOs: 1-9, the HIV CTL peptide derived from VIF protein of' SEQ ID NO: 10, a pathogen protein, or any combination thereof.

(3.) The HIV Th/CTL vaccine composition according to (2), wherein aj the Th/CTL peptide derived from the HIV Gag-pol protein is SEQ ID NOs: 1-9; b).the Th peptide derived from a pathogen protein is selected from the group consisting ofSEQ ID NO: 1 1.

(4) The HIV Th/CTL vaccine coinposition according to (3), further comprising a mixture of Th/CTL peptides of SEQ ID NOs: 1-1 1.

(5) The HIV Th/CTL vaccine composition according to (4), wherein each of the Th/CTL peptides are present in the mixture in equal-weight amounts.

(6) The HIV Th/CTL vaccine composition according to (5), wherein the ration (w:w) of the gpl60-region based protein to the total weight of the mixture of Th/CTL peptides is 89: 11 .

(7) The HIV Th/CTL vaccine composition according to (5), wherein the pharmaceutically acceptable excipient is an adjuvant, buffer, surfactant, emulsifier, pH adjuster, saline solution, preservative, solvent, or any combination thereof.

(8) The HIV Th/CTL vaccine composition according to (5), wherein the pharmaceutically acceptable excipient is selected from the group consisting of a CpG oligonucleotide, ADJIJPHOS (aluminum phosphate), histidine, histidine HCbfhO, arginine

HCI, TWEEN 80 (polyoxyethylene (20) sorbitan monooleate), hydrochloric acid, sodium chloride, 2-phenoxyethanol, water, and any combination thereof.

(9) A method for treating HIV infection and AIDS in a subject comprising administering a pharmaceutically effective amount of the HIV Th/CTL vaccine composition according to (8) to the subject .

(10) The method according to (9), wherein the pharmaceutically effective amount of the vaccine composition is administered to the subject in two doses.

(11) The method according to (10), wherein a first dose of the vaccine composition is administered to the subject and a second dose of the vaccine composition is administered to the subject about 4 weeks after the first dose.

(12) An HIV vaccine composition comprising the components in the amounts shown in Table 2.

(13) An HIV Th/CTL vaccine composition comprising: a) a gp 160-region based protein, b) a mixture of HIV WCTL peptides comprising; SEQ ID NOs; 1-10; and c) a UBITh® I a peptide (SEQ ID NO: 1 I).

( 14) An HIV Th/CTL vaccine composition comprising the protein according to (13) and a pharmaceutically acceptable carrier and/or adjuvant.

( 15) The HIV Th/CTL vaccine composition according to ( 14) further comprising a HIV Th/CTL peptide selected from the group consisting of: SEQ ID NOs; 1-10; and any combination thereof.

(16) The HIV Th/CTL vaccine composition according to (15) further comprising a UBITh®! a peptide (SEQ ID NO: 1 1 ).

(17) The HIV Th/CTL vaccine composition according to (16) further comprising: a) a HIV Th/CTL peptide selected from the group consisting of: SEQ ID NOs: 1-10, and any combination thereof; and b) a UBIThSla peptide (SEQ ID NO: 11).

( 18) The HIV Th/CTL vaccine composition according to any one of (13-18), wherein the pharmaceutically acceptable carrier and/or adjuvant is CpG I (SEQ ID NO* 12).

(19) A Global HIV T vaccine composition comprising a Th/CTL peptide.

(20) The Global HIV T vaccine composition according to (19), wherein the Th/CTL peptide is derived from the HIV Gag-pol protein, the HIV VIF protein, and a pathogen protein, or any combination thereof

(21 ) The Global HIV T vaccine composition according to (20), wherein a), the Th/CTL peptide derived from the HIV Gag-Pol is SEQ ID NO: 1-9; b). the Th/CTL peptide derived from the HIV VIF protein is selected from the group consisting of SEQ ID NO: 10; c). the Th/CTL peptide derived from a pathogen protein is selected from the group consisting of SEQ ID NOs: 11. (22) The Global HIV T vaccine composition according to (21), wherein the Th/CTL peptides is a mixture of SEQ ID NOs: 1-11.

