Induction of durable protective immune responses is the main goal of prophylactic vaccines, and adjuvants play a role as drivers of such responses. Despite advances in vaccine strategies, development of a safe and effective HIV vaccine remains a significant challenge. Use of an appropriate adjuvant is crucial to the success of HIV vaccines. Here we assessed the saponin/MPLA nanoparticle (SMNP) adjuvant with an HIV envelope (Env) trimer, evaluating the safety and effect of multiple variables — including adjuvant dose (16-fold dose range), immunization route, and adjuvant composition — on the establishment of Env-specific memory T and B cell (T _Mem and B _Mem ) responses and long-lived plasma cells in nonhuman primates (NHPs). Robust B _Mem were detected in all groups, but a 6-fold increase was observed in the highest- versus the lowest-SMNP-dose group. Similarly, stronger vaccine responses were induced by the highest SMNP dose in CD40L ⁺ OX40 ⁺ CD4 ⁺ T _Mem (11-fold), IFN-γ ⁺ CD4 ⁺ T _Mem (15-fold), IL21 ⁺ CD4 ⁺ T _Mem (9-fold), circulating T follicular helper cells (T _FH ; 3.6-fold), BM plasma cells (7-fold), and binding IgG (1.3-fold). Substantial tier 2 neutralizing antibodies were only observed in the higher-SMNP-dose groups. These investigations highlight the dose-dependent potency of SMNP and its relevance for human use and next-generation vaccines.

Background/Objectives: Despite progress in antiretroviral therapy, HIV remains a major global health challenge with over one million new infections annually. An effective vaccine is urgently needed. Germline-targeting immunogens show promise in initiating broadly neutralizing antibody (bNAb) precursors. This study developed a scalable, cGMP-compliant process to manufacture the HIV vaccine booster immunogen HxB2.WT.Core-C4b, a nanoparticle designed to direct bNAb precursor maturation after priming. Methods: A CHO cell platform was established through single-cell cloning from a high-producing stable pool. Upstream and downstream processes were optimized for scalability and yield. Three scales were tested 10 L, 40 L, and 400 L. Key parameters (pH, temperature, feeding, metabolite profiles) were systematically refined. Analytical characterization included glycosylation profiling, electron microscopy, and antigenicity testing. Viral clearance was evaluated per ICH Q5A guidelines. Results: Optimization ensured consistent yields above 130 mg/L, with titers up to 250 mg/L. The selected clone (4E22) demonstrated strong growth, viability, and reproducibility. Glycan occupancy at 18 N-linked sites, including bNAb epitopes (N276, N332), was stable across scales. Over 70% of self-assembling nanoparticle were fully assembled at the GMP level. Antigenicity and purity met cGMP release criteria. Viral clearance achieved >13-log reduction for enveloped and >7-log for non-enveloped viruses. Conclusions: This work establishes a robust, scalable platform for HIV nanoparticle immunogens. Consistent quality and yield across scales support clinical development of HxB2.WT.Core-C4b and provide a model for other glycosylated nanoparticle vaccines. The immunogen is being evaluated in clinical study HVTN 320 (NCT06796686), enabling early testing of next-generation vaccines designed to elicit broadly neutralizing antibodies.

The successful translation of rationally designed HIV-1 immunogens into effective vaccines requires manufacturing platforms that maintain structural conformity while meeting clinical-grade quality standards. We developed and scaled a robust, cGMP-compliant process for N332-GT5 gp140, a germline-targeting envelope trimer designed to initiate broadly neutralizing antibody responses, which is now undergoing first-in-human evaluation in HVTN144. Starting with a stable CHO cell line developed using Leap-In® transposon technology, we established a production clone exhibiting high-titer expression (>200 mg/L) and genetic stability through 60 population doublings. The manufacturing process scaled efficiently from Ambr® 250 miniature bioreactors to 200-L single-use systems, delivering consistent product quality across multiple cGMP batches. A streamlined three-step purification strategy—affinity capture, multimodal polishing, and viral clearance- yielded >99% trimeric purity with preserved quaternary structure and native-like antigenicity. Orthogonal LC-MS analyses confirmed site-specific glycan occupancy matching design specifications, while robust viral clearance exceeded 18-log and 11-log reductions for model retroviruses. Clinical material manufactured through this platform has been successfully administered in HVTN144. This work establishes a scalable, reproducible manufacturing paradigm for structurally complex HIV-1 envelope immunogens, advancing the field toward rational vaccine design based on germline-targeting principles.

Background/Objectives: The rapid emergence of SARS-CoV-2 variants that evade neutralizing antibodies underscores the need for new antiviral biologics that integrate precise molecular design with scalable, cost-effective manufacturing. Computationally designed miniproteins targeting the spike protein’s receptor-binding domain (RBD) offer a promising alternative to monoclonal antibodies because of their small size, high thermal stability, and compatibility with microbial expression systems. Here, we detail the full development and cGMP manufacturing of IPD-52520, a novel antiviral miniprotein produced using an optimized E. coli platform. Methods: Two candidate miniproteins, a homotrimeric construct (Trimer, IPD-52520, 17 kDa) and a tandem fusion construct (Daisy, IPD-52521, 25 kDa), were evaluated in parallel through systematic optimization of strain selection, media formulation, fed-batch fermentation, inclusion-body solubilization, refolding, and chromatographic purification. The leading candidate was scaled from 5 L to 50 L under cGMP conditions, and biophysical and stability analyses were conducted to support nonclinical and Phase 1 clinical development. Results: The Trimer was selected as the lead molecule based on superior preclinical efficacy, favorable pharmacokinetics, and higher volumetric yields. The optimized process yields about 2 g/L of purified protein at over 90% purity. Scale-up from 5 L to 50 L demonstrated excellent batch consistency across six independent runs. Biophysical studies confirmed a well-folded, predominantly alpha-helical trimer (Tm = 73.4 °C; polydispersity = 1.005) with an intact primary structure and strong target binding (KD < 1 pM). Stability studies show the drug remains stable at 2–8 °C for at least 12 months, with ongoing long-term studies. Conclusions: These findings demonstrate that computationally designed antiviral miniproteins can be translated into scalable biologics and establish a platform for rapid therapeutic development against current and future pandemics.