Engineering a serum-resistant and thermostable vesicular stomatitis virus G glycoprotein for pseudotyping retroviral and lentiviral vectors

Abstract

Vesicular stomatitis virus G glycoprotein (VSV-G) is the most widely used envelope protein for retroviral and lentiviral vector pseudotyping; however, serum inactivation of VSV-G pseudotyped vectors is a significant challenge for in vivo gene delivery. To address this problem, we conducted directed evolution of VSV-G to increase its resistance to human serum neutralization. After six selection cycles, numerous common mutations were present. On the basis of their location within VSV-G, we analyzed whether substitutions in several surface exposed residues could endow viral vectors with higher resistance to serum. S162T, T230N and T368A mutations enhanced serum resistance, and additionally K66T, T368A and E380K substitutions increased the thermostability of VSV-G pseudotyped retroviral vectors, an advantageous byproduct of the selection strategy. Analysis of a number of combined mutants revealed that VSV-G harboring T230N+T368A or K66T+S162T+T230N+T368A mutations exhibited both higher in vitro resistance to human serum and higher thermostability, as well as enhanced resistance to rabbit and mouse serum. Finally, lentiviral vectors pseudotyped with these variants were more resistant to human serum in a murine model. These serum-resistant and thermostable VSV-G variants may aid the application of retroviral and lentiviral vectors to gene therapy.

Figure 1. Common mutations following directed evolution of VSV-G

(a) The frequency of mutation at each amino acid residue of VSV-G among 36 randomly chosen VSV-G clones after 5 or 6 selection steps. (b) The location of each apparent ‘hot spot mutation’ in the crystal structure of the prefusion form of VSV-G (PDB ID: 2J6J). Figure was made using PyMol (http://www.pymol.org). Each monomer of VSV-G was colored in green, purple, and sky-blue, respectively. Green, blue, and red balls represent carbon, nitrogen, and oxygen atoms, respectively.

Figure 2. Genomic and infectious titers of VSV-G chimeric retroviral vectors

Murine retroviral vector was packaged with the pCLPIT GFP vector plasmid, pCMV gag-pol, and pcDNA IVS VSV-G helper plasmid containing individual VSV-G variants. (a) Vector genomic titers were measured by real-time qPCR. (b) Transduction efficiencies on 293T cells were determined by flow cytometry analysis of retroviral vector mediated GFP expression. (c) Relative ratios of infectious titers to genomic titers (grey bars) titers and VSV-G ELISA data to genomic titers (black bars) of the retroviral vectors were calculated with the ratio of wild type VSV-G pseudotyped retroviral vector as 1, respectively. Error bars denote SD (n = 3). * and ** indicate statistical differences of P < 0.05 and P < 0.01, respectively, compared to infectivity of wild type VSV-G, as determined using an one-way ANOVA.

Figure 2. Genomic and infectious titers of VSV-G chimeric retroviral vectors

Murine retroviral vector was packaged with the pCLPIT GFP vector plasmid, pCMV gag-pol, and pcDNA IVS VSV-G helper plasmid containing individual VSV-G variants. (a) Vector genomic titers were measured by real-time qPCR. (b) Transduction efficiencies on 293T cells were determined by flow cytometry analysis of retroviral vector mediated GFP expression. (c) Relative ratios of infectious titers to genomic titers (grey bars) titers and VSV-G ELISA data to genomic titers (black bars) of the retroviral vectors were calculated with the ratio of wild type VSV-G pseudotyped retroviral vector as 1, respectively. Error bars denote SD (n = 3). * and ** indicate statistical differences of P < 0.05 and P < 0.01, respectively, compared to infectivity of wild type VSV-G, as determined using an one-way ANOVA.

Figure 3. Human serum resistance and thermostability of retroviral vectors pseudotyped with VSV-G mutants

The amounts of viral vectors were normalized based on VSV-G ELISA assay. (a) Human serum neutralization was quantified by measuring vector titers after incubation with human serum at 37°C for 1 hr, relative to those after incubation with PBS at 37°C for 1 hr. (b) Thermal effects were determined by quantifying relative titers after incubation with PBS at 37°C for 1 hr compared to those without incubation at 37°C. (c) Human serum neutralization and thermal effects were determined by calculation of relative titers after incubation with human serum at 37°C for 1 hr compared to those without incubation at 37°C. Error bars denote SD (n = 4). * and ** indicate statistical differences of P < 0.05 and P < 0.01, respectively, compared to infectivity of wild type VSV-G, as determined using an one-way ANOVA.

Figure 3. Human serum resistance and thermostability of retroviral vectors pseudotyped with VSV-G mutants

The amounts of viral vectors were normalized based on VSV-G ELISA assay. (a) Human serum neutralization was quantified by measuring vector titers after incubation with human serum at 37°C for 1 hr, relative to those after incubation with PBS at 37°C for 1 hr. (b) Thermal effects were determined by quantifying relative titers after incubation with PBS at 37°C for 1 hr compared to those without incubation at 37°C. (c) Human serum neutralization and thermal effects were determined by calculation of relative titers after incubation with human serum at 37°C for 1 hr compared to those without incubation at 37°C. Error bars denote SD (n = 4). * and ** indicate statistical differences of P < 0.05 and P < 0.01, respectively, compared to infectivity of wild type VSV-G, as determined using an one-way ANOVA.

