Compositions and methods comprising targeted liposomal therapeutic vaccines

Abstract

Methods and compositions for the treatment of viral infections, such as HIV infection, are disclosed herein. In certain embodiments, a molecular conjugate comprising a viral capture domain and an immunostimulant molecule is provided.

Description

[0001] Compositions and Methods Comprising Targeted Liposomal Therapeutic Vaccines By

[0002] Peter Deak

[0003] Elias El Haddad

[0004] Irwin Morris Chaiken

[0005] Cross-Reference to Related Applications

[0006] This application claims the benefit of the priority of US Provisional Patent Application No. 63 / 711,886, filed October 25, 2025, which is incorporated herein by reference.

[0007] Background of the Invention

[0008] Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated by reference herein as though set forth in full.

[0009] Latent and mutagenic viruses leading to chronic infection are a serious health concern in the United States. Thousands of deaths occur from chronic viral infections in the US.1HIV is perhaps the most well-known lifelong chronic viral infection, infecting mainly T cells and weakening the immune system without treatment.2Indeed, there are over 1.2 million HIV cases in the US, accounting for a tremendous healthcare burden, with over 34,000 new cases documented annually.2Effective treatments are available, but no cure or vaccine has been developed. A major challenge for development of HIV cures is viral immune evasion due to Env subunits gpl20 and gp41 glycosylation and Env rapid mutations, particularly within gp 120.3,4HIV also generates latent viral reservoirs in various tissues.5

[0010] HCV is another well-known chronic virus, which affects 2.4 million individuals in the US and both actively and chronically infects liver cells.6While not a classic chronic virus, mounting evidence is demonstrating that SARS-CoV-2 infection can sometimes lead to chronic viral infection. Studies show that viral mRNA can be found in fecal samples of a small but identifiable population (8-12%) of patients at 4 months post infection and that “Long-COVID” is often associated with persistent viral antigens.7Even if not fatal, chronic latent viral infections can reduce quality of life dramatically.8 Effective cures for these viral infections would have tremendous significance on patients but also reduce the burden on the healthcare system. Vaccines are a cornerstone of public health, yet effective vaccines are still lacking for many chronic viral infections due to two primary viral mechanisms. First, viruses, like SARS-CoV-2, can have high genetic variation, preventing an accurate prediction of which antigen to include in the vaccine.9Second, viruses, like hepatitis C (HCV). evade the immune response.10There are many mechanisms of evasion, but the most potent is viral latency, where inactive or mostly inactive virus lingers in peripheral tissue until active immunity wanes, leading to chronic infection.11Another example, HIV, uses both of these mechanisms to great effect and has frustrated vaccine development for over 40 years.12The treatment for chronic viral infection is typically antiviral therapy, i.e. small molecules known to inhibit one or more pathways of viral infection or replication. These can often be highly effective. Antiviral therapies, however, carry a myriad of side effects, ranging from nausea to liver toxicity.13For HIV, over 25% of patients to discontinue their therapies due to side effects.14More importantly, these antiviral therapies must be administered chronically and do not generate antiviral immunity or eliminate viral reservoirs.15

[0011] Accordingly, a new kind of therapeutic vaccine for treatment of viral infections, such as HIV, is urgently needed.

[0012] Summary of the Invention

[0013] In one aspect of the invention, a molecular conjugate comprising a viral capture domain and an immuno stimulant molecule is provided. In certain embodiments, the molecular conjugate further comprises a linker molecule. In certain embodiments, the viral capture domain is specific for a viral surface protein of HIV, SARS-CoV-2, or HCV. In certain embodiments, the viral capture domain targets a viral surface protein of a virus selected from Human Immunodeficiency Virus (HIV-1). Human Immunodeficiency Virus (HIV-2), Influenza A virus. Influenza B virus. Respiratory Syncytial Virus (RSV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), SARS-CoV (SARS-CoV-1), Measles virus, Dengue virus, West Nile virus, Ebola virus, Marburg virus, Nipah virus, Human Parainfluenza virus, Hendra virus, Chikungunya virus, Zika virus, Yellow fever virus. Human herpes simplex virus 1 (HSV-1), Human herpes simplex virus 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), Human adenovirus, or rabies virus. In certain embodiments, the viral capture domain is a pan- viral inhibitor. In certain embodiments, the viral capture domain is one of fostemsavir, temsavir, CJF-III-288, BMN-III-170, enfuvirtide, maraviroc, presatovir, umifenovir, clofazimine, nelfinavir mesylate, ziresovir, sifuvirtide, posaconazole, cilnidipine, and MG-II-20.

[0014] In certain embodiments, the immunostimulant molecule is a Thlskewing adjuvant or a CTL skewing adjuvant. In certain embodiments, the immuno stimulant molecule is selected from CpG1018, a TLR-agonist, or a TLR7 / 8agonist. In certain embodiments, the immunostimulant molecule is selected from CpG1018, R848, pixatimod, ISG15, 2’3’-c’diAM(PS)2, Poly I:C, or cGAS. In certain embodiments, the linker comprises a charged amino acid, an ethylene glycol spacer, and a reactive moiety. In certain embodiments, the charged amino acid is Arg or Lys. In certain embodiments, the reactive moiety is an amine, carboxylic acid or DBCO group. In certain embodiments, the linker is selected from the linkers of Table 2.

[0015] In certain embodiments, the molecular conjugate has a chemical structure of TK-9, TK14, TK-16, TK15, or TK20.

[0016] In another aspect of the invention, a liposomal formulation comprising the molecular conjugate disclosed herein is provided. In certain embodiments, the liposomal formulation comprises at least one of a PEG-lipid conjugate and a bulk lipid. In certain embodiments the molecular conjugate comprises at least 0.01%-20% of the lipids present in the liposome. In certain embodiments, the PEG-lipid comprises between 1 %-20% of the lipids present in the liposome. In certain embodiments, the bulk lipids comprise between 80%-99% of the lipids present in the liposome. In certain embodiments, the liposome comprises about 2% molecular conjugate, about 5% PEG-lipid conjugate, and about 93% bulk lipid. In certain embodiments, the liposomal formulation further comprises cholesterol. In certain embodiments, the size of the liposome is between 50nm-500nm, 100nm-300nm, or 140nm-170nm.

[0017] In another aspect of the invention, methods of treating a viral infection in a subject in need thereof is provided. In certain embodiments, the methods comprise administering an effective amount of the liposomal formulation described herein. In certain embodiments, the viral infection is caused by Human Immunodeficiency Virus (HIV-1), Human Immunodeficiency Virus (HIV-2), Influenza A virus. Influenza B virus. Respiratory Syncytial Virus (RSV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), SARS-CoV (SARS-CoV-1), Measles virus, Dengue virus, West Nile virus, Ebola virus, Marburg virus, Nipah virus, Human Parainfluenza virus, Hendra virus, Chikungunya virus, Zika virus, Yellow fever virus, Human herpes simplex virus 1 (HSV-1). Human herpes simplex virus 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), Human adenovirus, or rabies virus.

[0018] In certain embodiments, the method reduces or suppresses at least one symptom of the viral infection. In certain embodiments, the symptom is selected from fatigue, feeling tired, weakness, brain fog (problems concentrating or thinking), headaches, tremor, rapid or pounding heartbeat, feeling of skipped heartbeats (palpitations), dizziness upon standing, symptoms that worsen after physical or mental activity (known as post-exertional malaise, PEM), gastrointestinal symptoms including stomach pain, diarrhea, and / or constipation, loss of or change in smell and / or taste, thirst (for instance, dry mouth), cough, changes in comfort or capacity for sex and / or desire for sex, chest pain, tightness, or pressure, hearing problems, including hearing loss or ringing in the ears (tinnitus), shortness of breath, muscle and / or joint pain, back pain, sleep apnea, fever, sweats, and / or chills, hair loss, sleep problems, including insomnia, bladder problems, including difficulty urinating or incontinence, vision problems, such as blurry vision, sensitivity to light, floaters, flashing lights, or difficulty reading or focusing eyes, depression, anxiety, swelling of the legs, problems with teeth, foot pain, skin rash, abnormal movements, skin color changes (for instance, skin that is red, white, or purple), and changes in menstrual cycle. In certain embodiments, the methods further comprise detecting inhibition of viral infection and / or propagation.

[0019] Still other aspects and advantages of these compositions and methods for making the compositions and using the compositions are described further in the following detailed description of the preferred embodiments thereof.

[0020] Brief Description of the Drawings

[0021] Fig. 1: Comparison of HIV infection in chronic patient, acutely infected patients with early intervention and Elite Controllers.

[0022] Fig. 2: Schematic of NTV Design. Fig. 3A-3B: Schematic for Generating HIV Targeted Liposome Formulations (Fig 3A) and an exemplary liposome (Fig. 3B) is provided.

[0023] Fig. 4: Exemplary HIV binding Molecules.

[0024] Fig. 5: NTV infection inhibition. 100 nm NTVs loaded with 1% Arg-CJF-lipid (liposome) or free CJF at varying concentrations were incubated with 10 pg / mL p24 of BaL.Ol and GHOST cells. GFP expression indicates infection. N=3 (Fig. 5A).

[0025] Fig. 6A-6D: CJF-lipid Synthesis and Purification (RP-HPLC and MALDI-TOF) for TK-9.

[0026] Fig. 7A-7D: CJF-lipid Synthesis and Purification (RP-HPLC and MALDI-TOF) for TK-14.

[0027] Fig. 8A-8D: CJF-lipid Synthesis and Purification (RP-HPLC and MALDI-TOF) for TK-16.

[0028] Fig. 9A-9D: cPT-lipid Synthesis and Purification (RP-HPLC and MALDI-TOF) for TK-15.

[0029] Fig. 10A-10C: cPT-lipid Synthesis and Purification (RP-HPLC and MALDI-TOF) for TK-20.

[0030] Fig. 11A-1 IE: Chemical structure for TK-9 (also identified as NTV-9)(Fig. 11 A), TK-14(also identified as NTV-14) (Fig. 11B), TK-16 (also identified as NTV-16) (Fig. 11C), TK-15 (also identified as NTV-15) (Fig. 11D), and TK-20 (also identified as NTV-20) (Fig. HE).

[0031] Fig. 12A-12B: Solid-phase peptide synthesis schematic.

[0032] Fig. 13: SRP Binding Data for lipid conjugates.

[0033] Fig. 14A-14C: Dynamic light Scattering analysis(Fig. 14A), Zeta potential analysis(Fig. 14B), and incorporation efficiency analysis(Fig. 14C) of NTV synthesis.

[0034] Fig. 15: Schematic for infection analysis in non-humanized mouse model of HIV.

[0035] Fig. 16A-I6G: In vivo NTV injection analysis. C57BL / 6 mice (N=4) were injected sequentially with pseudovirus and R848 loaded NTV (NTV), R848 blank liposome (Blank) or free R848 (Free). (Fig. 16A) Analysis of IL-6 and TNFa 1 h post injection via cytokine bead array. (Fig.

[0036] 16B) Analysis of serum p24 via ELISA. (Fig. 16C) RBC depleted blood was analyzed via flow cytometry for liposomes. RBC Depleted blood (Fig. 16D) or disaggregated spleen cells (Fig. 16E) were analyzed for % liposome+ cells in the following cell populations: CD1 lc+, pDC (CDllc+, B220-), monocytes (CDllc-, CD68-, CD14+), CD14+ macrophages (CD14+, CD68+), CD14- macrophages (CD14-, CD68+), B cells (CDllc-. B220+) or T cells (CD3+). n.d.= not detected with corresponding number of samples. Schematic of in vivo experiment (Fig.

[0037] 16F). Analysis of Liposome + cells of all CD45+ cells in free R848, liposome and NTV group. (Fig. 16G).

[0038] Fig. 17: THP-1 co-culture improves infection inhibition of NTVs. 10k THP-1 and 50k GHOST cells (red) or GHOST alone (blue) were incubated with 100 nm DiD labeled NTVs (1% Arg-CJF-lipid) at varying concentrations and 2 pg of p24 / mL BaL.Ol. THPs were removed selectively after 1 h. After 72 h, GHOST cells were analyzed for GFP expression, indicating infection. N=3.

[0039] Fig. 18: DLS data of loading of R848.

[0040] Fig. 19: DLS data shows that NTV-HIV pseudotyped virus form complexes. NTVs loaded with 1% TK-16 or blank liposomes (DSPC+ 5% cholesterol, 5% DSPE-mPEG2000+0.2% DiD) were incubated with pseudotyped viruses containing gpl60 from HXB2 viruses (approximately 1 ng / mL p24). Samples were incubated for 10, 20 or 30 minutes then analyzed via DLS (NanoPhox, Sympatec) for complex formation. Graphs show relative sizes (top left) or timepoints. Controls include pseudovirus only (Blank PV), NTV only, blank Liposome (Bl. Lipo) or Blank liposome plus PV (Bl. Lipo +NTV, mislabeled)

[0041] Fig. 20: Binding profile of NTV 10 pM total lipid, loaded at 1% TK-16, to 500RU gpl20 monomer, YU2 variant (red trace), 10 min injection followed by 10 min wash with PBS running buffer. Blank liposome injection is shown (blue trace) among the buffer injection traces, indicating there is no binding of unmodified liposomes in absence of CJF-III-288.

