(183b) Engineered HIV-Capturing Liposomes to Facilitate Immune Uptake

Additionally, HIV remains incurable due to its high mutation rate and antigenic variation, which allow it to evade immune recognition and develop resistance to immune responses. Since HIV primarily targets and destroys CD4+ T cells, the immune system collapses if the virus is left untreated. These unique and challenging characteristics of HIV have made finding a definitive cure a persistent challenge, despite 40 years of dedicated research and effort.

However, we propose a new therapeutic vaccine design, the Nanotrap Therapeutic Vaccine (NTV), to overcome the limitations of current ARTs. NTVs are engineered liposomal platforms that multivalently present HIV-targeting molecules, such as cyclic peptide triazole (cPT) or CJF-III-288, to capture the primary surface glycoprotein, gp120, on the HIV envelope. These liposomes also contain immune adjuvants in the hydrophilic core. The goal of this design is to bind HIV particles in the bloodstream, be recognized and phagocytosed by antigen-presenting cells (APCs), and present HIV viral epitopes via MHC-I cross-presentation. This process would activate Th1 and CD8+ T cell responses, skewing the immune system toward a more effective defense against the virus.

HIV is a retrovirus with a complex structure optimized for infection and immune evasion. The virus has around 42 gp120 subunits on its surface, which capture CD4 receptors and coreceptors (CCR5 or CXCR4). gp41, a transmembrane subunit, is non-covalently attached to gp120, enabling fusion between the viral membrane and the CD4+ T cell membrane. Both cPT and CJF-III-288 are HIV strain-specific fusion inhibitors that bind strongly to gp120. However, their mechanisms differ: cPT blocks the gp120-CD4 binding site, preventing the conformational changes required for tight binding, while CJF-III-288 disrupts the gp120-coreceptor interaction sites, exposing viral epitopes for immune attack.

While most HIV-infected individuals experience a significant reduction in CD4+ T cell counts, elite controllers (ECs) can suppress the virus to undetectable levels without ARTs. Although ECs have an asymptomatic infection, there is still a potential for viral rebound. In contrast, exceptional elite controllers (EECs) can maintain EC-like advantages for over 25 years without disease progression, with inflammation levels similar to those of HIV-negative individuals. The most promising explanation for this control over HIV replication is a stronger CD8+ T cell response compared to chronic HIV progressors. These CD8+ T cells target HIV-infected cells, prevent HIV mutagenic progression, and help limit the formation of latent reservoirs.

The adjuvants loaded into NTV are expected to induce immune responses. By using commercially available toll-like receptors (TLRs) or TLR activators, NTVs will stimulate robust CD8+ T cell responses. This means that NTV will be able to induce HIV-specific effector T cell responses tailored to each patient’s unique viral strain. When co-administered with optimized background therapy (OBT) in HIV-infected individuals at an early stage of infection, this strategy will not only prevent viral replication but also help control the formation of viral reservoirs. This combination of reducing viral load and stimulating adaptive immune responses sets NTV apart from existing ARTs.

Another feature of NTV is its bulk lipid component, which enables the formation of liposomes. The lipid linker includes two types of amino acids (Arg/Lys), PEG spacers, and lipid tails. Using solid-phase peptide synthesis, three different CJF-III-288-conjugated NTV-lipids and two different cPT-conjugated NTV-lipids were synthesized, purified by HPLC, and validated using MALDI. For the CJF-III-288-conjugated lipids, gp120 binding affinity was assessed via surface plasmon resonance (SPR) to confirm that the chemical conjugation on a CJF-III-288 molecule did not compromise the gp120-binding ability and to determine which lipid linker design best preserved this capability. All 3 CJF-III-288 modified NTV candidates preserved gp120 binding after conjugation, and the candidate with a lipid linker containing three lysines exhibited the highest binding affinity among the three, with a Kd value comparable to that of the original CJF-III-288 molecule.

Afterward, 1% of the synthesized NTV lipids were incorporated into NTVs using thin lipid film hydration followed by extrusion. The remaining lipid components included DSPC (88.8%), mPEG2000 (5%), cholesterol (5%), and DiD (0.2%). The NTVs were formulated to be approximately 100 nm in size to facilitate mobility and recognition by antigen-presenting cells (APCs), and this was confirmed by dynamic light scattering (DLS). To evaluate their ability to inhibit viral infection, a pseudotyped HIV infection assay was performed using GHOST cells and flow cytometry. For this assay, two NTV formulations were tested: one conjugated with CJF-III-288 and another with MG-II-20, a representative cPT molecule. These were used to confirm that the ability to bind gp120 was preserved after lipid conjugation and liposome formation, and to validate the multivalent presentation of HIV-binding molecules for viral inhibition.

Both NTV candidates included an arginine residue in the lipid linker and demonstrated strong inhibitory effects against in vitro pseudotyped HIV infection. These results confirmed that converting NTV lipids into a liposomal platform did not compromise their ability to bind gp120. We then proceeded to an in vivo study to assess NTV persistence and uptake after injection. When pseudotyped HIV was co-administered with R848-loaded (a Th1-skewing adjuvant) NTVs in mice, no significant side effects were observed based on serum IL-6 and TNF-α levels. ELISA-based detection of serum p24 indicated that binding between NTV and the pseudovirus occurred. NTVs were also detected in systemic circulation, and non-specific binding persisted for up to 24 hours. Furthermore, in both the bloodstream and spleen, data suggested that NTVs bound to gp120 in vivo remained stable in circulation and were eventually phagocytosed by APCs.

In conclusion, we successfully developed three CJF-III-288-conjugated and two cPT-conjugated NTV candidates. All CJF-III-288-conjugated NTV lipids retained their gp120-binding capacity after chemical conjugation. Both types of multivalently presented NTVs (cPT and CJF-III-288) effectively inhibited HIV infection following liposome formation. In an in vivo mouse model, R848-loaded NTVs suggested that NTVs can bind circulating HIV particles and remain in the bloodstream long enough to be phagocytosed by APCs without leading to HIV infection of the APCs.

Moving forward, we plan to test NTVs against various HIV-1 strains to assess their ability to respond to viral mutations. We also aim to evaluate different Th1-skewing adjuvants to determine whether NTVs can induce HIV patient-specific CD8+ T cell responses using samples from HIV-infected individuals.