(23) The Global HIV T vaccine composition according to (22), wherein each of the Th/CTL peptides are present in the mixture in equal-weight amounts,

(24) The Global HIV T vaccine composition according to (23), further comprising a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is an adjuvant, buffer, pH adjuster, saline solution, preservative, solvent, or any combination thereof

(25) The Global HIV T vaccine composition according to (24), wherein the pharmaceutically acceptable excipient is selected from the group consisting of a CpG oligonucleotide, ALUM (aluminum phosphate or Alhydrogel), hydrochloric acid, sodium chloride, 2 -phenoxyethanol, water, and any combination thereof

(26) A Global HIV T vaccine composition comprising; a), a Th/CTL peptide selected from the group consisting of SEQ ID NOs: 1-11 and any combination thereof; b). a pharmaceutically acceptable excipient.

(27) The Global HIV T vaccine composition according to (26), wherein the Th/CTL peptides in (a) is selected from the group consisting of SEQ ID NOs: 1-11 and any combination thereof

(28) The Global HIV T vaccine composition according to (27, wherein each of the Th/CTL peptides are present in equal-weight amounts,

(29) The Global HIV T vaccine composition according to (28), wherein the pharmaceutically acceptable excipient is an adjuvant, buffer, pH adjuster, saline solution, preservative, solvent, or any combination thereof.

(30) The Global HIV T vaccine composition according to (29), wherein the pharmaceutically acceptable excipient is selected from the group consisting of a CpG oligonucleotide, ALUM (aluminum phosphate or aluminum hydroxide), hydrochloric acid, sodium chloride, 2-phenoxyethanol, water, and any combination thereof.

(31) The Global HIV T vaccine composition according to (30), wherein the Th/CTL peptide is a mixture of SEQ ID NOs: 1-11, wherein each peptide is present in the mixture in equal-weight amounts; the pharmaceutically acceptable excipient is a combination of a CpGl oligonucleotide, ALUM(aluminum phosphate or aluminum hydroxide), hydrochloric acid, sodium chloride, and 2-phenoxyethanol in water.

(32) A method for preventing and treating HIV infection in a subject comprising administering a pharmaceutically effective amount of the vaccine composition according to (31) to the subject.

(33) An HIV Th/CTL vaccine composition comprising the components in the amounts shown in Table 2. EXAMPLE 1

Synthesis of HI V related peptides

Methods for - synthesizing HIV related Th and CTL peptides as immunogens- for vaccine development are described. The peptides can be synthesized in small-scale amounts that are useful for serological assays, laboratory pilot studies, and field studies, as well as in large-scale (kilogram) amounts for use in commercial production of pharmaceutical compositions. A large repertoire of HIV related Th/CTL epitope peptides having sequences with lengths from approximately 9 to 40 amino acids were designed and selected as peptide immunogen constructs for use in vaccine formulations.

Table 1 provides the sequences of Th/CTL peptides (SEQ ID NOs: 1-10) derived from HIV Gag-pol and VIF proteins with known MHC binding activities as designer peptides (e.g. with KKK. as a linker at the N-terminus to increase its positive charges for belter formulation) along with their respective HPLC profiles (Figures 1A-1 J) for inclusion in the final HIV vaccine formulations.

All peptides that can be used for immunogenicity studies or related tests were synthesized on a small-scale using F-moc chemistry by peptide synthesizers of Applied

BioSystems Models 430A, 431 and/or 433. Each peptide was produced by an independent synthesis on a solid-phase support, with F-moc protection at the N-terminus and side chain protecting groups of trifunctional amino acids. After synthesis, the peptides were cleaved from the solid support with side chain protecting groups removed with 90% Trifluoroacetic acid ( I t A r Synthetic peptide preparations were evaluated by Matrix-Assisted Laser Desorption/lonization-Time-Of-Flight (MALDI-TOF) Mass Spectrometry to ensure correct molecular weights and amino acid content. Each synthetic peptide was evaluated by Reverse Phase HPLC (RP-HPLC) to confirm the synthesis profile and concentration of the preparation. Despite rigorous control of the synthesis process including stepwise monitoring of the coupling efficiency, peptide analogues were also produced due to unintended events during elongation cycles, including amino acid insertion, deletion, substitution, and premature termination. Thus, synthesized preparations typically included multiple peptide analogues, though in minute amounts, along with the targeted peptide.

Despite the inclusion of such unintended peptide analogues, the resulting synthesized peptide preparations were nonetheless suitable for use in immunological applications and as peptide immunogens. Typically, such peptide analogues were frequently as effective as the purified peptide, as long as a discerning QC procedure is developed to monitor both the manufacturing and the evaluation processes to assure the reproducibility and efficacy of the final product employ ing these peptides. Large scale peptide syntheses in the multi-hundred to kilo gram quantities were conducted on a customized automated peptide synthesizer UBI2003 at 15 mmole to 150 mmole scale.