[0042] Fig 21: Assessment of NTV binding by comparing gpl20+TF228 cells and gp 120 Jurkat cells.

[0043] Fig. 22: Co-culture of TF-288 cells with Jurkat.

[0044] Fig. 23: Binding of NTVs to gpl20-expressing TF228 cells was evaluated using DiD fluorescence as a readout for liposomal association. Fig. 24: DiD fluorescence analysis of gpl20 binding.

[0045] Fig. 25: Analysis of NTV effect on the antiviral activity of Fostemsavir, Lenacapavir, and Emtricitabine evaluated in HIV pseudovirus infection assays.

[0046] Fig. 26A-26B: BMDCs incubated with DiO labeled PV (blue) or unlabeled (red) showing that DiO can label PV (Fig. 26A). Murine bone marrow-derived dendritic cells (BMDCs) were incubated for 2 h at 37 °C with pseudotyped HIV (PV, DiO-labeled) that had been pre-incubated for 30 min with either NTV 16 (DiD-labeled) or blank liposomes. Cellular uptake was quantified by flow cytometry based on dual DiO and DiD fluorescence. NTV-PV complexes showed significantly higher uptake compared with blank liposomes across lipid concentrations (1-100 pM), indicating that NTV enhances dendritic cell internalization of pseudotyped HIV through gpl20-specific interactions. (Fig. 26B)

[0047] Fig. 27A-27D: Confocal microscopy images show the intracellular distribution of NTV (20 pM, Fig. 27 A) and blank liposomes (20 pM, Fig. 27B) in murine bone marrow-derived dendritic cells. Nuclei (blue), lysosomes (yellow), NTV or blank liposome (red), and pseudotyped HIV (cyan) were visualized after 30 min pre-incubation of NTV or liposomes with PV followed by cellular exposure. Merged images demonstrate that NTV exhibited strong co-localization with both lysosomes and pseudotyped HIV, indicating efficient cellular uptake and gp 120- specific binding. In contrast, blank liposomes showed minimal overlap with lysosomes or pseudovirus, confirming that NTV facilitates targeted interaction with HIV particles within endolysosomal compartments. This experiment was replicated in THP1 DCs in Figs. 27C-27D.

[0048] Fig. 28A-28B: Murine bone marrow-derived dendritic cells were treated with R848-loaded NTV, blank NTV, or blank liposomes at varying lipid concentrations (0-100 pM) for 24 h at 37 °C. Surface marker expression (CD80, MHC II) was analyzed by flow cytometry (left panels), and cytokine secretion (TNF-a, IL-6) was quantified by ELISA (right panels). R848-loaded NTV markedly upregulated CD80 and MHC II expression and induced robust secretion of TNF-a and IL-6 in a dose-dependent manner, comparable to free R848 treatment. In contrast, blank NTV and blank liposomes showed minimal activation, confirming that the R848 encapsulated within NTV effectively stimulates dendritic cell maturation (Fig. 28A). The analysis of the release of inflammatory cytokines is shown in Fig. 28B. Fig. 29A-29G: NTVs generate anti-HTV immunity in a temporary mouse infection model. (Fig.

[0049] 29A) 8 week old, C57B1 / 6 mice were injected i.p with 0.1 ng p24 of HXB2 pseudovirus on the left side. 5 minutes later, mice were injected i.p. on right side with either PBS (PV only), 160 pM total lipid of blank DSPC liposome loaded with 15pg of R848 (Bl. Lipo) or similar amount of NTV (including 1% TK-16 total lipid). Additional control was a single injection of 10 pg of both p24 and gpl20 protein from HXB2 mixed with 25 pg R848 in PBS (Free R848). Both injections were repeated on day 14. Splenocytes were isolated and analyzed by (Fig. 29B-29D) ICS and T cell proliferation (Fig. 29E-29G). For ICS, cells were stimulated with peptide pools from 1 pg / mL Vpr, Pol, Gag, Env or Nef for 1 h, then blocked with brefeldin A for 5 h. Cells were fixed, stained and analyzed by flow cytometry for intracellular IFNy. Controls include PMA / Ionomycin treated samples or cells treated with no peptide. (Fig. 29B) Representative flow plots of SSC (y axis) by IFNy signal (x axis). (Fig. 29C) IFNy+ CD8+ T cells, (Fig. 29D) IFNy+ CD4+ T cells. For T cell proliferation, splenocytes were treated with same peptide pools plus 0.2 pg / mL anti-CD28. Positive controls were treated with 1 pg / mL of both anti-CD3 / CD28 antibodies. Cells were stained with proliferation dye and incubated for 72 h, then analyzed by flow cytometry. N=5 mice per group, significance determined by two-way ANOVA.

[0050] Fig. 30: Serum samples collected from mice immunized with pseudotyped HIV alone or in combination with R848. blank liposomes, or NTV were analyzed by antigen-specific ELISA using recombinant HIV p24 and HXB2 gp 120 proteins. Area under the curve (AUC) values were calculated from serial serum dilutions to quantify antibody binding. NTV-immunized mice showed the highest AUC value for p24 and the second highest for gpl20. indicating that NTV enhances antigen-specific antibody generation compared to pseudotyped HIV alone or other control formulations.

[0051] Fig. 31: HIV pseudovirus infection was quantified by GFP mean fluorescence intensity (MFI) and the percentage of GFP+infected cells. As shown in the left panels, NTV treatment markedly reduced both MFI and GFP+% compared to PV alone, demonstrating effective inhibition of viral infection. The antiviral response was comparable to that of R848 or BL + PV, indicating that NTV formulation maintained potent antiviral activity while incorporating immunostimulatory components. In the right panels, lenacapavir served as a positive control, showing a strong dosedependent reduction in GFP expression, whereas the untreated control group exhibited consistently high infection levels. Together, these results confirm that NTV effectively suppresses HIV pseudovirus infection, consistent with the inhibitory profile of lenacapavir.

[0052] Fig. 32: MTV schematic.

[0053] Detailed Description of the Invention

[0054] HIV remains a major public health issue in the US, with thousands of new infections every year. While advancements in anti-retroviral therapies (ART) have increased patient lifespans, ART does not cure HIV infection, leaving patients reliant upon ARTs. Here, we propose a new strategy for therapeutic HIV vaccines, the Nanotrap Therapeutic Vaccine (NTV). NTVs are modified liposomes consisting of HIV targeting molecules that bind gpl20 and adjuvants that stimulate innate immune responses. This is essentially a targeted adjuvant which can improve initial immune responses to HIV. NTVs are designed to be injected after HIV infection, but concurrent with ART therapy when circulating viral loads are high. NTV will bind circulating HIV and be phagocytosed by local APCs, thereby co-delivering both HIV viral particle and adjuvant to APCs, activating them.

[0055] While the overall goal of developing NTVs is to generate early and potent anti-HIV responses, the data presented herein show the initial validation of NTVs for other viral infections. First, we have shown that NTVs can bind and facilitate HIV psuedoviruses uptake by local APCs. We have selected three gp!20 fusion inhibitors, typically used to inhibit HIV viral entry, which we displayed on liposomal surfaces, verified that these liposomes bind HIV and inactivate the virus from infecting other cells using SPR and in vitro infection assays. Second, we have demonstrated that HIV Pseudovirus can be captured in vivo using simple mouse models and be uptaken by murine innate immune cells. This invention is innovative because NTVs can generate immune responses specific to the circulating virus in each patient, overcoming HIV’s high mutability evasion strategy. NTVs are a new class of HIV vaccine which could reduce patient reliance upon continual ART.

[0056] Here, we investigate a novel therapeutic vaccine platform, called Viral Targeting Adjuvants (VTAs), that direct immuno stimulatory molecules to “tag” circulating viral particles, rendering them more immunogenic and stimulating adaptive immunity. VTAs consist of two domains, a viral capturing moiety and an immuno stimulatory adjuvant. The capture domain repurposes viral inhibitory molecules made to simulate viral entry receptors, effectively “baiting” the circulating viral particle and then tagging with an adjuvant to promote viral phagocytosis rather than infection. This versatile, tunable and modular platform has the potential to generate potent anti-viral immunity for several viruses. Herein, we target HIV, SARS-CoV-2 and HCV to demonstrate the adaptability of the platform and as models to optimize biophysical parameters of VTA constructs (surface charge, size and viral capture ligand valency).

[0057] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. In addition to definitions included in this sub-section, further definitions of terms are interspersed throughout the text.

[0058] In this invention, “a”, “or” and “an” can mean “at least one” or “one or more,” etc., unless clearly indicated otherwise by context. The term “or” means “and / or” unless stated otherwise. In the case of a multiple-dependent claim, however, use of the term “or” refers back to more than one preceding claim in the alternative only.

[0059] Furthermore, a compound “selected from the group consisting of’ refers to one or more of the compounds in the list that follows, including mixtures (i.e. combinations) of two or more of the compounds. According to the present invention, an isolated, or biologically pure molecule is a compound that has been removed from its natural milieu. As such, “isolated” and “biologically pure” do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using laboratory synthetic techniques or can be produced by any such chemical synthetic route.

[0060] It is also contemplated that the term “compound” or “compounds” refers to the compounds discussed herein and includes precursors and derivatives of the compounds, and pharmaceutically acceptable salts of the compounds, precursors, and derivatives.

[0061] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. Can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

[0062] Molecular Conjugates and Liposomal Formulations In one aspect of the invention, molecular conjugates for incorporation into liposomal formulations are provided.

[0063] The term “molecular conjugate,” a “Viral Targeting Adjuvant” or “VTA” refers to a chemical entity comprising a covalent or ono-covalent association between a lipid or lipidderivative and at least one functional molecule, moiety, or agent, such that the resulting conjugate exhibits modified physiochemical or biological properties relative to its unconjugated components. The functional molecule may be conjugated to the lipid or lipid-derivative through direct chemical linkage (such as, amide, ester, thioester, disulfide, or click chemistry linkage), through incorporation of functionalized lipids into the liposomal membrane (such as, DSPE-PEG-ligand conjugates), or via non-covalent interactions (such as electrostatic, hydrophobic, or affinity-based interactions). In certain embodiments, the functional molecule, moiety, or agent may include, without limitation, a viral capture domain, an immunostimulant molecule, and / or a linker molecule. In certain embodiments, the molecular conjugates includes a viral capture domain directly conjugated to an immunostimulant molecule. In certain embodiments, a viral capture domain is conjugated to an immunostimulant molecule using a linker molecule. In certain embodiments, the molecular conjugate is selected from TK-9, TK-14, TK-16, TK-15, TK-20, the chemical structures for which are provided in Figures 11 A-l IE.

[0064] The molecular conjugate is incorporated into the liposomal formulation, typically by being embedded within, or associated with, the lipid bilayer or surface of the liposome.

[0065] Incorporation of the molecular conjugate may impart modified physicochemical or biological properties to the liposome, including altered biodistribution, targeting, stability, or drug release characteristics.

[0066] The phrase “viral capture domain” refers to a molecular moiety, region, or structural element that is capable of specifically or non- specifically binding to, associating with, or otherwise interacting with one or more viral particles, viral surface proteins, or viral components. In certain embodiments, the viral capture domain is a component of a molecular conjugate that is incorporated into a liposomal formulation, such that the domain is presented on or accessible at the liposomal surface. The viral capture domain thereby facilitates the interaction, binding, or sequestration of viral particles by the liposomal construct.

[0067] A viral capture domain may comprise, for example, one or more of a peptide, protein, glycoprotein, antibody, antibody fragment, receptor, receptor-binding fragment, aptamer, carbohydrate, lipid, or synthetic ligand that exhibits affinity for a viral epitope or surface feature; a mimetic or analog thereof that structurally or functionally reproduces a viral binding interaction; or a chemical or biological moiety engineered to associate with viral membranes, capsids, or envelope glycoproteins. In certain embodiments, the viral capture domain targets a viral surface protein identified in Table 3 and / or Figure 4. In certain embodiments, the viral capture domain is specific for a viral surface protein of an enveloped virus. In certain embodiments, the viral capture domain is specific for a latent or chronic virus. In certain embodiments, the viral capture domain is specific for viruses that are not naturally controlled by the immune system. In certain embodiments, enveloped virus includes, without limitation, Human Immunodeficiency Virus (HIV-1), Human Immunodeficiency Virus (HIV-2), Influenza A virus. Influenza B virus, Respiratory Syncytial Virus (RSV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), SARS-CoV (SARS-CoV-1), Measles virus, Dengue virus, West Nile virus, Ebola virus, Marburg virus, Nipah virus, Human Parainfluenza virus, Hendra virus. Chikungunya virus, Zika virus, Yellow fever virus, Human herpes simplex virus 1 (HSV-1), Human herpes simplex virus 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), Human adenovirus, or rabies virus. In certain embodiments, the viral capture domain is specific for a viral surface protein of HIV, SARS-CoV-2, or HCV. In certain embodiments, the viral capture domain is pan-viral capture domain which is capable of binding to more than one type of virus. In certain embodiments, the viral capture moieties are repurposed fusion inhibitors. In certain embodiments, the viral capture domain is selected from the molecules identified in Table 3.

