A hollow-fiber bioreactor for expanding HIV-1 in human lymphocytes used in preparing an inactivated vaccine candidate

Abstract The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems. Key points • Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1 • Biolayer interferometry allows target protein quantitation in expression media • High production yields in the bioreactor do not impair Netrin-1 proteoglycan quality Graphical abstract

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In vivo delivery of a multiepitope peptide and Nef protein using novel cell-penetrating peptides for development of HIV-1 vaccine candidate

Biotechnology Letters ◽

10.1007/s10529-020-03060-3 ◽

2021 ◽

Author(s):

Saba Davoodi ◽

Azam Bolhassani ◽

Fatemeh Namazi

Keyword(s):

Vaccine Candidate ◽

Cell Penetrating Peptides ◽

Cell Penetrating ◽

In Vivo Delivery ◽

Hiv 1

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Plant cell immobilization in a dual hollow fiber bioreactor

Biotechnology Techniques ◽

10.1007/bf01875568 ◽

1989 ◽

Vol 3 (2) ◽

pp. 139-144 ◽

Cited By ~ 11

Author(s):

Dong Jin Kim ◽

Ho Nam Chang ◽

Jang Ryol Liu

Keyword(s):

Plant Cell ◽

Hollow Fiber ◽

Cell Immobilization ◽

Hollow Fiber Bioreactor ◽

Plant Cell Immobilization

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Replication-Competent NYVAC-KC Yields Improved Immunogenicity to HIV-1 Antigens in Rhesus Macaques Compared to Nonreplicating NYVAC

Journal of Virology ◽

10.1128/jvi.01513-18 ◽

2018 ◽

Vol 93 (3) ◽

Cited By ~ 5

Author(s):

Karen V. Kibler ◽

Benedikt Asbach ◽

Beatriz Perdiguero ◽

Juan García-Arriaza ◽

Nicole L. Yates ◽

...

Keyword(s):

Clinical Trial ◽

Vaccine Candidate ◽

Rhesus Macaques ◽

Phase Iii ◽

Effective Vaccine ◽

Phase Iii Clinical Trial ◽

Boost Regimen ◽

Clade C ◽

The One ◽

Hiv 1

ABSTRACT As part of the continuing effort to develop an effective HIV vaccine, we generated a poxviral vaccine vector (previously described) designed to improve on the results of the RV144 phase III clinical trial. The construct, NYVAC-KC, is a replication-competent, attenuated recombinant of the vaccinia virus strain NYVAC. NYVAC is a vector that has been used in many previous clinical studies but is replication deficient. Here, we report a side-by-side comparison of replication-restricted NYVAC and replication-competent NYVAC-KC in a nonhuman primate study, which utilized a prime-boost regimen similar to that of RV144. NYVAC-C and NYVAC-C-KC express the HIV-1 antigens gp140, and Gag/Gag-Pol-Nef-derived virus-like particles (VLPs) from clade C and were used as the prime, with recombinant virus plus envelope protein used as the boost. In nearly every T and B cell immune assay against HIV-1, including neutralization and antibody binding, NYVAC-C-KC induced a greater immune response than NYVAC-C, indicating that replication competence in a poxvirus may improve upon the modestly successful regimen used in the RV144 clinical trial. IMPORTANCE Though the RV144 phase III clinical trial showed promise that an effective vaccine against HIV-1 is possible, a successful vaccine will require improvement over the vaccine candidate (ALVAC) used in the RV144 study. With that goal in mind, we have tested in nonhuman primates an attenuated but replication-competent vector, NYVAC-KC, in direct comparison to its parental vector, NYVAC, which is replication restricted in human cells, similar to the ALVAC vector used in RV144. We have utilized a prime-boost regimen for administration of the vaccine candidate that is similar to the one used in the RV144 study. The results of this study indicate that a replication-competent poxvirus vector may improve upon the effectiveness of the RV144 clinical trial vaccine candidate.

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