For active ingredients used in the final pharmaceutical composition for clinical trials or commercial use, peptide immunogen constructs were purified by preparative RP-HPLC under a shallow elution gradient and characterized by MALDI-TOF mass spectrometry, amino acid analysis and RP-HPLC for purity and identity.

EBMEtEl

Compounding process for the manufacturing of designer HIV T cell vaccines against HIV isolates

Figures 2A and 2B illustrate the components and manufacturing compounding processes of the protein/peptide HIV T cell vaccine. More specifically, Figure 2A illustrates the components of the HIV Th/CTL vaccine, with optional gpl60-region based protein as the B immunogen. The HIV T cell vaccine composition contains ten synthetic Th/CTL peptides (SEQ ID NOs: 1-10) for class I and II MHC molecules derived from HIV Th/CTL Gag/Pol/VIF proteins, and the UBITh® la peptide (SEQTD NO: 11) as a catalyst for T cell activation. These components are mixed with CpGl (SEQ ID NO: 12) which binds to the positively (designed) charged peptides by dipolar interactions and also serves as an adjuvant, which is then bound to Alum adjuvant to constitute the vaccine compositions. Figure 2B. illustrates the compounding processes for the manufacturing of HIV Th/CTL Vaccines against HIV isolates with optional inclusion of Gpl60-egion based protein as B immunogen. To produce the vaccine composition, sequential addition of peptides, CpG 1, alum adjuvant and finally the protein component is carried out. Specifically, the designer

Th/CTL peptides are added to WFI, followed by the addition of CpG I in the mixture to form the pepticles/CpGl complex. Thereafter, the protein bufler, Alum and NaCl are added to the solution which now contains peptides/CpGl/AtoniZNaCl. finally, the gpl60-region. based protein solution is added to the solution mixture to arrive at the final vaccine product.

HMBO

Special T cell assays: for assessment of Th and CTL activities in vaccines

T cell responses bv ELISPOT

Human peripheral blood mononuclear cells (PBMCs) were used in the detection of the T cell response. Antigen-specific interferon-gamma (IFN-y) measurement to assess cellular (T cell) immune response is performed by an ELISpot method using human IFN-y

ELISpotPLUS kit (ALP) (MABTECH). ELISpot assays can also be perfomed using the human li \-y l l -4 FluoroSpot¹ ’ ' " kit (MABTECH). Aliquots of 250,000 PBMCs are plated into each well and stimulated, respectively, with 10 pg/mL (each stimulator) of protein solution, Th/CTL, or Th/CTL pool without UBiThla (HIV Th/CTL peptides), and cultured in culture medium alone as negative controls for each plate for 24 hours at 37 °C with 5% COz. The analysis is conducted according to the manufacturer’s instructions. Spotforming units (SFU) per million cells is calculated by subtracting the negative control wells. IntraceIIuIarCytokineStaining(lCS)

Intracellular cytokine staining and flow cytometry are used to evaluate CD4⁺ and

CD8⁺ T cell responses. PBMCs are stimulated, respectively, with protein plus HIV Th/CTL peptide pool, HIV Th/CTL peptide pool only, PMA + Inonmycin (as positive controls), or cultured in culture medium alone as negative controls for 6 hours at 37°C with 5% COa, Following stimulation, cells are washed and stained with viability dye for 20 minutes at room temperature, followed by surface stain for 20 minutes at room temperature, cell fixation and permeabilization with the BD cytofix/cytoperm kit (Catalog # 554714) for 20 minutes at room temperature, and then intracellular stain for 20 minutes at room temperature. Intracellular cytokine staining of DFN-y, IL-2 and IL-4 is used to evaluate CD4⁺ T cell response. Intracellular cytokine staining of IFN-y, IL-2, CD 107a and Granzyme B is used to evaluate CD8⁺ T cell responses. Upon completion of staining, cells are analyzed in a FACSCanto II flow cytometry (BD Biosciences) .using BD FACSDiva software.

EXAMPLE 4

High precision designer HI V T cell vaccine to treat HIV infection and AIDS employing HIV Th/CTL epitope peptides and optionally a gp!60 region based- protein as the B immunogen.