[0068] The term “fusion inhibitor” or “entry inhibitor” refers to any molecule, compound, peptide, protein, nucleic acid, lipid, or synthetic construct that prevents, inhibits, or reduces the fusion of a viral membrane with a host cell membrane, thereby impeding viral entry or infection of the host cell. In certain embodiments, the fusion inhibitor acts by interfering with viral fusion proteins, blocking receptor-mediated fusion mechanisms, or altering membrane properties required for the fusion process. The inhibition may occur at any stage of viral entry, including attachment, conformational rearrangement of viral glycoproteins, or membrane merging.

[0069] Exemplary fusion inhibitors include, without limitation, Fostemsavir, temsavir, CJF-III-288, BMN-III-170, MG-II-20, enfuvirtide, maraviroc, presatovir, umifenovir, clofazimine, nelfinavir mesylate, ziresovir, sifuvirtide, posaconazole, and cilnidipine. The term “immunostimulant molecule” refers to any compound, molecule, or chemical entity that is capable of enhancing, activating or modulating the immune response of a subject. In certain embodiments, the immunostimulant molecule activates the innate immunity of the subject. Such immunostimulant molecules stimulate pattern recognition receptors (PRRs) such as toll-like receptors, RIG-I-like receptors, NOD-like receptors, or other pathogen recognitions pathways. In certain embodiments, the immunostimulant molecule enhances the adaptive immunity of the subject by enhancing activation, proliferation, or differentiation of T cells, B cells, natural killer (NK) cells, or antigen-presenting cells. In certain embodiments, the immunostimulant molecule induces cytokine or chemokine production to potentiate immune cell recruitment or effector function. In certain embodiments, the immunostimulant molecule is an adjuvant that leads to an increased immunogenic response when co-administered with an antigen or therapeutic agent. In certain embodiments, the immunostimulant molecule is a Thl skewing adjuvant or a CTL skewing adjuvant. In certain embodiments, the adjuvant is a TLR-9 agonist or a TLR 7 / 8 agonist such as CpG1018 or R848. In certain embodiments, the adjuvant is a small molecule, such as pixatimod. Other immunostimulatory molecules include without limitation ISG15 (protein)16, 2'3'-c'diAM(PS)217, Poly I:C17andcGAS5.

[0070] The term “linker” or “linker molecule” refers to any chemical moiety, spacer, or molecular entity that connects two or more molecular components within a molecular conjugate. Linkers included, without limitation, peptide linkers, polymer linkers, polyethylene glycol (PEG) chains, oligonucleotides, lipid-based linkers, and other synthetic or naturally derived chemical linkers. In certain embodiments, the linker comprises a charged amino acid, an ethylene glycol spacer, and a reactive moiety. In certain embodiments, the charged amino acid is an Arg or Lys. In certain embodiments, the reactive moiety is an amine, carboxylic acid or dibenzocyclooctyne (DBCO) group. In certain embodiments, the linker is selected from the linkers of Table 2.

[0071] As used herein, the term “incorporated” refers to embedding a molecular conjugate as described herein in the double membrane of the liposome. Because the double membrane of liposomes is lipophilic, compounds with high lipophilicity can be trapped within the double membrane of the liposome.

[0072] The term “liposome” or “liposomal composition” is a broad definition for vesicles composed of lipid bilayers enclosing aqueous compartments. The membrane-forming lipids are amphiphilic and accordingly contain a polar and an apolar region. The polar region typically consist of a phosphate group, an acidic group and / or tertiary or quaternary ammonium salts and can either have a net negative (anionic), neutral or positive (cationic) surface charge at physiological pH, depending on the composition of the lipid head groups. The pH is preferably adjusted to physiological pH such as by dispersion adjusted to pH 5.0-8.0 in Tris or histidine buffer, most preferably adjusted to pH 6.5-7.5. The apolar region typically consists of one or more fatty acid chains with at least 8 carbons and / or cholesterol. The lipids constituting the vesicular bilayer membranes are organized such that the apolar hydrocarbon “tails” are oriented toward the center of the bilayer while the polar “heads” orient towards the in- and outside aqueous phase, respectively.

[0073] Thus, “liposome” or “liposomal” is defined as closed vesicle structures made up of one or more lipid bilayers surrounding an aqueous core. Each lipid bilayer is composed of two lipid monolayers, each of which has a hydrophobic “tail” region and a hydrophilic polar “head” region. In the lipid bilayer, the hydrophobic “tails” of the lipid monolayers orient toward the inside of the bilayer, while the hydrophilic “heads” orient toward the outside of the bilayer. Liposomes can have a variety of physicochemical properties such as size, lipid composition, surface charge, fluidity and number of bilayer membranes. According to the number of lipid bilayers, liposomes can be categorized as unilamellar vesicles (UV) or small unilamellar vesicles (SUV) comprising a single lipid bilayer or multilamellar vesicles (MLV) comprising two or more concentric bilayers each separated from the next by a layer of water. Water soluble compounds are entrapped within the aqueous phases / core of the liposomes opposed to lipophilic compounds, which are trapped in the core / center of the lipid bilayer membranes.

[0074] In certain embodiments, the liposome contains the viral targeting molecule on its surface and the immunostimulatory molecule in its core. The size of the liposome can be altered to optimize its therapeutic application to the specific viral infection being treated. In certain embodiments, the liposome is between 50-500nm in size. In certain embodiments, the liposome is between 100-300nm in size. In certain embodiments, the liposome is between 140-170nm in size. In certain embodiments, the liposome comprises a molecular conjugate, as described herein, a PEG-lipid conjugate, and a bulk lipid. In certain embodiments, the molecular conjugate comprises at least or at most 0.1%, 0.2%, 0.3%, 0.4%, 0.5%. 0.6%. 0.7%. 0.8%. 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the lipids present in the liposome. In certain embodiments, the molecular conjugate comprises between 0.01%-20% of the lipids present in the liposome. In certain embodiments, the liposome comprises the structure identified in Fig. 3B.

[0075] The term “PEG-lipid conjugate” refers to composition comprising a compound, molecule, or chemical entity that included a lipid portion that anchors the compound in the liposomal bilayer, and PEG portion which is a hydrophilic polymer chain that extends into the aqueous environment. PEG-lipid conjugates improve the stability of the liposome and increases circulation time for interacting with the virus. In certain embodiments, the PEG-lipid conjugate comprises at least or at most 1%, 2%, 3%, 4%, or 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the lipids present in the liposome. In certain embodiments, the PEG-lipid conjugate comprises between l%-20% of the lipids present in the liposome.

[0076] The term “bulk lipid” refers to the primary lipid component of the liposomal bilayer. The bulk lipids provide structural integrity for the liposome. Bulk lipids are phospholipids which include, without limitation, at least one of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In certain embodiments, cholesterol is included with the bulk lipids to modulate membrane fluidity, rigidity and permeability. In certain embodiments, the bulk lipids comprises at least or at most 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the lipids present in the liposome.

[0077] Methods of Treating a Viral Infection

[0078] In certain embodiments, the molecular conjugate or liposomal formulations described herein may be administered to a subject for the treatment, prevention, or inhibition of a disorder or disease. In certain embodiments, an effective amount of the molecular conjugate or liposomal formulation is administer to a subject for the treatment, prevention, or inhibition of a viral infection.

[0079] The terms “subject” and “patient” are, unless otherwise noted, used interchangeably to refer to the target or recipient of a treatment or composition described herein. These terms include, unless otherwise specified, all vertebrates, including all mammals, including humans. Unless otherwise noted, an embodiment using the term “subject” and “patient” is intended to include an embodiment directed solely to solely to mammals, solely to humans, solely to non- human mammals, solely to companion mammals, solely to companion vertebrates, solely to companion mammals, solely to non-human animals, and solely to non-human mammals.

[0080] The term “preventing” as used herein refers to administering a compound prior to the onset of clinical symptoms of a disease or conditions so as to prevent a physical manifestation of aberrations associated with the disease or condition.

[0081] The term “in need of treatment” as used herein refers to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals) that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a care giver's expertise, but that includes the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the disclosed compounds.

[0082] By “treatment” and “treating” is meant the medical management of a subject with the intent to cure, ameliorate, or stabilize, a pathological condition or disorder. This term includes active treatment, that is. treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. It is understood that treatment, while intended to cure, ameliorate, or stabilize, a disease, pathological condition, or disorder, need not actually result in the cure, amelioration, or stabilization. The effects of treatment can be measured or assessed as described herein and as known in the art as is suitable for the disease, pathological condition, or disorder involved. Such measurements and assessments can be made in qualitative and / or quantitative terms. Thus, for example, characteristics or features of a disease, pathological condition, or disorder and / or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount.

[0083] As used herein, the term “viral infection” or “disease caused by virus” describes a diseased state in which a virus invades healthy cells, uses the cell's reproductive machinery to multiply or replicate and ultimately lyse the cell resulting in cell death, release of viral particles and the infection of other cells by the newly produced progeny viruses. Latent infection by certain viruses is also a possible result of viral infection.

[0084] Viral infections that can be treated or prevented by the compounds of the present disclosure can be any infection caused by a virus, e.g., a virus from the Human Immunodeficiency Virus (HIV-1), Human Immunodeficiency Virus (HIV-2), Influenza A virus, Influenza B virus, Respiratory Syncytial Virus (RSV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), SARS-CoV (SARS-CoV-1), Measles virus, Dengue virus, West Nile virus. Ebola virus, Marburg virus, Nipah virus, Human Parainfluenza virus, Hendra virus, Chikungunya virus, Zika virus, Yellow fever virus, Human herpes simplex virus 1 (HSV-1), Human herpes simplex virus 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), Human adenovirus, or rabies virus. In preferred embodiments, the viral infection is a HIV, SARS-CoV-2, or HCV infection.

[0085] Symptoms vary according to the location and type of viral infection being treated. In certain embodiments, symptoms of viral infection include, fever, chills, head and body aches, fatigue, sore throat, cough, sneezing, runny or stuffy nose, nausea, vomiting, diarrhea, Rashes, sores, blisters, warts, weakness, night sweats, mouth ulcers, swelling, swollen lymph nodes and no symptoms at all.

[0086] The compounds described herein can be formulated for parenteral administration. For example, parenteral administration may include administration to a patient intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally. intravitreally, intratumorally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, intrapericardially, intraumbilically, by injection, and by infusion.

[0087] Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Typically, such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and microemulsions thereof, liposomes, or emulsomes. For intravenous administration, the compositions may be packaged in solutions of sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent. The components of the composition are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or concentrated solution in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent. If the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water or saline can be provided so that the ingredients may be mixed prior to injection.

[0088] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, com oil, sesame oil, etc.), and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and / or by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

[0089] Solutions and dispersions of the active compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, viscosity modifying agents, and combination thereof.

[0090] Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface-active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions.

[0091] The formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent(s).

[0092] The formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers. Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above.

[0093] The term “effective amount,” unless otherwise noted, means an amount which provides a therapeutic benefit to a subject. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact “effective amount.” However, an appropriate effective amount can be determined by one of ordinary skill in the art using only routine experimentation. The dosages or amounts of the compounds described herein are large enough to produce the desired effect in the method by which delivery occurs. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the subject and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician based on the clinical condition of the subject involved. The dose, schedule of doses and route of administration can be varied.

[0094] The compositions are administered in an effective amount and for a period of time effect to reduce one or more symptoms associated with the disease to be treated. It should be understood that the “effective amount” for a composition having anti-viral proliferation properties may vary. In one embodiment an effective amount includes without limitation about 0.001 to about 25 mg / kg subject body weight. In one embodiment, the range of effective amount is 0.001 to 0.01 mg / kg body weight. In another embodiment, the range of effective amount is 0.001 to 0.1 mg / kg body weight. In another embodiment, the range of effective amount is 0.001 to 1 mg / kg body weight. In another embodiment, the range of effective amount is 0.001 to 10 mg / kg body weight. In another embodiment, the range of effective amount is 0.001 to 20 mg / kg body weight. In another embodiment, the range of effective amount is 0.01 to 25 mg / kg body weight. In another embodiment, the range of effective amount is 0.01 to 0.1 mg / kg body weight. In another embodiment, the range of effective amount is 0.01 to 1 mg / kg body weight. In another embodiment, the range of effective amount is 0.01 to 10 mg / kg body weight. In another embodiment, the range of effective amount is 0.01 to 20 mg / kg body weight. Tn another embodiment, the range of effective amount is 0.1 to 25 mg / kg body weight. In another embodiment, the range of effective amount is 0.1 to 1 mg / kg body weight. In another embodiment, the range of effective amount is 0.1 to 10 mg / kg body weight. In another embodiment, the range of effective amount is 0.1 to 20 mg / kg body weight. In another embodiment, the range of effective amount is 1 to 25 mg / kg body weight. In another embodiment, the range of effective amount is 1 to 5 mg / kg body weight. In another embodiment, the range of effective amount is 1 to 10 mg / kg body weight. In another embodiment, the range of effective amount is 10 to 20 mg / kg body weight. In another embodiment, the range of effective amount is 20 to 30 mg / kg body weight. In another embodiment, the range of effective amount is 30 to 40 mg / kg body weight. In another embodiment, the range of effective amount is 40 to 50 mg / kg body weight. In another embodiment, the range of effective amount is 1 to 50 mg / kg body weight. Still other doses falling within these ranges are expected to be useful.

