Stabilization and formulation of a recombinant Human Cytomegalovirus vector for use as a candidate HIV-1 vaccine

Abstract

Live attenuated viral vaccine/vector candidates are inherently unstable and infectivity titer losses can readily occur without defining appropriate formulations, storage conditions and clinical handling practices. During initial process development of a candidate vaccine against HIV-1 using a recombinant Human Cytomegalovirus vector (rHCMV-1), large vector titer losses were observed after storage at 4 °C and after undergoing freeze-thaw. Thus, the goal of this work was to develop candidate frozen liquid formulations of rHCMV-1 with improved freeze-thaw and short-term liquid stability for potential use in early clinical trials. To this end, a virus stability screening protocol was developed including use of a rapid, in vitro cell-based immunofluorescence focus assay to quantitate viral titers. A library of ∼50 pharmaceutical excipients (from various known classes of additives) were evaluated for their effect on vector stability after freeze-thaw cycling or incubation at 4 °C for several days. Certain additives including sugars and polymers (e.g., trehalose, sucrose, sorbitol, hydrolyzed gelatin, dextran 40) as well as removal of NaCl (lower ionic strength) protected rHCMV-1 against freeze-thaw mediated losses in viral titers. Optimized solution conditions (e.g., solution pH, buffers and sugar type) slowed the rate of rHCMV-1 titer losses in the liquid state at 4 °C. After evaluating various excipient combinations, three new candidate formulations were designed and rHCMV-1 stability was benchmarked against both the currently-used and a previously reported formulation. The new candidate formulations were significantly more stable in terms of reducing rHCMV-1 titer losses after 5 freeze-thaw cycles or incubation at 4 °C for 30 days. This case study highlights the utility of semi-empirical design of frozen liquid formulations of a live viral vaccine candidate, where protection against infectivity titer losses due to freeze-thaw and short-term liquid storage are sufficient to enable more rapid initiation of early clinical trials.

Epidemiology and biology of a herpesvirus in rabies endemic vampire bat populations

“…Genome annotation showed highest protein similarity by BLASTx to the bat Miniopterus schreibersii BHV B7D8 (223kbp) genome, the only available complete bat-derived BHV genome, although 24/114 predicted proteins had insufficient BLAST similarity to be assigned (ORFs predicted by GeneMarkS). Recombinant BHV vaccines have inserted genes into several locations along the vector genome, including UL82 40 (encoding for IE protein pp71) in HCMV, IE2 41 in MCMV, and RhCMV ORF Rh211 25,42,43 (US27), Rh107 43 , Rh110 42 , and Rh186-9 25 (US8-11). Whilst similar coding regions within DrBHV could not be identified for all of the above proteins, a homologue for UL82 in DrBHV could serve as a potential site for VBRV glycoprotein insertion.…”

Efficient clinical-grade γ-retroviral vector purification by high-speed centrifugation for CAR T cell manufacturing

“…Future work is required to determine whether optimization of a final buffer formulation might confer enhanced stability as it is impacted by a variety of chemical 53 and physical 54 factors. A comprehensive examination of the effects of different excipients 55 on the stability of γ-RV may increase process flexibility further and is worthy of future study.…”

Exploring the Potential of Cytomegalovirus-Based Vectors: A Review

“…Live viral vaccine vectors are typically difficult to preserve and prone to instability, especially if exposed to high temperatures and multiple freeze-thaw cycles [ 118 , 119 ]. A recombinant HCMV vector (rHCMV-1) experienced large vector titer losses after being stored at 4 °C and undergoing a freeze-thaw cycle [ 120 ]. Further investigation revealed that the removal of NaCl, which decreased the ionic strength, and the incorporation of additives including sugars and polymers decreased viral titer losses caused by freeze-thaw cycles, while optimized solution pH, buffers, and sugar types protected rHCMV-1 titer losses against prolonged storage at 4 °C in liquid state [ 120 ].…”

“…A recombinant HCMV vector (rHCMV-1) experienced large vector titer losses after being stored at 4 °C and undergoing a freeze-thaw cycle [ 120 ]. Further investigation revealed that the removal of NaCl, which decreased the ionic strength, and the incorporation of additives including sugars and polymers decreased viral titer losses caused by freeze-thaw cycles, while optimized solution pH, buffers, and sugar types protected rHCMV-1 titer losses against prolonged storage at 4 °C in liquid state [ 120 ]. However, maintaining the stability of live CMV-based vectors remains a problem for its long-term storage and widespread use and distribution.…”