Rationale of UBI HIV multitope Th/CTL T cell vaccine

Vaccine approach has the potential to prevent HIV infection and cure AIDS in humans. However, despite near 40 years of extensive global research effort, there is currently 110 vaccine available io eflectively -prevent HIV infection, treat HIV infection or

AIDS. HIV vaccine researchers have long been focused on (1) elicitation of broadly neutralizing antibodies (bNAbs) by targeting HIV gp!60 Env to directly block viral entry thus prevent- infection by interfering with engagement of host cell receptor (CD4) or coreceptors (OCRS or CXCR4); or (2) passi ve administration of bNAbs to protect against heterologous HIV -infection. HIV bNAb induction, however, is particularly challenging: bNAbs take years to develop in natural HIV infections as they often have high levels of somatic mutation, insertions and deletions, with unusually long CDRH3 regions. Thus, even if vaccine elicitation of bNAbs is achieved, response to any one bNAb epitope is unlikely to provide universal sterilizing protection against circulating HIV-L ^<2)

Several lines of evidence, including genome-wide association studies in humans and experimental studies with Simian Immunodeficiency Virus (SIV) in macaques support the notion that CD8+ T cells play an essential role in mediating viral control in elite controllers:

(1) The emergence of HIV-specific CD8+ T cell responses has consistently been associated with reduction in peak virus replication during primary infection; (2) Rapid escape from HIV-specific CD8+ T cell responses has been observed in acute infection, indicating antiviral function for at least a subset of these cells; (3) In an animal model of AIDS, elimination of CD8+ lymphocytes in SIV infected rhesus monkeys during chronic SIV infection resulted in a rapid and marked increase in viremia that was again suppressed coincidently with the reappearance of SI V-specific CD8+ T cells. These results confirm the importance of cell-mediated immunity in controlling HIV-1 infection and support the exploration of vaccination approaches to elicit these effector cell-mediated immunity ^<3).

HIV controllers can suppress viral replication without antiretroviral medications. HIV- specific CD8+ T cells from controllers, compared with those from progressors, have greater capacity to proliferate and develop cytolytic potential upon in vitro antigenic stimulation. By contrast, CD8+ T cells from progressors often exhibit strong ex vivo activation but fail to proliferate or acquire cytolytic capacity due to exhaustion and necroptotic cell death, and these deficiencies are not restored despite prolonged ART. In comparison with progressors, CD8+ T cell responses in HIV controllers show an overall increased ability to maintain long- term memory and effector potential, with the ability to kill infected cells before progeny virions are produced, providing a clear rationale for exploiting these features for treatment, prevention and cure. ⁽⁴⁾ Since specific CD8+ T cells are capable of limiting HIV-1 replication in acute infection or in untreated controllers, an effective HIV vaccine should therefore harness CD8+ T cells against HIV infection. In light of the extensive genetic diversity of HIV, antigenic diversification is therefore essentia! in the design of an effective T cell vaccine to prevent, escape from immune selection and therapy; a major obstacle in current global HIV vaccine development strategy.

Vaccine immunogen design is important to elicit enhanced CU8+ T-cell immune and humoral responses. We assessed closely two important studies conducted lately including one study using vaccine containing four small peptides from the conserved HIV-1 protein regions comprising less than one-fifth of the corresponding whole Gag/Env/NEF protein contained in a control vaccine. The peptide based vaccine was able to elicit in vaccinees equivalent total magnitudes of both antigen specific CD4+ and CD8+ T lymphocyte responses when compared to the corresponding whole Gag/'En.v/NEF protein contained in a control vaccine. These data demonstrate the immunogenicity of these small peptides selected from the conserved regions can function as effective immunogens in a vaccine to steer innnunodominance toward conserved epitopes. ^if” A second study also involves a second-generation conserved region vaccine, lHlVconsvX vaccine, which employed computational strategies to determine the potential of multiple T cell epitopes selected from across the HIV proteome as the T-cell mosaic vaccine immunogens. Importantly. tHIVconsvX vaccine, comprised of six peptides derived from the conserved Gag and Pol regions, has demonstrated that the T-cell responses to peptides derived from these vaccine immunogens were found in vaccinces to be significantly associated with lower plasma viral load and higher CD4+ T-cell count in HIV- 1 -infected, treatment-naive Japanese individuals in a clinical trial. In this study, T ceils specific for 5 Gag and 6 Pol conserved epitopes in the IHIVconsvX have demonstrated their ability to suppress replication of circulating viruses in HIV- 1 -infected individuals with improved clinical outcome in 221 HIV- 1 -infected individuals, incl tiding detailed comparison of responders and non-responders having the same restricting HLA alleles. ⁽ ' These clinical trial results indicated that xtHIVconsvX vaccine is able to induce Gag and Pol specific cytotoxicity T lymphocyte (CTL) responses that are associated with control of HIV-1 replication and increase of CD4+ T cell count in infected individuals