[0095] In another embodiment, the range of effective amount is 0.001 mg to 10g. In another embodiment, the range of effective amount is 0.01 mg to 1 g. In another embodiment, the range of effective amount is 0.01 mg to 100 mg. In another embodiment, the range of effective amount is 0.1 mg to 100 mg. In another embodiment, the range of effective amount is 0.1 mg to 500 mg. In another embodiment, the range of effective amount is 1 mg to 100 mg. In another embodiment, the range of effective amount is 10 mg to 500 mg. In another embodiment, the range of effective amount is 10 mg to 750 mg. In another embodiment, the range of effective amount is 0.01 mg to 100 mg. In another embodiment, the range of effective amount is 1 mg to 500 mg.

[0096] In certain embodiments, the compositions described herein is administered via intraperitoneal administration. In these embodiments, the effective amount of the compositions described herein may be between 2-20mg / kg, 0-20mg / kg, about Img / kg, about 2mg / kg, about 3mg / kg, about 4mg / kg, about 5mg / kg, about 6mg / kg, about 7mg / kg, about 8mg / kg, about 9mg / kg, about lOmg / kg, about llmg / kg, about 12mg / kg, about 13mg / kg, about 14mg / kg, about 15mg / kg, about 16mg / kg, about 17mg / kg, about 18mg / kg, about 19mg / kg, or about 20mg / kg.

[0097] In certain embodiments, the compositions described herein is administered via systemic or intravenous administration. In these embodiments, the effective amount of the compositions described herein may be between 2-10mg / kg, 2-20mg / kg, 0-20mg / kg, 14-50mg / kg, 12.5- lOOmg / kg, or at least about Img / kg, about 2mg / kg, about 3mg / kg, about 4mg / kg, about 5mg / kg, about 6mg / kg, about 7mg / kg, about 8mg / kg, about 9mg / kg, about lOmg / kg, about llmg / kg, about 12mg / kg, about 13mg / kg, about 14mg / kg, about 15mg / kg, about 16mg / kg, about 17mg / kg, about 18mg / kg, about 19mg / kg, about 20mg / kg, about 21mg / kg, about 22mg / kg, about 23mg / kg, about 24mg / kg, about 25mg / kg, about 26mg / kg, about 27mg / kg, about 28mg / kg, about 29mg / kg, about 30mg / kg, about 31mg / kg, about 32mg / kg, about 33mg / kg, about 34mg / kg, about 35mg / kg, about 36mg / kg, about 37mg / kg, about 38mg / kg, about 39mg / kg, about 40mg / kg, about 41mg / kg, about 42mg / kg, about 43mg / kg, about 44mg / kg, about 45mg / kg, about 46mg / kg, about 47mg / kg, about 48mg / kg, about 49mg / kg, about 50mg / kg, about 5 Img / kg, about 52mg / kg, about 53mg / kg, about 54mg / kg, about 55mg / kg, about 56mg / kg, about 57mg / kg, about 58mg / kg, about 59mg / kg, about 60mg / kg, about 61mg / kg, about 62mg / kg, about 63mg / kg, about 64mg / kg, about 65mg / kg, about 66mg / kg, about 67mg / kg, about 68mg / kg, about 69mg / kg, about 70mg / kg, about 71mg / kg, about 72mg / kg, about 73mg / kg, about 74mg / kg, about 75mg / kg, about 76mg / kg, about 77mg / kg, about 78mg / kg, about 79mg / kg, about 80mg / kg, about 81mg / kg, about 82mg / kg, about 83mg / kg, about 84mg / kg, about 85mg / kg, about 86mg / kg, about 87mg / kg, about 88mg / kg, about 89mg / kg, about 90mg / kg, about 91mg / kg, about 92mg / kg, about 93mg / kg, about 94mg / kg, about 95mg / kg, about 96mg / kg, about 97mg / kg, about 98mg / kg, about 99mg / kg, or about lOOmg / kg.

[0098] In certain embodiments, the compositions described herein are administered via oral administration. In these embodiments, the effective amount of the compositions described herein may be between 2-10mg / kg, 2-20mg / kg, 0-20mg / kg, 0-40mg / kg, 2-40mg / kg, 10-40mg / kg, 10-20mg / kg, 20-40mg / kg, about Img / kg, about 2mg / kg, about 3mg / kg, about 4mg / kg, about 5mg / kg, about 6mg / kg, about 7mg / kg, about 8mg / kg, about 9mg / kg, about lOmg / kg, about llmg / kg, about 12mg / kg, about 13mg / kg, about 14mg / kg, about 15mg / kg, about 16mg / kg, about 17mg / kg, about 18mg / kg, about 19mg / kg, or about 20mg / kg, about 21mg / kg, about 22mg / kg, about 23mg / kg, about 24mg / kg, about 25mg / kg, about 26mg / kg, about 27mg / kg, about 28mg / kg, about 29mg / kg, about 30mg / kg, about 3 Img / kg, about 32mg / kg, about 33mg / kg, about 34mg / kg, about 35mg / kg, about 36mg / kg, about 37mg / kg, about 38mg / kg, about 39mg / kg, or about 40mg / kg.

[0099] Still further provided are kits comprising one of more of the molecular conjugates or liposomes described herein in one or more vials, tubes, or other suitable vessels. The kit may further comprise a suitable vessel to deliver the molecular conjugate or liposome to a subject. The materials and methods below are provided to facilitate the practice of the present invention.

[0100] All solvents were reagent or high-performance liquid chromatography (HPLC) grade. Anhydrous CH2CI2 and THF were obtained from the Pure Solve™ PS -400 system under argon atmosphere. All reagents were purchased from commercially available sources and sued as received. Reactions were magnetically stirred under a nitrogen or argon atmosphere, unless otherwise noted and reactions were monitored by Thin layer chromatography (TLC) was performed on pre-coated silica gel 60 F-254 plates (40-55 micron, 230-400 mesh) and visualized by UV light. Yields refer to chromatographically and spectroscopically pure compounds. Optical rotations were measured on a JASCO P-2000 polarimeter. Proton (JH) and carbon (13C) NMR spectra were recorded on a Bruker Avance III 500-MHz spectrometer or a Broker NEO600600-MHz spectrometer. Chemical shifts (8) are reported in parts per million (ppm) relative to chloroform (67.26). or methanol (83.31) for}H NMR, and chloroform (877.2) or methanol (8 49.15) for13C NMR. High resolution mass spectra (HRMS) were recorded at the University of Pennsylvania Mass Spectroscopy Service Center on either a VG Micromass 70 / 70H or VG ZAB-E spectrometer. Analytical HPLC was performed with a Waters HPLC-MS system consisting of a 515 pump and Sunfire C18 reverse phase column (20 pL injection volume, 5 pm packing material, 4.5 x 50 mm column dimensions) with detection accomplished by a Micromass ZQ mass spectrometer and 2996 PDA detector. Preparative-scale HPLC was carried out on a Waters AutoPurification system (Milford, MA) equipped with a 3100 mass detector, a 2767 sample manager and a 2489 UV / visible detector. Purification was done on a 19x100mm SunFire® Prep C18 OBD 5pm column using a binary solvent gradient with mobile phase A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile). HPLC grade water and acetonitrile as well as Optima™ LC / MS -grade formic acid were purchased from Fisher Scientific and used without further purification. Fraction collection was triggered using the mass detector. The purity of new compounds was judged by NMR and LCMS (>95%).

[0101]

[0102] benzyl (2R,3S)-6-(((tert-butoxycarbonyl)(methyl)amino)methyl)-3-(2-((4-chloro-3-fluorophenyl)amino)-2-oxoacetamido)-2-(((E)-l,2,3-tris(tert-butoxycarbonyl)guanidino)methyl)indoline-l -carboxylate (1): Was synthesized according to literature procedure, and spectral data was compared to known references to confirm identity of the product.

[0103]

[0104] (2R,3S)-6-(((tert-butoxycarbonyl)(methyl)amino)methyl)-3-(2-((4-chloro-3-fluorophenyl)amino)-2-oxoacetamido)-2-(((E)-l,2,3-tris(tert-butoxycarbonyl)guanidino)methyl)indoline-l -carbonyl chloride (2): Compound (1) (200 mg,

[0105] 0.204 mmol, 1.0 equiv) was added to a microwave vial followed by platinum on graphitized carbon, 20% loading (20 mg, 10% w / w), and anhydrous methanol (2.04 mL, lOml / mmol).

[0106] Subsequently, NaBFL (8.48 mg, 0.224 mmol, 1.1 equiv) was added and an aluminum crimp seal top was added on top of the microwave vial and immediately sealed. Upon addition of the

[0107] NaBH4 the solution bubbled indicated production of H2 (g). This vessel was transferred to an aluminum block and heated at 40 °C for 1 hour, at which time UPLCMS indicated complete consumption of the starting material. The reaction was then diluted in methanol and filtered

[0108] through a pad of celite, washed with methanol (2 mL) and concentrated in vacuo. The

[0109] concentrated product was once again taken up in EtOAc (2 mL), and extracted with water (2 x 2 mL), washed with brine (2 mL), dried over Na2SO4, and concentrated in vacuo to yield a white solid. The crude material was then immediately passed through a plug of silica pre-treated with 10% EtOAc / hexanes with a 1% NEts buffer and eluted with the same solvent system until elution of the desired product 1.1 was observed by TLC (142 mg, 84% yield).

[0110] The material (1.1) was then redissolved in anhydrous CH2CI2 (0.84 mL, 5 mL / mmol), followed by the addition of diisopropylamine (0.0354 mL, 0.251 mmol, 1.5 equiv). Separately, triphosgene (17.35 mg, 0.058 mmol, 0.15 equiv) was dissolved in anhydrous CH2CI2 (0.290 mL, 5 mL / mmol). The triphosgene solution was then added to the starting material solution dropwise at 0 °C and stirred for 3 hours at which time UPLCMS analysis indicated consumption of the starting material. The reaction was then quenched with NH4CI (1 mL), and the aqueous layer was extracted with CH2CI2 (3 x 2 mL). The organic layers were separated, dried with Na2SC>4, filtered and concentrated in vacuo. The crude carbamoyl chloride (2) was then purified by flash chromatography (20% EtOAc / hexanes) to yield 2 as a white powder (160 mg, 83% yield over 2 steps).

[0111] 'H NMR (500 MHz. MeOD) 87.83 - 7.80 (bs, 1H), 7.82 (dd, J= 11.4, 2.4 Hz, 1H), 7.53 - 7.29 (m, 4H), 5.48 - 5.18 (m. 2H), 4.44 (d, J = 17.7 Hz, 2H), 4.18 - 4.06 (m. 1H), 3.97 (dt, J = 14.7, 8.2 Hz, 1H), 2.83 (s, 3H), 1.56 - 1.39 (m, 36H).

[0112] 13C NMR (126 MHz, MeOD) 8 160.92, 160.08 (d, J = 245.22 Hz), 159.62, 154.86, 146.32, 139.04 (d, J = 9.89 Hz), 131.66, 129.61, 118.05, 117.05 (d, J = 17.41 Hz), 109.60 (d, J = 25.89 Hz), 85.92, 61.54, 53.66, 53.16, 28.73, 28.47, 28.30, 20.86, 14.46.

[0113] HRMS (ESI) m / z [M+H]+ calcd for C41H55CI2FN7O11 : 910.3321. found 910.3319;

[0114] [«]D24+68.2 (r 0.50, MeOH).

