Expression of SHIV Antigens in Mammalian Expression Vectors
Frazier, Thomas H.; Compans, Richard; Bu, Zhigao; and Yang, Chinglai

Abstract

Preliminary results for HIV DNA vaccination reveal both the feasibility of the approach, as well as, the requirement for more immunogenic alterations. While currently developing immunomodulatory strategies including codon optimization and the addition of genetic adjuvants seem to be conferring improvement, a more complete knowledge of optimal mammalian expression vectors for SHIV antigens was needed. In light of this, the intent of this study was to analyze by Western Blot the expression of SIV gag and codon optimized versions of HIV envelope proteins syngp120 and syngp140 within various mammalian expression vectors. Vectors utilized included pCI (Promega) which exploits a CMV promoter, Simian Virus 40 polyadenylation sequence, chimeric intron, and also the T7 promoter; pSI (Promega) which is identical to pCI except in its Simian virus 40 promoter; JR-FL which utilizes a CMV promoter; and finally pBudCE4 (Invitrogen) a potential dual expression plasmid complete with two promoters (CMV and Human Elongation Factor-1a), poly A sequences (Bovine Growth Hormone and SV40), and multiple cloning sites. pBudCE4 also makes use of the T7 promoter. The construction and expression of gag-pCI, pSI, pBudCE4; syngp120-pCI, pSI, pBudCE4, JR-FL; syngp140-pCI, pSI, pBudCE4, JR-FL; as well as gag-gp120-pBudCE4 and gag-gp140-pBudCE4 (dual-expression vectors) resulted in vital knowledge regarding the comparison of expression systems as they pertained to SHIV antigen production. Neither pBudCE4 nor Promega vectors were conducive to high envelope expression, while the JR-FL-envelope constructs (obtained from Brian Seed) were highly expressed. Of the SIV gag constructs, only pCI indicated promise. While the results are preliminary, the implication of such understanding allows for optimal SHIV expression systems to be more easily developed and the possibility of more potent vaccine design.

Introduction/Background

DNA vaccinations have proven feasible alternatives to conventional live-attenuated or protein-based vaccines. Utilizing recombinant DNA technology, genes encoding for antigenic proteins are cloned into plasmids containing mammalian expression systems (e.g. promoter, intron, and polyadenylation sequence). Similar to viral infection, the genes are then transcribed and translated endogenously. This endogenous production of protein is conducive to a more complete immunogenic response than protein-based counterparts, eliciting both CD4 and CD8 restricted immune mechanisms. In addition, because the genes themselves are not pathogenic, the safety is much more reliable than live-attenuated versions of vaccination. Finally, DNA vaccines are inexpensive, easy to transport/store, and relatively easy to produce. For these reasons DNA vaccinations have been studied for a wide variety of pathogens and diseases including HIV. The promise of DNA vaccine development for HIV has been shown in numerous animal studies and a few human trials. However, these studies have indicated the need for a more complete, broad, and potent immunogenic vaccine. By replacing HIV wild type sequences with more highly expressed mammalian alternatives (known as codon optimization), increased expression and subsequent immune response is induced. Another method of increasing a vaccine’s immunogenic properties involves the addition of chemical adjuvants such as cationic liposomes and/or genetic adjuvants such as known immunomodulator Listeriolysin O. Finally, the expression vector utilized is also crucial to the endogenous expression and subsequent immune response of an antigen. Commonly used mammalian expression promoters include the human cytomegalovirus (CMV), elongation factor-1a (EF-1a), and simian virus 40 (SV40), but compatibility with certain antigens and therefore expression seems to vary. Other factors that appear to affect levels of expression are introns (e.g. CMV intron A), as well as , polyadenylation sequences such as Bovine Growth Hormone(BGH) or SV 40 versions. By developing compatible expression systems for HIV and/or SIV, long strides can be made in developing a more adequate vaccine for this pathogen

Methods

Transfection: COS-7 (African green monkey kidney cells) were transfected with 4g DNA/ Lipofectin (GIBCO-BRL)/ Dulbeco’s Modification of Eagle’s Medium (DMEM). Medium was changed 5-16 hours post-transfection to10%serum, %antibiotic in DMEM. Cells were harvested 72 hours post-transfection. Selected controls were infected with vaccinia virus 1-2 hours pre-transfection, transfected, and harvested after 16 hours using similar conditions. Cells were resuspended in 1ml DMEM, separated into 500l aliquots, and stored at -20C. Protein Analysis by Western Blot: One aliquot of each cell/DMEM sample was readied for blot using either 30l lysis buffer, centrifugation, 30l 2X loading buffer, and 5 minute 95 boiling or simply 30l 2X loading buffer and 5 minute 95 boiling. 15-20l was loaded onto gel. Gels were transferred to nitrocellulose membranes and blotted at 20volts for 2 h. The blots were washed with TTBS (0.1 M Tris-HCL [pH 7.5], 0.5 M NaCl, 0.1% Tween 20) and blocked in 5%dry milk/TTBS overnight. SIV gag was detected using primary antibody (monkey anti-SIV serum) followed by secondary antibody (HRP-conjugated rabbit anti-monkey antibody). HIV envelope proteins were detected using primary antibody (combined serum from HIV infected patients) followed by secondary antibody (HRP-conjugated mouse anti-human antibody). Primary antibodies were administered 1-2 hours, followed by wash, and 1-2 h secondary antibody administration. Cloning Strategy