To develop a T-cell-based vaccine against HIV, we have designed a set of 10 synthetic peptides containing immunodominant CTL. or Th epitopes of different lengths (9 - 43 amino acid) derived from Gag, Pol and VIF proteins. The epitope selection and peptide design were based on high sequence conservation and coverage of a wide range of HLA associations. A Lys-Lys-Lys sequence is added to the N terminus of each designer Th/CTL peptide thus increasing its positive charges to allow subsequent binding to the highly negatively charged CpG oligonucleotide molecule with adjuvants of Alhydrogel® or Adju-PhoS’W to form immunostimulatory complexes through “charge neutralization”, These peptides were further synthesized and analyzed for their scalability and amenability for ease in operations meeting Chemistry-Mauufacturing-Controi criteria under GMP conditions.

Final 10 designer peptides passing our rigorous screening processes with their respective sequences shown in Table 1 and respective HPLC profiles in Figures 1 A-l J are selected as peptide immunogen components for use in our HIV Th/CTL vaccine targeted for extensive clinical trials in Taiwan.

A similar multitope vaccine formulation approach incorporating SARS-CoV-2 Th/CTL epitope peptides was employed in our latest vaccine development effort (IJB612) to fend off SARS-CoV-2 and has been found with great success in safety, manufacturability, and clinical efficacy. Overall, in the combined three clinical trials of UB612, including an extended booster third-dose, we have demonstrated that UB-612 vaccination can induce substantial viral neuiralizine antibodies with a long half-life (> 187 days, best in its class) that go in parallel with a long-lasting cellular immunity. As memory B and T cells are critical in secondary responses to infection, a successful vaccine must generate and maintain immunological memory and mount a rapid recall of effective humoral and cellular responses upon natural exposure or vaccine boosting. UB-612 has demonstrated such important vaccine design features through these clinical studies. Of special note, there are five precision-designed T -cell epitope peptides representing the helper T-cell (Th) and cytotoxic T-eell (CTL) epitopes from Sarbecovirus regions of the N, M and S2 proteins in UB612. These epitope peptides are highly conserved across all Variants of Concern including Delta and Omicron and are promiscuous epitopes allowing for induction in a broad population of memory recall, T-ceil activation and effector functions. Thus, the long-lasting and robust T cell immunity could be efficacious against all VbCs including Omicron, in addition to a potent anti-Delta and anti-Omicron effect upon a booster 3^rf-dose of UB-612

General design

Au effective immune response against viral infections depends on both humoral and cellular immunity. More specifically, the potential of a high precision designer preventative vaccine would employ designer immunogens, either peptides or proteins, as active pharmaceutical ingredients for ( 1) induction of neutralizing antibodies through the employment of B cell epitopes on the viral protein that is involved in the binding of the vims to its receptor on the target cell; (2) induction of cellular responses, including primary and memory B cell and CDS' T cell responses, against invading viral antigens through the employment of endogenous Th and CTL epitopes. Such vaccines can be formulated with adjuvants such as ALHYDROGEI,, ADJUPHOS, M0NTAN1DE ISA, CpG, etc. and other excipients to enhance the immunogenicity of the high-precision designer immunogens.

A representative designer HIV Th/CTL vaccine employs optionally a Gpl60 -region based protein to function as the B -immunogen. This protein was designed and prepared to elicit neutralizing antibodies against HIV infection. However, the important role of this HI V Th/CTL vaccine is to employ a mixture of designer peptides incorporating endogenous HIV Th and CTL epitope peptides capable of promoting host specific Th cell mediated immunity to facilitate the viral-specific primary and memory B cel! and CTL responses towards the HIV, for the prevention and treatment of HIV infection, An effective vaccine needs to prime the memory T cells and B cells to allow rapid recall upon viral mfection/challenge. To improve the effectiveness of the disclosed designer immunogens, two representative adjuvant formulations are employed ALUM (ALIfYDROGEUCpG, ADJU-PHOSWCpG and MONTANIDE^IM ISA/CpG) for induction of optimal anti-HIV B and T immune responses.