[0115] F

[0116] ci NBoc 1) HO N3NHBoc

[0117] 2) TFA

[0118] 2

[0119]

[0120] (DY-IV-040)

[0121] 3-azidopropyl (2R,3S)-3-(2-((4-chloro-3-fluorophenyl)amino)-2-oxoacetamido)-2-(guanidinomethyl)-6-((methylamino)methyl)indoline-l-carboxylate (DY-IV-040) (3):

[0122] Compound 2 was dissolved in CH2CI2 (0.1 M), followed by addition of 3-azidopropan-l-ol (1.1 equiv.). N,N-dimethylaminopyridine (1.2 equiv.) was then added in one portion, and the mixture was allowed to stir overnight, at which time UPLCMS analysis indicated consumption of 2. The reaction was quenched with sat. aq. NH4CI, and the aqueous layer was extracted 3 x CH2O2. The organic layers were separated, dried with Na2SC>4, filtered and concentrated in vacuo. The crude 3 (1.0 equiv.) was then taken up in CH2Q2 (0.1 M) and cooled to 0 °C. Trifluoroacetic acid (28 equiv.) was added and the mixture was allowed to warm to rt. The reaction was allowed to stir for 18 hours at which time UPLCMS analysis indicated consumption of the starting material. Upon completion, solvent was removed in vacuo and the product was taken up in 1:1 MeCN / fUO and purified via Waters AutoPur with mass directed HPLC with a flow rate of 32 mL / min and the gradient program as follows: 0-0.5min 15% B, l-9min linear from 15% to 35% B, 9-9.5min linear from 35% to 95% B, 9.5-11.5min 95% B and 11.5-12min 10% B. Fractions were collected based on a mass trigger and was lyophilized to yield the final product, 3 (DY-IV-040) as a colorless oil.

[0123] ‘H NMR (500 MHz, MeOD) 57.94 (bs, 1H), 7.83 (dd, J = 11.4, 2.2 Hz, 1H), 7.54 - 7.41 (m, 3H), 7.23 (dd, J = 7.8, 1.6 Hz, 1H), 5.24 (d, J= 2.4 Hz, 1H). 4.58 (bm, 1H), 4.45 (m, 1H), 4.23 (s. 2H), 3.63 - 3.47 (m, 4H), 2.73 (s, 3H), 2.04 (m, 2H);

[0124] 13C NMR (126 MHz, MeOD) 5 162.90 (q, JCF = 35.57 Hz, TFA), 161.31, 161.28, 159.94 (d, JCF = 246.54 Hz), 159.29, 159.23, 138.96 (d, JCF = .T1 Hz), 134.57, 131.75, 128.00.

[0125] 126.45, 118.31, 118.08 (d, JC = 3.49 Hz), 117.41 (d, JCF = 18.13 HZ), 109.85 (d, J = 25.71 Hz), 66.71, 65.62, 54.44, 53.55, 49.57, 44.48, 33.11, 29.38;

[0126] HRMS (ESI) m / z 575.2043 [calcd for C24H29CIFN10O4 (M+H)+ 575.2039].

[0127] The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.

[0128] Example I: Nanotrap Therapeutic Vaccine for HIV

[0129] To date, clinical treatment of HIV is limited to antiretroviral therapies (ART), which suppress viral replication but do not generate anti-HIV immunity or eliminate latent reseviors.15ART therapies also carry a myriad of side effects, ranging from nausea to liver toxicity13, leading 25% of ART patients to discontinue their therapies.14Strategies to reduce ART reliance, therefore, are highly significant. A promising HIV cure strategy is a therapeutic vaccine, where vaccines are administered after viral exposure to stimulate anti-HIV immunity.18To date. however, no therapeutic vaccines have been generated that significantly reduce viral loads, most likely a result of high genetic variation in HIV preventing effective antigen selection.12Here, we propose a new HIV vaccine that leverages the natural immunity of “Elite Controllers” (ECs). ECs are a small (<0.5%) population of patients who have been infected with HIV, show an initial viral infection, but then naturally suppress the infection.19While EC immunity is still not completely understood, it is known that EC immunity correlates with protective HLA alleles and type I interferon (IFN) responses from APCs.20-21Recapitulating even one aspect of the EC-like immunity during vaccination would likely have strong HIV protective effects. (Fig. 1)

[0130] In contrast to existing therapies, such as ART that require lifelong administration contrast, there is corroborating evidence that EC also generate strong CD8+T cell responses against HIV, which could effectively suppress HIV.

[0131] Here, we provide a novel therapeutic vaccine, termed nanotrap therapeutic vaccines (NTVs). NTVs are modified liposomes which contain HIV targeting molecules on their surfaces and adjuvants in their cores to skew downstream adaptive immunity towards CD8+T cells (Fig 2). The data presented herein indicate that NTVs bind and inactivate circulating HIV particles in early infection when viral loads are high. Then NTVs are phagocytosed by local APCs, skewed into Thl responses using Thl skewing adjuvants, or into cytotoxic T lymphocytes (CTLs) using CTL-skewing adjuvants, and therefore generate strong CD8+T cell responses against the unique HIV strain of each patient. Furthermore, during early infection CD4 T cell counts remain relatively high and would still be able to participate in CD8+T cell activation and APC licensing.22

[0132] Our data indicates that NTVs can be administered concurrently with ART, when viral loads are high and CD4 T cell counts are still high, to reprogram the initial immune response. The data presented herein demonstrates that NTVs can 1) capture circulating HIV and 2) facilitate uptake of HIV viral particles without infection.

[0133] Development of High Avidity HIV Binding Nanoparticles to Develop Patient- Specific Anti-HIV Immunity:

[0134] NTVs are designed to bind and inactivate a wide range of HIV strains with high sensitivity. NTVs repurpose previously developed HIV fusion inhibitors, such as CD4 mimetics, and use them instead to capture HIV viral particles on the surfaces of liposomes. Liposomes are highly modular nanoparticles; this allows for tuning of fusion inhibitor valency, liposomal size, and surface charge. These can be optimized to develop highly selective NTVs.

[0135] The fusion inhibitors which bind gpl20 with lipid tails (termed ligand- lipid conjugates) are chemically modified to facilitate their insertion into liposomes at controlled ratios of inhibitors per liposome (Fig. 2). This allows precise control over fusion inhibitor valency and overall HIV-NTV avidity. Several options are available for fusion inhibitors to display on NTVs and use in combination to engineer the highest avidity and broadly binding NTV.

[0136] One major challenge in the design of HIV therapeutic vaccines is viral escape to the chosen inoculation antigen.18Here, NTVs offer a significant advantage; by capturing the prevalent circulating HIV strain as the “antigen” of the vaccine, viral escape is less likely. NTVs repurpose viral entry inhibitors which selectively bind Env proteins, mimic CD4 binding sites and bind a broad spectrum of HIV strains.23These inhibitors have been shown to have functionality against a diversity of HIV strains.24Our data indicates NTVs reduce the incidence of viral escape via multivalency. Even if Env mutations reduce the monovalent affinity of viral entry inhibitors, we can modulate the overall avidity of NTVs through increasing valency; furthermore, we can use combinations of entry inhibitors.25

[0137] NTV Mediated Control of APC-HIV Immune Responses: Our data indicates that NTVs capture HIV and then facilitate uptake by local APCs. Our vision is that CTL or Thl skewing adjuvants can be loaded into liposomal cores and facilitate robust EC-like CD8+T cell responses. There is evidence that CD8+T cells control HIV.26,27NTVs can be used as a platform to test this by selectively delivering Thl skewing adjuvant with HIV to APCs.

[0138] Demonstration of NTVs binding HIV viral particles and preventing viral infection in vitro:

[0139] We first validated that NTVs can be synthesized and effectively bind HIV in vitro at low concentrations and inhibit infection due to their multivalent display of fusion inhibitors.

[0140] NTVs can be synthesized and optimized by conjugating small molecule gp!20 binding fusion inhibitors to lipids (ligand-lipid conjugates), facilitating incorporation into liposomes to generate NTVs, which bind HIV viral particles (Fig. 3). There are many developed fusion inhibitors that bind either gpl20 or gp41 in either clinical use or preclinical testing.28Here, we

[0141] T1 focused on three fusion inhibitors that target gp!20. The first is CJF-III-288, a small molecule CD4 mimetic developed by the Smith group.29We have modified CJF-III-288 with an azide to facilitate lipid conjugation using click chemistry.30Next, we used an optimized cyclic peptide triazole (cPT) called MG-II-20, which was developed by the Chaiken group (a Co-I on this project) and designed to tightly bind gpl20 and trigger its shedding from HIV.31The cPT molecule contains a free amine that can be used as a conjugation handle. Lastly, we modified the prodrug FDA fusion inhibitor, fostemsavir, another CD4 mimic, via free phosphate groups.32All three molecules have documented HIV replication inhibition (Table 1).

[0142] Table 1: gpl20 Fusion Inhibitors for NTVs

[0143] Viral Entry Inhibitor IC50* Synthesis Handle Ref

[0144] CJF-III-288 200nM Azide 24

[0145] cPT (GM-II-20) 43nM Primary amine 26

[0146] Fostemsavir lOOnM Phosphate 28

[0147]

[0148] * viral inhibition assays were performed on JRFL wild type Pseuc ovirus

[0149] These three molecules were chosen for this initial study because 1) they each have high binding to gpl20, 2) are readily available, 3) have documented capacity to bind and inhibit HIV function, and 4) function against a wide range of HIV clades. Other potential HIV binding molecules are identified in Fig. 4.

[0150] NTV Design and Synthesis:

[0151] To generate NTVs, we first modified the gpl20 binding ligands with lipids, then synthesize liposomes using these modified lipids. We modified the lipids prior to synthesis (ligand-lipid conjugate), facilitating precise control over ligand density on the surface (Fig. 3). Modified lipids are synthesized to display ethylene glycol spacers and a charged amino acid side chain to increase surface availability of gpl20 binding molecules.33NTVs are then synthesized using the ligand-lipid conjugates. NTV size, HIV binding ligands, combinations of ligands and valency can be varied.

[0152] Ligand-Lipid Conjugate Lipid Synthesis: Palmitic acid (Cl 6) lipid tails are conjugated to linker moieties containing arginine to increase polarity and ethylene glycol spacers using standard Fmoc solid phase peptide syntheses (Fmoc-SPPS).34NTV lipids were synthesized sequentially coupling amino acids, PEG spacers, and lipid tails via HBTU activation. The HTV-binding moiety was conjugated through copper-free click chemistry to a DBCO group near the lipid head. Final products were cleaved with 88% TFA for purification. Linkers contain three modulatory moieties: 1) a charged amino acid (typically Arg or Lys) which facilitate proper display on liposomal surfaces33, 2) an ethylene glycol (PEG) spacer of variable length and 3) a reactive moiety (amine, carboxylic acid or DBCO group).

[0153] Lipid-linkers are cleaved from solid phase and purified using reverse phase HPLC (RP-HPLC) using a C8 column and a linear acetonitrile / water (0.1% TFA) gradient. Each compound showed a sharp peak at ~10 minutes under UV detection at 280 nm, confirming high purity and efficient synthesis. Purified NTV lipids were then analyzed using matrix assisted laser desorption / ionization time of flight mass spectrometry (MALDLTOF) to verify molecular identity. The spectra displayed dominant peaks corresponding to the theoretical molecular weights of each construct, confirming successful conjugation and high structural fidelity.

[0154] Lipid-linkers are then reacted with azide modified CJF-III-288 using click chemistry, cPT using amide chemistry or fostemsavir using phosphate-amine reactions.30,35The ligand-lipid conjugates are purified via RP-HPLC and analyzed via MALDI. TK-9 (CJF-lipid with Arg linker) HPLC and MALDI-TOF data are shown in Fig. 6. TK-14(CJF-lipid with Lys linker) HPLC and MALDI-TOF data are shown in Fig. 7. TK-16 (CJF-lipid with Lys x3 linker) HPLC and MALDI-TOF data are shown in Fig. 8. TK-15 (MG-II-20-lipid with Arg linker) HPLC and MALDI-TOF data are shown in Fig. 9. TK-20 (N2-lipid with Arg linker) HPLC and MALDI-TOF data are shown in Fig. 10. The chemical structure of each of these conjugates are shown in Fig. 11 A- HE.

[0155] NTV Synthesis: NTVs bearing ligand-lipid conjugates are generated using solid-phase peptide synthesis (SPPS) using standard Fmoc chemistry. A schematic of this synthesis is shown in Fig. 12. Sequential coupling and deprotection cycles with 20% piperidine in DMF enabled controlled peptide chain elongation on resin, while PEG spacers or specific amino acids were incorporated to enhance hydrophilicity and flexibility. After the peptide backbone assembly, the ivDde protecting group on lysine was selectively removed, and the free amine was coupled with DBCO-TFP ester to introduce an alkyne functionality. Subsequently, the azide-functionalized ligand CJF-III-288 was conjugated via copper-free click chemistry, yielding the final lipid ligand-peptide construct. The completed product was cleaved from the resin using a TFA / phenol / water / triisopropylsilane (88:5:5:2) mixture, followed by precipitation and purification by HPLC for subsequent characterization and liposomal incorporation.

[0156] Liposomal assembly: The hydrodynamic diameters of various NTV formulations were measured by dynamic light scattering (DLS). (Fig. 14A) All formulations exhibited uniform particle sizes ranging from 140 to 170 nm, depending on the incorporated peptide-lipid composition, indicating stable liposomal assembly.

