Therapeutic protein drugs are an important class of medicines serving patients most in need of novel therapies. Recently approved recombinant protein therapeutics have been developed to treat a wide variety of clinical indications, including cancers, autoimmunity/inflammation, exposure to infectious agents, and genetic disorders. The latest advances in protein-engineering technologies have allowed drug developers and manufacturers to fine-tune and exploit desirable functional characteristics of proteins of interest while maintaining (and in some cases enhancing) product safety or efficacy or both. In this review, we highlight the emerging trends and approaches in protein drug development by using examples of therapeutic proteins approved by the U.S. Food and Drug Administration over the previous five years (2011–2016, namely January 1, 2011, through August 31, 2016).

A major obstacle in human immunodeficiency virus type 1 (HIV-1) eradication is the ability of the virus to remain latent in a subpopulation of the cells it infects. Latently infected cells can escape the viral immune response and persist for long periods of time, despite the presence of successful highly active antiretroviral therapy (HAART). Given the appropriate stimulus, latently infected cells can reactivate and start producing infectious virions. The susceptibility of these cell populations to HIV-1, their life span, their proliferative capacity, and their ability to periodically produce infectious virus subsequent to alterations in cellular physiology and/or immunologic controls are critical issues which determine the contribution of these cells to viral persistence. Memory CD4+ T cells due to the long life span, which may be several years, and their ability to reactivate upon encounter with their cognate antigen or other stimulation, are considered a critical reservoir for maintenance of latent HIV-1 proviral DNA. Cells of the monocyte-macrophage lineage, which originate in the bone marrow (BM), are of particular importance in HIV-1 persistence due to their ability to cross the blood-brain barrier (BBB) and spread HIV-1 infection in the immunoprivileged central nervous system (CNS). Hematopoietic progenitor cells (HPCs) are also a potential HIV-1 reservoir, as several studies have shown that CD34+ HPCs carrying proviral DNA can be found in vivo in a subpopulation of HIV-1-infected patients. The ability of HPCs to proliferate and potentially generate clonal populations of infected cells of the monocyte-macrophage lineage may be crucial in HIV-1 dissemination. The contribution of these and other cell populations in HIV-1 persistence, as well as the possible strategies to eliminate latently infected cells are critically examined in this review.

Due to the degeneracy of the genetic code, most amino acids can be encoded by multiple synonymous codons. Synonymous codons naturally occur with different frequencies in different organisms. The choice of codons may affect protein expression, structure, and function. Recombinant gene technologies commonly take advantage of the former effect by implementing a technique termed codon optimization, in which codons are replaced with synonymous ones in order to increase protein expression. This technique relies on the accurate knowledge of codon usage frequencies. Accurately quantifying codon usage bias for different organisms is useful not only for codon optimization, but also for evolutionary and translation studies: phylogenetic relations of organisms, and host-pathogen co-evolution relationships, may be explored through their codon usage similarities. Furthermore, codon usage has been shown to affect protein structure and function through interfering with translation kinetics, and cotranslational protein folding. Despite the obvious need for accurate codon usage tables, currently available resources are either limited in scope, encompassing only organisms from specific domains of life, or greatly outdated. Taking advantage of the exponential growth of GenBank and the creation of NCBI’s RefSeq database, we have developed a new database, the High-performance Integrated Virtual Environment-Codon Usage Tables (HIVE-CUTs), to present and analyse codon usage tables for every organism with publicly available sequencing data. Compared to existing databases, this new database is more comprehensive, addresses concerns that limited the accuracy of earlier databases, and provides several new functionalities, such as the ability to view and compare codon usage between individual organisms and across taxonomical clades, through graphical representation or through commonly used indices. In addition, it is being routinely updated to keep up with the continuous flow of new data in GenBank and RefSeq. Given the impact of codon usage bias on recombinant gene technologies, this database will facilitate effective development and review of recombinant drug products and will be instrumental in a wide area of biological research. The database is available at hive.biochemistry.gwu.edu/review/codon .

Patients with HIV-1 often present with a wide range of hematopoietic abnormalities, some of which may be due to the presence of opportunistic infections and to therapeutic drug treatments. However, many of these abnormalities are directly related to HIV-1 replication in the bone marrow (BM). Although the most primitive hematopoietic progenitor cells (HPCs) are resistant to HIV-1 infection, once these cells begin to differentiate and become committed HPCs they become increasingly susceptible to HIV-1 infection and permissive to viral gene expression and infectious virus production. Trafficking of BM-derived HIV-1-infected monocytes has been shown to be involved in the dissemination of HIV-1 into the central nervous system (CNS), and it is possible that HIV-1 replication in the BM and infection of BM HPCs may be involved in the early steps leading to the development of HIV-1-associated dementia (HAD) as an end result of this cellular trafficking process. In addition, the growth and development of HPCs in the BM of patients with HIV-1 has also been shown to be impaired due to the presence of HIV-1 proteins and changes in the cytokine milieu, potentially leading to an altered maturation process and to increased cell death within one or more BM cell lineages. Changes in the growth and differentiation process of HPCs may be involved in the generation of monocyte populations that are more susceptible and/or permissive to HIV-1, and have potentially altered trafficking profiles to several organs, including the CNS. A monocyte subpopulation with these features has been shown to expand during the course of HIV-1 disease, particularly in HAD patients, and is characterized by low CD14 expression and the presence of cell surface CD16.

Synonymous codons occur with different frequencies in different organisms, a phenomenon termed codon usage bias. Codon optimization, a common term for a variety of approaches used widely by the biopharmaceutical industry, involves synonymous substitutions to increase protein expression. It had long been presumed that synonymous variants, which, by definition, do not alter the primary amino acid sequence, have no effect on protein structure and function. However, a critical mass of reports suggests that synonymous codon variations may impact protein conformation. To investigate the impact of synonymous codons usage on protein expression and function, we designed an optimized coagulation factor IX (FIX) variant and used multiple methods to compare its properties to the wild-type FIX upon expression in HEK293T cells. We found that the two variants differ in their conformation, even when controlling for the difference in expression levels. Using ribosome profiling, we identified robust changes in the translational kinetics of the two variants and were able to identify a region in the gene that may have a role in altering the conformation of the protein. Our data have direct implications for codon optimization strategies, for production of recombinant proteins and gene therapies.