ALUM (ADJUPHOS and ALHYDROGEL) is generally accepted as an adjuvant for human vaccines. This adjuvant induces a Th.2 response by improving the attraction and uptake of designer immunogens by antigen presenting cells (APCs). MONTAN IDE™ ISA 51 is an oil which forms an emulsion when mixed with the water phase designer pepride/protein immunogens to elicit potent immune responses to HIV. CpGs Oligonucleotides are TLR9 agonists that improve antigen presentation and the induction of vaccine-specific cellular and humoral responses. In general, the negatively charged CpG molecule is combined with positively charged designer immunogens to form immuuosrimulatory complexes amenable for antigen presentation to further enhance the immune responses .

The disclosed high precision designer HIV T cell vaccine (with optionally included gp!60-region based protein as the B immunogen) has the advantage of producing highly specific immune responses compared to weak or inappropriate antibody presentation of vaccines with a more complicated immunogen content employing inactivated viral lysate or other less characterized immunogens. In addition, there are potential pitfalls in HIV vaccine development that are related to a mechanism named antibody-dependent enhancement (ADE). Specifically. ADE is a phenomenon in which binding of a virus to non-neutralizing antibodies enhances its entry into host cells, and sometimes also its replication. This mechanism leading to both increased infectivity and virulence has been observed with mosquito-borne flavivintses and coronaviruses. The disclosed high precision designer HIV vaccine is designed to avoid vaccine-induced disease enhancement by monitoring the quality and quantity of the antibody responses (by inclusion of a site directed focused gpI60-region based B immunogen) or by administration of monoclonal antibodies like UB421, highly effective in HIV entry inhibition of CD4+ T cells), they would dictate an effective functional outcomes.

Table I

HIV CTL/Th Epitope Peptides for use in HIV TH/CTL vaccine formulation

Table 2

Composition of Global HIV Th/CTL Vaccine 11 μg/mL

⁵ Materials to be used for clinical trials are manufactured to cGMP

Claims

1 . An HIV Th/CTL vaccine composition comprising a Th/CTL peptide.

2. The HIV Th/CTL vaccine composition according io claim 1, wherein the

Th/CTL peptide is derived from the HIV Gag-pol protein of SEQ ID NOs: 1-9, the

HIV CTL peptide derived from VIF protein of SEQ ID NO: 10, a pathogen protein, or any combination thereof.

The HIV Th/CTL vaccine composition according to claim 2, wherein a), the Th/CTL peptide derived from the HIV Gag-pel protein is SEQ ID NOs: 1-

9; and b). the Th peptide derived from a pathogen protein is selected .from the group consisting of SEQ ID NO: 1.1.

4. The HIV WCTL vaccine composition according to claim 3, further comprising a mixture of Th/CTL peptides of SEQ ID NOs: 1 -1 L

5. The HIV Th/CTL vaccine composition according to claim 4, wherein each of the Th/CTL peptides are present in the mixture in equal-weight amounts.

6. The HIV Th/CTL vaccine composition according to claim 5, wherein the ration (\v:w) of the gpl 60-region based protein to the total weight of the mixture of

Th/CTL peptides is 89: 1 1.

7. The HIV Th/CTL vaccine composition according io claim 5, wherein the pharmaceutically acceptable excipient is an adjuvant, buffer, surfactant, emulsifier, pH adjuster, saline solution, preservative, solvent, or any combination thereof

8. The HIV Th/CTL vaccine composition according to claim 5, wherein the pharmaceutically acceptable excipient is selected from the group consisting of a CpG oligonucleotide, ADJUPHOS (aluminum phosphate), histidine, histidine HCI4L0, arginine HC1, TWEEN 80 (polyoxyethylene (20) sorbitan monooleate), hydrochloric acid, sodium chloride, 2-phenoxyethanol, water, and any combination thereof.

9. A method for treating HIV infection and AIDS in a subject comprising administering a pharmaceutically effective amount of the HIV Th/CTL vaccine composition according to claim 8 to the subject.

10. The method according to claim 9, wherein the pharmaceutically effective amount of the vaccine composition is administered to the subject in two doses. H , The method according to claim 10, wherein a first dose of the vaccine composition is administered to the subject and a second dose of the vaccine composition is administered io the subject about 4 weeks after the first dose.