[0157] The poly dispersity index (PDI) of various NTV formulations (NTV-9, NTV-14, NTV-15, NTV-16, and NTV-20) was measured at 10 pM in PBS to assess nanoparticle uniformity and dispersion stability. All formulations exhibited low PDI values (< 0.25), indicating monodisperse particle populations with minimal aggregation. Among them, NTV-14 showed the highest PDI (-0.22), while NTV-16 maintained the narrowest distribution (-0.10), suggesting enhanced structural homogeneity. These results confirm that the NTV formulations remain stable and well-dispersed under physiological buffer conditions, suitable for downstream biological studies.

[0158] Zeta potential measurements of NTV formulations revealed negative surface charges between -20 and -30 mV. (Fig. 14B) The consistent negative (^-potential values reflect colloidal stability, which helps prevent aggregation and enhance formulation stability in physiological conditions.

[0159] To evaluate the incorporation efficiency of NTV lipids into NTV liposomes, equal concentrations of synthesized NTV and corresponding free NTV lipids were analyzed using an analytical C8 HPLC column. (Fig. 14C) After diluting both samples in acetonitrile, chromatograms were compared based on peak area differences between the intact NTV lipid (red) and degraded form (blue). Both NTV lipid and degraded NTV peak exhibited a retention time around 10 min. Quantitative analysis revealed that only 34.53% of the total NTV lipid was successfully incorporated into the liposomal formulation. This result highlights the need for optimization of lipid composition and incorporation parameters to improve the overall NTV assembly efficiency.

[0160] NTV Binding and inhibiting HIV virus in vitro:

[0161] After synthesizing NTVs, we down selected NTVs that show poor gpl20 binding or HIV infection inhibition. For binding affinity, the binding affinity (Kd) of ligand-lipid conjugate (i.e. not in liposomes) and the apparent Kd of the multivalent interaction between gpl20 and NTVs is measured using surface plasmon resonance (SPR). Additionally, NTV prevention of infection is confirmed using standard in vitro infection assays with reporter cell lines and quantitative PCR (qPCR).

[0162] Surface Plasmon Resonance (SPR) Analysis: SPR is used to verify that ligand-lipid conjugates still retain binding for gpl20. For SPR analysis the Drexel Biacore S200 SPR Shared Instrument Laboratory was used. For all experiments we measure binding of our modified lipids and NTVs against gpl20 from HIV-1 Env variants from subtype B, namely YU2, BaL.Ol and JRFL bound to sensor chip surfaces using literature procedures.31SPR is also used to measure competitive binding in the presence of soluble CD4 or CD4 binding site antibodies. Controls include binding site mutated gpl20 and blank liposomes. We have tested the three variations of CJF-lipid using SPR against 8000 RFU of surface bound YU2 gpl20 and observed the binding affinity (Kd) of each (Table 2).

[0163] TABLE 2: CJF-Lipid gpl20 binding affinity

[0164] Linker Kd (nM)

[0165] Arg (TK-9) 780 ± 20

[0166] Lys (TK-14) 75 ± 3

[0167] Lys x3 (TK-16) 6.1 + 3

[0168] Free CJF 6.4

[0169]

[0170] Our data shows that the modified CJF-lipids bind gpl20 via SPR, demonstrating that modification of amino acid linker improves binding affinity (Table 2). SPR data shows CJF-lipid molecules bind gpl20. 3000 RFU of gpl20 YU2 variant was attached to a SPR chip (Biacore S200) and concertation of either TK-9 (C9, arg linker), TK-14 (C14, Lys linker) or TK-16 (C16, Lysx3 linker) compared to free CJF-III-288. Kd was calculated. (Fig. 13)

[0171] HIV Pseudovirus and Live Virus Samples: The data provided herein was generated using both Pseudovirus samples and live human samples. We first validated binding using Env from BaL.Ol wild type (NIH BEI sample repository) and expressed on a non-infectious NL-4 backbone with a luciferase reporter (ARP-3418) or expressing GFP (HRP-12455), depending on the experiment.36We have chosen R5 binding Env as our model, as R5 is more common during early infection.37After down selecting NTVs that do not bind, we validated the breadth of binding against the 12-pseudo virus panel taken from the Duke HIV Global Reference Panel.38 GpI20 Shedding Analysis: The cPT was designed to inactivate HIV by triggering gpl20 shedding.39To investigate whether liposome bound cPT will still trigger this effect, we analyzed cPT bearing NTVs incubated with wild type BaL.Ol by HPLC analysis and western blot according to previously published protocols.40Controls include blank liposomes, Env deficient pseudovirus, and free cPTs. TK-16 was tested for binding against HIV-infected patient serum samples from Drexel CARES Cohort (Fig. 19).

[0172] Binding profile of NTV 10 M total lipid, loaded at 1% TK-16, to 500RU gpl20 monomer, YU2 variant (red trace), 10 min injection followed by 10 min wash with PBS running buffer. Blank liposome injection is shown (blue trace) among the buffer injection traces, indicating there is no binding of unmodified liposomes in absence of CJF-III-288. (Fig. 20) Specificity of NTV binding was then assessed by comparing gpl20+TF228 cells and gp 120 Jurkat cells. At 4 °C (endocytosis blocked), NTV + TF228 showed strong surface binding, while Jurkat + NTV and blank + TF228 controls produced minimal fluorescence. These results confirm that NTV binding is driven by gpl20 recognition rather than nonspecific adsorption. (Fig. 21) To confirm that NTVs selectively target gpl20 expressing cells, we co-cultured DiO labeled gpl20 expressing cells (TF-288) and non-gpl20 expressing cells (Jurkat). The cells were incubated with DiD labeled (0.2%) NTVs at different ratios of Jurkat:TF288 (1:1, 10, 100 or 1000) DiD-i- cells were

[0173] identified by flow cytometry and a enrichment ratio, DiD % cells / % TF288 cell, was calculated. (Fig. 22) Next, the binding of NTVs to gpl20-expressing TF228 cells was evaluated using DiD fluorescence as a readout for liposomal association.

[0174] NTVs incorporating gpl20-binding ligands (Arg, Lys. Lysx3) showed strong and concentration-dependent DiD signals, while blank and control lipids displayed minimal fluorescence. These results confirm specific gpl20-mediated binding of NTVs to TF228 cells at physiological temperature. (Fig. 23) To investigate how ligand density influences gpl20 binding, Lysx3-functionalized NTVs were formulated with varying incorporation ratios (0-2 %). DiD fluorescence increased proportionally with ligand content, indicating that higher surface presentation of gpl20-binding peptides enhances NTV-gpl20 interaction efficiency. (Fig. 24)

[0175] In Vitro Infection Inhibition Assays: Iterations of HIV infection inhibition assays are performed in vitro. The goal of these experiments is to show that NTVs can bind and inactivate HIV virus over a wide range of concentrations according to established literature protocols. We use two different reporter cell lines for HIV infection: 1) a luciferase reporter cell line TZM-bl and 2) a GFP expressing line. GHOST. TZM-bl is used for higher throughput initial screening of NTV via 96-well plate luminance reader.39GHOST cells are analyzed via flow cytometry, fluorescence microscopy and kinetic real-time infection analysis using an Incucyte live cell analyzer. Controls include wells with no inhibitor, no pseudovirus, and free fusion inhibitors. We loaded 1% of Arg-CJF-lipid into liposomes and showed inhibition like free CJF using GHOST cells (Fig. 5A).

[0176] The effect of NTV on the antiviral activity of Fostemsavir, Lenacapavir, and Emtricitabine was further evaluated in HIV pseudovirus infection assays. Each drug was tested across serial dilutions in the presence or absence of 10 uM NTV. Flow cytometry analysis revealed that co-treatment with NTV (red) did not significantly alter the inhibition profiles compared with PBS controls (blue), indicating that NTV is compatible with standard antiretroviral compounds and does not interfere with their antiviral efficacy. (Fig. 25).

[0177] Live Vims Infection Inhibition Assays: In a similar in vitro assay, we analyze NTV’s capacity to prevent infection from live virus in human PBMCs. This assay is used to assess 3-5 optimized NTV formulations and their ability to bind live HIV. We also validate NTVs capacity to bind and inhibit HIV at low concentrations (<1000 copies / mL), which reflects virus concentrations in recently infected but ART suppressed patients.41Serum samples from the Drexel CARES cohort are incubated at various dilutions with NTVs for 24-72 h and then incubated with commercially sourced human peripheral blood mononuclear cells (PBMCs). Cells are analyzed for HIV using either quantitative PCR (qPCR) for viral mRNA or using flow cytometry after staining for p24 using literature protocols.19Controls include no inhibitors, free fusion inhibitors, no virus, healthy patient serum and pseudovirus spiked controls.

[0178] Establishment that NTVs bind HIV viral particles in vivo and facilitate viral uptake and activation by local APCs

[0179] Next, we evaluated NTV-HIV binding with an in vivo system and analyzed how HIV binding facilitates phagocytosis by local APCs. Furthermore, we validated that NTV can trigger APC immune activation. All NTV formulations were loaded with one of two immune agonists, the toll-like-receptor (TLR) 7 / 8 agonist R848 or the TLR 9 agonist, CpG (either ODN 1826 for mouse in vivo studies or ODN 1018 for human in vitro studies).39,40R848-loaded NTVs, blank NTVs, and free R848 were analyzed by reverse-phase high-performance liquid chromatography (HPLC). An overlapping peak at -10 minutes observed for both NTV and free R848 confirms successful R848 encapsulation, with an estimated loading efficiency of -38%. (Fig. 18) Both of these agonists have been shown to both load into liposomes readily and induce Thl skewed immune activation of from APCs.

[0180] Next we developed a non-humanized mouse model of HIV by injecting wild-type mice with pseudovirus to simulate early HIV infection and immediately inject NTVs. We analyzed NTV-HIV binding in vivo using this model and observe cellular uptake of HIV-NTV complexes. Because HIV does not infect murine cells, any uptake of pseudovirus is due to NTVs. We used C57B1 / 6, as these mice have been shown to more likely generate Thl responses from APCs.42

[0181] Pseudoviral Mouse Model for In vivo NTV Binding: C57B1 / 6 mice are injected via retroorbital injection (r.o), i.p or s.c. with 0.5 or 5 ng / kg p24 of 1) blank pseudovirus or 2) luciferase expressing pseudovirus. 5 or 50 mg / kg total lipid of NTV is immediately

[0182] injected (either r.o., i.p., or s.c.), and blood drawn 30 mins after that to analyze circulating virus and blood cells. (Fig. 15) Spleens and lymph nodes are also be removed for

[0183] analysis.

[0184] In vivo NTV-HIV Binding Analysis: Mice are injected with pseudovirus and agonist loaded NTVs bearing biotin tags and blood will be taken after 30 minutes and analyzed for NTV-HIV binding in vivo. We isolated the HIV-NTV complex using affinity precipitation on streptavidin bearing magnetic microparticles, washed the particles, removed complexes and quantified pseudovirus bound to NTVs using qPCR or luciferase expression.43Controls include no pseudovirus, no NTV, non-biotin bearing NTVs and samples spiked with preincubated NTV and HIV. Our data shows that NTVs facilitate viral uptake by immune cells by flow cytometry and microscopy (Figs. 26-27). Our data further indicates that NTVs also active immune cells, triggering upregulation of immune receptors and release of inflammatory cytokines. (Fig. 28) PBMC, Spleen and Lymph Node Analysis: Mice sequentially injected with pseudovirus and fluorescently labeled, agonist loaded NTVs have PBMCs isolated from blood and spleens / lymph node proximal to the injection site and analyzed by qPCR (for virus), flow cytometry and microscopy at 24 h post injection. These experiments are done using GFP-expressing pseudovirus and infrared dye labeled NTVs. Cells are labeled with immunophenotyping for T cells (CD3+), B cells (CD19+), monocytes (CD14+), macrophages (CD68+), central DCs (CDllc+B220 ) or peripheral DCs (pDCs. CDllc+B220+).44. We also observed APC activation markers, CD80, CD86 and CD40. These studies are repeated using pseudovirus with a TAT controlled luciferase reporter and analyze cells for luciferase activity to ensure that NTV is facilitating viral uptake but not infection. Controls include no pseudovirus, no NTV, and no-agonist loaded NTVs.

[0185] The data generated from this experiment to validated that 1) NTVs are safe and tolerable in vivo and 2) NTV and pseudovirus circulate in vivo long enough to associate. 8 ng of p24 / kg mouse of BaL.Ol was injected r.o., then sequentially 50 mg liposome / kg mouse of DiD-tagged NTVs displaying 1% Lysx3-CJF-lipid and loaded with 10 pg R848 / kg mouse were injected i.p. into C57B1 / 6 mice (NTV). R848 loaded liposomes with no CJF molecules (blank) and free R848 (free) were included as controls. Blood was drawn at 5 min, 1 h, and 4 h post injection and mice were sacrificed, and spleen and blood harvested 24 h post injection. We observed a decrease in the systemic cytokines TNFa and IL-6 1 h post injection from liposomal formulations compared to free R848 and no major side effects, indicating that NTVs are likely safe and tolerable (Fig.