Figure 4. Human serum neutralization and thermostability of retroviral vectors pseudotyped with VSV-G variants that combine several ‘hot spot mutations’

VSV-G mutants with combined beneficial mutations were generated by site-directed mutagenesis. The amounts of viral vectors were normalized based on VSV-G ELISA assay. Thermal effects, human serum neutralization, and combined serum neutralization and thermal effects were determined by quantifying relative titers after incubation with PBS at 37°C for 1 hr compared to those without incubation at 37°C, after incubation with human serum at 37°C for 1 hr compared to those after incubation with PBS at 37°C for 1 hr, and after incubation with human serum at 37°C for 1 hr compared to those without incubation at 37°C, respectively. (a) Variants contained ‘hot spot mutations’ for human serum resistance. (b) K66T and (c) E380K were added to enhance the thermal stability of retroviral vectors. Error bars denote SD (n = 4). * and ** indicate statistical differences of P < 0.05 and P < 0.01, respectively, compared to the wild type VSV-G, as determined using an one-way ANOVA.

Figure 4. Human serum neutralization and thermostability of retroviral vectors pseudotyped with VSV-G variants that combine several ‘hot spot mutations’

VSV-G mutants with combined beneficial mutations were generated by site-directed mutagenesis. The amounts of viral vectors were normalized based on VSV-G ELISA assay. Thermal effects, human serum neutralization, and combined serum neutralization and thermal effects were determined by quantifying relative titers after incubation with PBS at 37°C for 1 hr compared to those without incubation at 37°C, after incubation with human serum at 37°C for 1 hr compared to those after incubation with PBS at 37°C for 1 hr, and after incubation with human serum at 37°C for 1 hr compared to those without incubation at 37°C, respectively. (a) Variants contained ‘hot spot mutations’ for human serum resistance. (b) K66T and (c) E380K were added to enhance the thermal stability of retroviral vectors. Error bars denote SD (n = 4). * and ** indicate statistical differences of P < 0.05 and P < 0.01, respectively, compared to the wild type VSV-G, as determined using an one-way ANOVA.

Figure 5. Human serum neutralization and thermostability of lentiviral vectors pseudotyped with VSV-G variants

A standard, GFP encoding lentiviral vector was packaged with five VSV-G mutants that appeared promising in the retroviral results. Five VSV-G variants (S162T + T230N, S162T + T368A, T230N + T368A, K66T + T368A + E380K, and K66T + S162T + T230N + T368A) showing higher resistance and thermal stability for retroviral vector packaging were selected. Thermal effects, human serum neutralization, and combined serum neutralization and thermal effects were determined by quantifying relative titers after incubation with PBS at 37°C for 1 hr compared to those without incubation at 37°C, after incubation with human serum at 37°C for 1 hr compared to those after incubation with PBS at 37°C for 1 hr, and after incubation with human serum at 37°C for 1 hr compared to those without incubation at 37°C, respectively. Error bars denote SD (n = 4). * and ** indicate statistical differences of P < 0.05 and P < 0.01, respectively, compared to the wild type VSV-G, as determined using an one-way ANOVA.

Figure 6. Aminal serum neutralization of retroviral and lentiviral vectors pseudotyped with VSV-G variants

For three variants (S162T+T230N, T230N +T368A and K66T + S162T + T230N + T368A) and wild type VSV-G, neutralization by animal sera was examined. (a) Retroviral vectors and (b) lentiviral vectors were diluted fivefold in animal sera and incubated at 37°C for 1 hr. Serum inactivation was determined by quantifying relative titers after incubation with human serum at 37°C for 1 hr compared to those after incubation with PBS at 37°C for 1 hr. Error bars denote SD (n = 4). * and ** indicate statistical differences of P < 0.05 and P < 0.01, respectively, compared to the wild type VSV-G, as determined using an one-way ANOVA.

Figure 7. In vitro transduction of multiple cell lines with retroviral vectors pseudotyped with VSV-G variants

Retroviral vectors expressing GFP were used to transduce a panel of cell lines: HEK 293T, HT1080 (human fibrosarcoma cell line), CHO K1, NIH 3T3 (mouse embryonic fibroblast cell line), and HeLa cells to assess the transduction profile of the novel VSV-G variants. Error bars denote SD (n = 3). ** indicates statistical differences of P < 0.01, as determined using an one-way ANOVA.

Figure 8. Human serum neutralization in vivo

For two variants (T230N +T368A and K66T + S162T + T230N + T368A) and wild type VSV-G, lentiviral vectors encoding luciferase were administered via tail vein injection to female BALB/c mice one hour after human serum or PBS introduction. After two weeks, levels of luciferase activity were determined and normalized to total protein for each sample analyzed. Relative luciferase expression in liver was determined by quantifying relative enzyme activity from human serum-primed mice relative to activity from naïve mice. Error bars denote SD (n = 4). * and ** indicate statistical differences of P < 0.05 and P < 0.01, respectively, compared with wild type VSV-G, as determined using an one-way ANOVA.