12. An HIV vaccine composition comprising the components in the amounts shown in Table 2,

13. An HIV Tb/CT L vaccine composition, comprising: a) a gpl60-region based protein. b) a mixture of HIV Th/CTL peptides comprising: SEQ ID NOs: 1.-10; and c) a LBn'h®la peptide (SEQ ID NO: 11 ).

14. An HIV Th/CTL vaccine composition comprising the protein according to claim .13 and a pharmaceutically acceptable carrier and/or adjuvant.

15. The HIV Th/CTL vaccine composition according to claim 14, further comprising a HIV Th/CTL peptide selected from the group consisting of: SEQ ID NOs: 1 - 10; and any combination thereof.

16. The HIV Th/CTL vaccine composition according to claim 15, further comprisi ng a USiTli®! a peptide (SEQ ID NO: 11),

17. The HIV Th/CTL vaccine composition according to claim 16, forther comprising: a) a HIV Th'CTT, peptide selected from the group consisting of: SEQ ID NOs: 1 -

10, and any combination thereof: and b) a UBITMMa peptide (SEQ ID NO: 1 1 ).

18. The HIV Th/CTL vaccine composition according to any one of claims 13-18, wherein the pharmaceutically acceptable carrier and/or adjuvant is CpGl (SEQ ID NO: 12).

19. A Global HI V T vaccine composition comprising a Th/CTL peptide

20, The Global HIV T vaccine composition according to claim 19, wherein the

Th/CTL peptide is derived from the HIV Gag-pol protein, the HIV VIF protein, and a pathogen protein, or any combination thereof.

21 . The Global HIV T vaccine composition according to claim 20, wherein a), the Th/CTL peptide derived from the HIV Gag-Fol is SEQ ID NO: 1-9; b). the Th/CTL peptide derived from the HIV VIF protein is selected from the group consisting of SEQ ID NO: 10; c). the Th/CTL peptide derived from a pathogen protein is selected from the group consisting of SEQ ID NOs: 11 .

The Global T vaccine composition according to claim 21, wherein the Th/CTL peptides is a mixture of SEQ ID NOs: 1-1 1

23. The Global HIV T vaccine composition according to claim 22, wherein each of the Th/CTL peptides are present in the mixture in equal-weight, amounts.

24. The Global HIV T vaccine composition according to claim 23, further comprising a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient is an adjuvant, buffer. pH adjuster, saline solution, preservative, solvent, or any combination thereof

25. The Global HIV T vaccine composition according to claim 24, wherein the pharmaceutically acceptable excipient is selected from the group consisting of a CpG oligonucleotide, ALUM (aluminum phosphate or Alhydrogel), hydrochloric acid, sodium chloride, 2-phenoxyethanol, water, and any combination thereof.

26. A Global HIV T vaccine composition, comprising: a), a Th/CTL peptide selected from the group consisting of SEQ ID NOs/1-11 and any combination thereof; and b). a pharmaceutically acceptable excipient.

27. The Global HIV T vaccine composition according io claim 26, wherein the Th/CTL. peptides in (a) is selected from the group consisting of SEQ ID NOs: l~l l and any combination thereof.

28. The Global HIV T vaccine composition according to claim 27, wherein each o f the Th/CTL peptides are present in equal-weight amounts.

29. The Global HIV T vaccine composition according to claim 28, wherein the pharmaceutically acceptable excipient is an adjuvant, buffer, pH adjuster, saline solution, preservative, solvent, or any combination thereof

30. The Global HIV T vaccine composition according io claim 29, wherein the pharmaceutically acceptable excipient is selected from the group consisting of a CpG oligonucleotide, ALUM (aluminum phosphate or aluminum hydroxide), hydrochloric acid, sodium chloride, 2-phenoxyethanol, water, and any combination thereof,

31. The Global HIV T vaccine composition according to claim 30, wherein the Th/CTL peptide is a mixture of SEQ ID NOs: 1 -Il , wherein each peptide is present in the mixture in equal-weight amounts; the pharmaceutically acceptable excipient is a combination of a CpGl oligonucleotide, ALUMtaiuminum phosphate or aluminum hydroxide), hydrochloric acid, sodium chloride, and 2-phenoxyethanol in water,

32. A method for preventing and treating HIV infection in a subject comprising administering a pharmaceutically effective amount of the vaccine composition according to claim 31 to the subject.

33. An HIV Th/CTL vaccine composition comprising the components in the amounts shown in Table 2.