[0186] 16 A). We observed that p24 levels in serum remain detectable for up to 4 h for free R848 groups but are lower for NTV group at 4 h (Fig. 16B), Liposome positive cells were observed via flow cytometry in red blood cell (RBC) depleted blood samples at 1 h and 4 h and increased after 24 h (Fig 16C). This indicates that liposomes and viruses are both present in systemic circulation between 1-4 h. We further analyzed RBC depleted blood and disaggregated spleen cells for liposome+ cells and observed that NTVs are taken up by pDCs and macrophages. This indicates that NTV and virus associate in vivo and are taken up by pDCs and macrophages.

[0187] APCs Selectively Uptake NTV-HIV complexes:

[0188] Next, we analyzed NTV-HIV complex cellular uptake by using co-culture systems of reporter cell lines and human primary immune cells. We observed how NTV-HIV complexes are selectively phagocytosed and processed by APCs, rather than infecting them, and NTV complexes will prevent uptake and / or infection of other cell types. Finally, we show that NTVs loaded with adjuvants will trigger APC activation and HIV processing.

[0189] In vitro APC Uptake Analysis: We investigated NTV-HIV complex uptake and immune stimulation. These experiments test our hypothesis that NTV-HIV complexes are preferentially phagocytosed by APCs and, when NTVs are agonist loaded, trigger immune activation.

[0190] These experiments consist of co-cultures of 1) HIV, 2) fluorescently labeled NTVs, 3) GHOST reporter cell lines and 4) APCs. We varied both the HIV source and APCs, using pseudovirus (both labeled and unlabeled) or live HIV and monocyte derived dendritic cells (moDCs), pDCs isolated from human blood or PBMCs, purchased from the UPenn Human Immunology Core. Cells are analyzed for NTV-HIV uptake by flow cytometry.

[0191] HIV infection is monitored using GHOST GFP expression, qPCR analysis and p24 ELISA. Controls include no viruses, no NTV and no APC. We also replaced the APCs with a non-phagocytic cell line as an additional control.

[0192] For an initial evaluation of NTV’s capacity to stimulate APCs, we loaded NTVs with the adjuvant CpG 1018, a TLR-9 agonist or R848, a TLR 7 / 8 agonist.45We

[0193] incubated agonist loaded NTV with either pseudovirus or live HIV, then incubated with isolated human pDCs or moDCs. We monitored inflammatory cytokines using Cytokine Bead Analysis (CBA) and changes in the cell surface markers HLA-DR, CD40, CD80, CD83 and CD86, indicating activation. Similar controls as in the previous section were used.

[0194] APC NTV-HIV Lysosomal Analysis: We further analyzed uptake of NTV-HIV complex into APC lysosomes using lysotracker dyes in similar experiments as the

[0195] previous section.46We monitored co-localization of lysosomal compartments with fluorescently stained NTVs and Pseudovirus with lysosomal staining dyes using fluorescence microscopy. We further tracked Pseudovirus degradation over the course of 24 h using p24 ELISAs. Similar controls as in the previous section are used.

[0196] Our data shows that GHOST cells incubated with THP-ls (a human monocyte cell line) decrease NTV mediated infection inhibition IC50 by lOOx (Fig. 17). We observed that labeled NTVs are most likely uptaken by pDCs in blood and spleen cells at 24 h. When this was repeated for longer time points (28 days) we observed increases in HIV specific T cells, antibodies and observed that these antibodies effectively prevented infection in vitro. (Fig. 29-31)

[0197] Example II: Nanotrap Therapeutic Vaccine for other viral infections In addition to latency, immune evasion and genetic diversity, another typical hallmark of chronic infections is lack of an effective vaccine. No approved vaccine exists for HIV or HCV, and SARS-CoV-2 vaccine efficacy is highly strain variable.7 12One promising cure strategy is therapeutic vaccination, where a vaccine is given after infection to generate appropriate protective immunity to latent or immune evasive infections.18There is a major knowledge gap, however, as highly mutagenic viruses do not allow for reliable prediction of antigens for use in therapeutic vaccination. There are many strategies currently pursued for therapeutic vaccination against viral chronic infections, including antigen loaded dendritic cells, high potency superagonists (such as N-803), highly multivalent antigen nanoparticles (eOD-GT8), and mRNA or cDNA based vaccines, typically delivered via lipid nanoparticles.10-18-47While these strategies have shown progress in generating broadly neutralizing antibodies and potent T cell responses, the primary challenge is to identify conserved antigens that can form protective responses against any strain of the virus. VTAs present a potential solution to this problem by using the circulating virus as the antigen with targeted adjuvants. Leveraging the show proof-of-concept for VTAs as both a nanoparticle-based design and a small molecule using HIV described above, we will also evaluate VTAs for HCV and SARS-CoV-2.

[0198] Most therapeutic vaccine strategies focus on strengthening the immune response and identifying conserved antigens. Instead, we take a radically different approach by capturing the circulating, immune evasive, viral particles and use the host viral proteins as the antigen. VTAs are designed generally to seek out circulating viral particles, tag them with immunogenic stimuli and facilitate their uptake by local antigen presenting cells (APCs). This effectively yields a “personalized” therapeutic vaccination. Additionally, we are investigating viral capture molecules, such as pixatimod or peptides derived from the antiviral protein Griffithsin, that have affinity for surface proteins expressed on several viruses (Table 3).15 1619

[0199] Table 3. List of potential viral capture domains

[0200] Virus Target Molecule KdIC50 Type Synthesis Source Ref nM nM Handle

[0201] HIV gp120 CJF-lll-288 6.4 200 Small Azide Huryn 29 (Azide molecule Lab modified)

[0202] HIV gp120 MG-ll-20 126 43 Cyclic Primary Chaiken 31 peptide amine Lab

[0203] (cPT)

[0204] HIV p24 Lenacapavir 1.4 0.03 Small Alkene Vendor 48 molecule

[0205] SARS- Spike VE607 N.D. 1000 Small Hydroxyl Vendor 49

[0206]

[0207] CoV-2 (RBD) molecule SARS- Spike P10 peptide 0.03 42 Peptide Peptide In house 50 CoV-2 synthesis

[0208] SARS- Spike S1b3inL1 50 300 Cyclic Peptide In house 51 CoV-2 peptide synthesis

[0209] HCV E2 Peptide 19 N.D Peptide Peptide In house 52 PE2D synthesis

[0210] HCV E1 / E2 Peptide E2- N.D. 1 Peptide Peptide In house 53

[0211] 42 synthesis

[0212] Multi- Many Suramin N.D. Low Heparin Sulfate Vendor 54-57 viral pM derivative group

[0213] Multi- Many Grifonin-1 N.D 190 Griffithsin Peptide In house 58 viral (HIV) peptide synthesis

[0214] Multi- Many Pixatimod N.D. Low Heparan Cholesterol Vendor 59,60 viral pM derivative group

[0215] attached

[0216] 0.15- HBV NTCP Bulevirtide 8 Peptide acyl / amide Vendor 61 0.2

[0217] HBc Compound Small

[0218] HBV n.d. 900 amine In house 62 protein 16 molecule

[0219] ~4- Macrocyclic

[0220] HBV NTCP L5 / L6 n.d. amine In house 63

[0221] 50 peptide

[0222] low- DNA / RNA DNA Custom HBV HBsAg HBs-A22 n.d. 64 nM aptamer conjugation oligo

[0223] DNA DNA DNA Custom HSV-1 gD aptamer 50 n.d. 65 aptamer conjugation oligo

[0224] (45-nt)

[0225] Entry

[0226] via 3-0- 3-0- HSV- 1

[0227] sulfated G2 Peptide NR sulfated HS Peptide In house 66 1 / 2 mg / mL

[0228] heparan peptides

[0229] sulfate

[0230]

[0231] Abbreviations:

[0232] HBV, Hepatitis B Virus; HCV, Hepatitis C Virus; HSV-1 / 2, Herpes Simplex Virus Type 1 and 2; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; NTCP, Sodium Taurocholate Cotransporting Polypeptide (HBV entry receptor); HBc, Hepatitis B core protein; HBsAg, Hepatitis B surface antigen; gD, HSV envelope glycoprotein D; E1 / E2, HCV envelope glycoproteins 1 and 2; RBD, Receptor Binding Domain of the SARS-CoV-2 spike protein; n.d., not determined; NR, not reported; pM, micromolar; Kd, equilibrium dissociation constant (affinity); ICso. half maximal inhibitory concentration; DNA / RNA aptamer, short structured oligonucleotide that binds a specific target; macrocyclic peptide, chemically cyclized peptide with enhanced binding and stability; heparin derivative, sulfated polysaccharide analog of heparan sulfate; Griffithsin peptide, lectin from Griffithsia sp. with broad and viral activity; Pixatimod, synthetic sulfated cholestanol glycoside (heparan sulfate mimetic); PE2D and E2-42, HCV E2 -region derived peptides; G2 peptide, synthetic mimic of 3-0-sulfated heparan sulfate binding motif; PIO peptide, SARS-CoV-2 spike fusion inhibitory peptide; Slb3inLl, cyclic peptide binder to SARS-CoV-2 spike SI domain; Compound 16, HBc small-molecule inhibitor from Sato et al.; Bulevirtide, HBV entry inhibitor (Myrcludex B, preSl lipopeptide).

[0233] It should be noted that these pan- viral inhibitors typically have lower monovalent affinities, making our highly multivalent display platform of NTVs ideal. This allows for generation of a universal antiviral therapy designed to improve immune responses without regard to strain or virus type. We also note that novel broad spectrum viral inhibitors are being developed, although concerns over synthesis feasibility make peptides and pixatimod more favorable.20These exemplary candidate viral capture moieties, originally designed as fusion inhibitors, that we can generate in house or purchase commercially and conjugate onto VTA.

[0234] Additionally, there are a number of small molecule adjuvants that have been identified that known to generate potent antiviral immunity driven by CD8+ T cells.26,67,68We can use molecules known to bind multiple types of viruses, such as Pixatimod, known to have pleiotropic antiviral effects, to develop a more versatile VTA.60We can adjust this adjuvant moiety to alter adaptive immune response, as it has been shown that adjusting the type of adjuvant and pattern recognition receptors stimulated can modulate not only the intensity of the immune responses but also the type (Thl vs Th2, etc.) and even peptides displayed on APCs.69Our platform is innovative because we can readily tune both the virus targeted and the type of adaptive immune responses by changing these moieties. We can also tune the viral capture ligand multivalency and the biophysical characteristics of the VTA (size, surface charge, etc.).

[0235] We can also leverage this platform to develop VTAs to both extracellular and intracellular viral antigens. Adjuvant loaded liposome bearing viral capture moieties, called nanotrap therapeutic vaccine (NTV), and a small molecule variant directly conjugating viral capture to adjuvant moiety, molecular therapeutic vaccine (MTV) can be generated. A translational concern is that viral particles are in relatively low concentrations in the blood during chronic or latent infections and mainly persist in diverse tissue sites.70Furthermore, for many chronic infections, the number of circulating viral particles is relatively low during the chronic infection phase, and viral material is sequestered intracellularly or on cell surfaces.71The benefit of using NTVs (nanoparticles) is that viral capture domains can be displayed in a multivalent fashion, improving overall selectivity to rare viral particles.72Conversely, nanoparticles often struggle to diffuse into distal tissue locations and cannot reach intracellular viral antigens. To address this potential issue, we can also generate small molecule variants, MTVs, with a higher tissue and cell permeability. For example, a MTVs that target p24 antigen, a capsid protein typically sequestered either within viral membranes or intracellularly in infected cells, by conjugating TLR agonists to the high affinity p24 binding molecule lenacapavir.48This innovative approach allows us to target rare viral particles with high selectivity (NTVs) and target intracellular viral material potentially in diverse peripheral tissue sites in vivo (MTVs).

[0236] MTVs target intracellular antigens, thereby expanding VTA functionality. We have chosen a conjugate of the high affinity capsid inhibitor and p24 binding molecule lenacapavir73to a chemically reactive variant of the TLR7 / 8 agonists R848, known as 2Bxy.74We can link these two functional moieties with polyethylene glycol (PEG) spacers. MTVs can then diffuse and bind p24 assemblies within infected cells (Fig. 32).

[0237] MTV synthesis: Lenacapavir contains an alkyne group that can be selective conjugated with azide containing molecules via click chemistry.30From structural binding analysis, this alkyne is unlikely to be critical for binding.752Bxy contains a reactive amine that can be conjugated via TFP esters. A commercially available TFP-PEG-azide molecule with varying PEG spaces of 4, 8 and 12, synthesizing 3 different conjugates can be utilized. Additionally, conjugates that replace 2Bxy with a biotin moiety to validate p24 binding can be generated. Molecules are purified with HPLC and validated via MALDI. All MTV assays include unmodified lenacapavir controls.