Cloning Strategy:

1. Subclone SIV gag, syngp120, and syngp140 from original plasmids into pBluescript II KS +/- (pBlue) using EcoRI and HindIII for gag and BamHI and HindIII for envelope genes.
2. Clone SIV gag from pBlue into pBudCE4 using SalI and SmaI for insert and HindIII followed by Klenow treatment and SalI for pBudCE4 (gag-pBud clone#1).
3. Clone SIV gag into pBlue using NotI and XhoI (gag-pBud clone #2).
4. Clone envelope genes into pBud using HindIII and BamHI.
5. Clone SIV gag from pBlue into pBud-syngp120 construct using KpnI and NotI.
6. Clone Clone SIV gag from pBlue into pBud-syngp120 construct using KpnI and NotI.
7. Clone SIV gag into pCI and pSI using EcoRI and SalI.
8. Clone envelope genes into pCI and pSI form pBlue using NotI and SalI.

List of Constructs:

• pBudCE4/ gag1(cloned into pBluescript II KS+/- using EcoRI and HindIII; cloned into pBudCE4 using smaI and sal I for the insert and HindIII, then Klenow treatment and SalI for pBudCE4)
• pBudCE4/ gag2 (cloned into pBluescript II KS+/- using EcoRI and HindIII; cloned into pBud using NotI and XhoI)
• pBudCE4/ syngp120 (cloned using HindIII, BamHI, BglI on insert and BamHI and HindIII on vector)
• pBudCE4/ syngp140 (cloned using HindIII and BamHI) PBudCE4/ syngp120/ gag (cloned gag from pBluescript into pBudCE4/ syngp120 using KpnI and NotI) PBudCE4/ syngp140/ gag (cloned gag from pBluescript into pBudCE4/ syngp140 using KpnI and NotI)
• PBudCE4/ lacZ (control plasmid)
• PSyn gp 120 JR-FL (original plasmid obtained from Brian Seed)
• PSyn gp 140 JR-FL (original plasmid obtained from Brian Seed)
• pBluescript II KS+/- / gag ( cloned using EcoRI and HindIII)
• pBluescript II KS+/- / syngp120 (cloned using HindII and BamHI)
• pBluescript II KS+/- / syngp140(cloned using HindII and BamHI)
• PSI/gag ( cloned from pBluescript II KS +/- using EcoRI and SalI)
• PSI/syngp120 (cloned from pBluescript II KS+/- / syngp120 using SalI and NotI)
• PSI/syngp140 (cloned from pBluescript II KS+/- / syngp140 using SalI and NotI)
• PCI/gag PCI/syngp120 (cloned from pBluescript II KS+/- / syngp120 using SalI and NotI)
• PCI/syngp140(cloned from pBluescript II KS+/- / syngp140 using SalI and NotI)

Conclusions

Preliminary results indicate there are fundamental differences between the JR-FL codon optimized envelope constructs and constructs developed with pBudCE4, pCI, pSI. SIV gag constructs with pCI were highly expressed under vaccinia virus expression system, but not under normal mammalian expression promoters.

Future Directions

• More complete expression analysis including Western blot, immunoprecipitation and radiolabeling.
• Chimeric syngp120 and gp140 / Literiolysin genes cloned into expression vectors
• DNA immunization studies and CTL analysis in mice

Acknowledgements

Funding provided by Hughes Science Initiatives (SURE program) and National Institute of Health (grant # 20465). Thanks to Dr. Compans’ lab for all the assistance and friendship.

In My Own Words

Thomas Frazier spent the summer cloning antigenic genes derived from HIV and SIV into various bacterial plasmids (vectors). These plasmids contained expression systems that are conducive to high expression mammalian cells. Once clones were obtained, the recombinant vectors were administered and taken up by COS-7 cells (African green monkey kidney cells) otherwise known as transfection. After tranfection, proteins were isolated from cells and Western blot analysis was completed. By comparing expression levels. Thomas was able to deduce optimal conditions for SHIV antigen expression. This information will now be used to clone and produce DNA vaccines for HIV.