[0238] MTV p24 ELISA: Biotin labeled MTVs is incubated with plate bound purified p24 capsid assemblies and then analyzed via ELISA for biotin. Binding in lysates from infected cells is also tested.

[0239] Inhibition Assay. The GHOST reporter cell assay can assess infection inhibition of MTVs or unmodified lenacapavir. GHOST cells express GFP under the control of a Tat promoter which can be detected via flow cytometry.76This determines whether MTVs still can bind intracellular p24 during infection.

[0240] APC In vitro Activation: HIV pseudoviruses are incubated with MTVs and then incubated with APCs (pDCs, moDCs or THP1 model cells) as disclosed above. APC activation are similarly be analyzed via CD80, CD86, CD40, TNFa, IL- 12 and IL- 15 expression. Controls include lenacapavir and free TLR agonist. REFERENCES

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[0312] While certain features of the invention have been described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the scope of the invention.

[0313] EMBODIMENTS

[0314] 1. A molecular conjugate comprising

[0315] a. a viral capture domain;

[0316] b. and an immuno stimulant molecule. 2. The molecular conjugate of embodiment 1 , further comprising a linker molecule.

[0317] 3. The molecular conjugate of any one of embodiments 1 or 2, wherein the viral capture domain is specific for a viral surface protein of HIV, SARS-CoV-2, or HCV.

[0318] 4. The molecular conjugate of any one of embodiments 1-3, wherein the viral capture domain is a pan-viral inhibitor.

[0319] 5. The molecular conjugate of any one of embodiments 1-4, wherein the viral capture domain targets a viral surface protein of a virus selected from Human Immunodeficiency Virus (HIV-1), Human Immunodeficiency Virus (HIV-2), Influenza A virus, Influenza B virus, Respiratory Syncytial Virus (RSV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), SARS-CoV (SARS-CoV-1), Measles virus. Dengue virus. West Nile virus, Ebola virus, Marburg virus, Nipah virus, Human Parainfluenza virus, Hendra virus, Chikungunya virus, Zika virus, Yellow fever virus, Human herpes simplex virus 1 (HSV-1), Human herpes simplex virus 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), Human adenovirus, or rabies virus.

[0320] 6. The molecular conjugate of any one of embodiments 1-5, wherein the viral capture domain is one of fostemsavir, temsavir, CJF-III-288, BMN-III-170, enfuvirtide, maraviroc, presatovir, umifenovir, clofazimine, nelfinavir mesylate, ziresovir, sifuvirtide, posaconazole, cilnidipine, and MG-II-20.

[0321] 7. The molecular conjugate of anyone of embodiments 1-6, wherein the immunostimulant molecule is a Th 1 skewing adjuvant or a CTL skewing adjuvant.

[0322] 8. The molecular conjugate of anyone of the preceding embodiments, wherein the immunostimulant molecule is selected from CpG1018, a TLR-agonist, or a TLR7 / 8agonist. 9. The molecular conjugate of anyone of the preceding embodiments, wherein the immunostimulant molecule is selected from CpG1018, R848, pixatimod, ISG15. 2’3’-c’diAM(PS)2, Poly EC, or cGAS.

[0323] 10. The molecular conjugate of any one of embodiments 2-9, wherein the linker comprises a charged amino acid, an ethylene glycol spacer, and a reactive moiety. 11. The molecular conjugate of embodiment 10, wherein the charged amino acid is Arg or Lys.

[0324] 12. The molecular conjugate of embodiment 10 or 11, wherein the reactive moiety is an amine, carboxylic acid or DBCO group.

[0325] 13. The molecular conjugate of any one of embodiments 10-12, wherein the linker is selected from the linkers of Table 2.

[0326] 14. The molecular conjugate of any one of the preceding embodiments, having a chemical structure of TK-9, TK14, TK-16, TK15, or TK20.

[0327] 15. A molecular conjugate having a chemical structure of TK-9. TK14, TK-16, TK15. or TK20.

[0328] 16. A liposomal formulation comprising the molecular conjugate of any one of the preceding embodiments.

[0329] 17. The liposomal formulation of embodiment 16, further comprising at least one of a PEG-lipid conjugate and a bulk lipid.

[0330] 18. The liposomal formulation of embodiment 16 or 17, wherein the molecular conjugate comprises at least 0.01%-20% of the lipids present in the liposome.

[0331] 19. The liposomal formulation of any one of embodiments 17-18, wherein the PEG-lipid comprises between 1 %-20% of the lipids present in the liposome.

[0332] 20. The liposomal formulation of any one of embodiments 17-19, wherein the bulk lipids comprise between 80%-99% of the lipids present in the liposome.

[0333] 21. The liposomal formulation of any one of embodiments 16-20, wherein the liposome comprises about 2% molecular conjugate, about 5% PEG-lipid conjugate, and about 93% bulk lipid.

[0334] 22. The liposomal formulation of anyone of embodiments 16-21 , further comprising cholesterol.

[0335] 23. The liposomal formulation of anyone of embodiments 16-22, wherein the size of the liposome is between 50nm-500nm, 100nm-300nm, or 140nm-170nm. 24. A method of treating a viral infection in a subject in need thereof, the method comprising administering an effective amount of the liposomal formulation of any one of embodiments 15-23.

[0336] 25. The method of embodiment 24, wherein the viral infection is caused by Human Immunodeficiency Virus (HIV-1), Human Immunodeficiency Virus (HIV-2), Influenza A virus, Influenza B virus, Respiratory Syncytial Virus (RSV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), SARS-CoV (SARS-CoV-1), Measles virus, Dengue virus, West Nile virus, Ebola virus, Marburg virus, Nipah virus, Human Parainfluenza virus, Hendra virus, Chikungunya virus, Zika virus, Yellow fever virus, Human herpes simplex virus 1 (HSV-1), Human herpes simplex virus 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), Human adenovirus, or rabies virus.

[0337] 26. The method of embodiments 24 or 25. wherein the treatment reduces or suppresses at least one symptom of the viral infection.

[0338] 27. The method of embodiment 26, wherein the symptom is selected from fatigue, feeling tired, weakness, brain fog (problems concentrating or thinking), headaches, tremor, rapid or pounding heartbeat, feeling of skipped heartbeats (palpitations), dizziness upon standing, symptoms that worsen after physical or mental activity (known as post-exertional malaise, PEM), gastrointestinal symptoms including stomach pain, diarrhea, and / or constipation, loss of or change in smell and / or taste, thirst (for instance, dry mouth), cough, changes in comfort or capacity for sex and / or desire for sex, chest pain, tightness, or pressure, hearing problems, including hearing loss or ringing in the ears (tinnitus), shortness of breath, muscle and / or joint pain, back pain, sleep apnea, fever, sweats, and / or chills, hair loss, sleep problems, including insomnia, bladder problems, including difficulty urinating or incontinence, vision problems, such as blurry vision, sensitivity to light, floaters, flashing lights, or difficulty reading or focusing eyes, depression, anxiety, swelling of the legs, problems with teeth, foot pain, skin rash, abnormal movements, skin color changes (for instance, skin that is red, white, or purple), and changes in menstrual cycle.

[0339] 28. The method of any one of the preceding embodiments, further comprising detecting inhibition of viral infection and / or propagation.

Claims

What is claimed is:

1. A molecular conjugate having a chemical structure of TK-9. TK14, TK-16, TK15. or TK20.

2. A molecular conjugate comprisinga. a viral capture domain;b. and an immunostimulant molecule.

3. The molecular conjugate of claim 2, further comprising a linker molecule.

4. The molecular conjugate of claims 3, wherein the viral capture domain is specific for a viral surface protein of HIV, SARS-CoV-2, or HCV.

5. The molecular conjugate of claim 3, wherein the viral capture domain is a pan-viral inhibitor.

6. The molecular conjugate of claim 3, wherein the viral capture domain targets a viral surface protein of a virus selected from Human Immunodeficiency Virus (HIV-1), Human Immunodeficiency Virus (HIV-2), Influenza A virus, Influenza B virus. Respiratory Syncytial Virus (RSV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), SARS-CoV (SARS-CoV-1), Measles virus, Dengue virus, West Nile virus, Ebola virus, Marburg virus, Nipah virus, Human Parainfluenza virus, Hendra virus, Chikungunya virus, Zika virus, Yellow fever virus, Human herpes simplex virus 1 (HSV-1), Human herpes simplex virus 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), Human adenovirus, or rabies virus.

7. The molecular conjugate of claim 3, wherein the viral capture domain is one of fostemsavir, temsavir, CJF-III-288, BMN-III-170, enfuvirtide, maraviroc, presatovir, umifenovir, clofazimine, nelfinavir mesylate, ziresovir, sifuvirtide, posaconazole, cilnidipine, and MG-II-20.

8. The molecular conjugate of claim 3, wherein the immunostimulant molecule is aTh 1 skewing adjuvant or a CTL skewing adjuvant.

9. The molecular conjugate of claim 3, wherein the immunostimulant molecule is selected from CpG1018, a TLR-agonist, or a TLR7 / 8agonist.

10. The molecular conjugate of claim 3, wherein the immunostimulant molecule is selected from CpG1018, R848, pixatimod, ISG15, 2’3’-c’diAM(PS)2, Poly I:C, or cGAS.

11. The molecular conjugate of claim 3, wherein the linker comprises a charged amino acid, an ethylene glycol spacer, and a reactive moiety.

12. The molecular conjugate of claim 11, wherein the charged amino acid is Arg or Lys.

13. The molecular conjugate of claim 11, wherein the reactive moiety is an amine, carboxylic acid or DBCO group.

14. The molecular conjugate of claim 11, wherein the linker is selected from the linkers of Table 2.

15. The molecular conjugate of claim 3, having a chemical structure of TK.-9, TK14, TK-16, TK15, or TK20.

16. A liposomal formulation comprising the molecular conjugate of any one of the preceding claims.

17. The liposomal formulation of claim 16, further comprising at least one of a PEG-lipid conjugate and a bulk lipid.

18. The liposomal formulation of claim 17, wherein the molecular conjugate comprises at least 0.01%-20% of the lipids present in the liposome.

19. The liposomal formulation of claim 17, wherein the PEG-lipid comprises between 1%-20% of the lipids present in the liposome.

20. The liposomal formulation of claim 17, wherein the bulk lipids comprise between 80%-99% of the lipids present in the liposome.

21. The liposomal formulation of claim 17, wherein the liposome comprises about 2% molecular conjugate, about 5% PEG-lipid conjugate, and about 93% bulk lipid.

22. The liposomal formulation of claim 17, further comprising cholesterol.

23. The liposomal formulation of claim 17, wherein the size of the liposome is between 50nm-500nm, 100nm-300nm, or 140nm-170nm.

24. A method of treating a viral infection in a subject in need thereof, the method comprising administering an effective amount of the liposomal formulation of claim 16.

25. The method of claim 24, wherein the viral infection is caused by Human Immunodeficiency Virus (HIV-1), Human Immunodeficiency Virus (HIV-2), Influenza A virus, Influenza B virus, Respiratory Syncytial Virus (RSV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), SARS-CoV (SARS-CoV-1), Measles virus, Dengue virus, West Nile virus, Ebola virus, Marburg virus, Nipah virus, Human Parainfluenza virus, Hendra virus, Chikungunya virus, Zika virus, Yellow fever virus, Human herpes simplex virus 1 (HSV-1), Human herpes simplex virus 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), Human adenovirus, or rabies virus.

26. The method of claims 24, wherein the treatment reduces or suppresses at least one symptom of the viral infection.

27. The method of claim 26, wherein the symptom is selected from fatigue, feeling tired, weakness, brain fog (problems concentrating or thinking), headaches, tremor, rapid or pounding heartbeat, feeling of skipped heartbeats (palpitations), dizziness upon standing, symptoms that worsen after physical or mental activity (known as post-exertional malaise, PEM), gastrointestinal symptoms including stomach pain, diarrhea, and / or constipation, loss of or change in smell and / or taste, thirst (for instance, dry mouth), cough, changes in comfort or capacity for sex and / or desire for sex, chest pain, tightness, or pressure, hearing problems, including hearing loss or ringing in the ears (tinnitus), shortness of breath, muscle and / or joint pain, back pain, sleep apnea, fever, sweats, and / or chills, hair loss, sleep problems, including insomnia, bladder problems, including difficulty urinating or incontinence, vision problems, such as blurry vision, sensitivity to light, floaters, flashing lights, or difficulty reading or focusing eyes, depression, anxiety, swelling of the legs, problems with teeth, foot pain, skin rash, abnormal movements, skin color changes (for instance, skin that is red, white, or purple), and changes in menstrual cycle.

28. The method of claim 24, further comprising detecting inhibition of viral infection and / or propagation.

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