Rabies DNA vaccine: no impact of MHC class I and class II targeting sequences on immune response and protection against lethal challenge

Abstract

Rabies is progressive fatal encephalitis. WHO estimates 55,000 rabies deaths and more than 10 million PEP every year world-wide. A variety of cell-culture derived vaccines are available for prophylaxis against rabies. However, their high cost restricts their usage in developing countries, where such cases are most often encountered. This is driving the quest for newer vaccine formulations; DNA vaccines being most promising amongst them. Here, we explored strategies of antigen trafficking to various cellular compartments aiming at improving both humoral and cellular immunity. These strategies include use of signal sequences namely Tissue Plasminogen Activator (TPA), Ubiquitin (UQ) and Lysosomal-Associated Membrane Protein-1 (LAMP-1). TPA, LAMP-1 and their combination were aimed at enhancing the CD4(+) T cell and antibody response. In contrast, the UQ tag was utilized for enhancing CD8(+) response. The potency of modified DNA vaccines assessed by total antibody response, antibody isotypes, cytokine profile, neutralizing antibody titer and protection conferred against in vivo challenge; was enhanced in comparison to native unmodified vaccine, but the response elicited did not pertain to the type of target sequence and the directed arm of immunity. Interestingly, the DNA vaccines that had been designed to generate different type of immune responses yielded in effect similar response. In conclusion, our data indicate that the directing target sequence is not the exclusive deciding factor for type and extent of immune response elicited and emphasizes on the antigen dependence of immune enhancement strategies.

Fig. 1

Schematic representation of plasmid DNA constructs encoding the rabies virus glycoprotein. The DNA encoding the full length RV-G (1575 nt), was amplified from pTargeT.rabgp, using SapI restriction site in both forward and reverse primer. rRV-G was cloned in DNA vaccine vectors and designated as: pgp: without any target sequence (native); pT.gp.L: downstream of TPA and upstream of LAMP-1 signal sequences; pT.gp: downstream of TPA signal sequence; pU.gp: downstream of UQ signal sequence and pgp.L: upstream LAMP-1 signal sequence. RV-G: rabies virus glycoprotein; CMV: cytomegalovirus promoter/enhancer; TPA: human tissue plasminogen activator signal sequence; LAMP-1: human lysosomal-associated membrane protein-1 signal sequence; UQ: mouse ubiquitin A76 signal sequence.

Fig. 2

(a) Flow cytometric analysis of cells expressing rabies glycoprotein. BHK-21 cells transfected with various plasmid DNA constructs were stained with anti-rabies hyperimmune sera as the primary antibody. The number of cells showing fluorescence, after staining with Alexa Fluor 488 labeled secondary antibody were analyzed using FL1 and displayed as histograms, which are means ± S.D. were obtained from duplicate cultures. Actin was used as positive control. (b) The address tags efficiently target glycoprotein to various sub cellular locations. Cell lysates, lysosomal fractions, concentrated culture supernatant and membrane fractions were prepared 40 h post-transfection. Subsequently, the protein samples were resolved on 12% SDS-PAGE under reducing conditions and transferred onto nitrocellulose membrane. Presence of rabies glycoprotein was detected using mouse polyclonal anti-rabies hyperimmune sera followed by alkaline phosphatase-conjugated anti-mouse IgG. The blot was developed using BCIP/NBT as substrate.

Fig. 3

Humoral immune response in mice vaccinated with various RV-G plasmid DNA constructs. Female BALB/c mice (four- to six-week-old) were immunized i.m. with plasmid DNA constructs encoding RV-G (100 μg/mice), vector (100 μg/mice) or PBS on days 0, 21 and 42. On days 20, 41 and 62, mice were bled and the sera were prepared; and subsequently analyzed for anti-RV-G antibodies by ELISA. Microtitration plates were coated with bacterially expressed, recombinant glycoprotein (500 ng/well) and incubated with 1:50 dilutions of immune sera samples. ELISA antibody titers are presented as the mean from all mice in each group.

Fig. 4

The isotype profile of the RV-G-specific IgG1 (Black bars) and IgG2a (Gray bars) titers in mice immunized by different protocols. Each group of mice (n = 10) was immunized respectively by DNA, vector or PBS. Mice were bled at three weeks after the last immunization and glycoprotein-specific IgG1 and IgG2a titers were detected by ELISA. Optical density was measured at 450 nm. Data shown represent geometric mean titers and standard deviations for each group of animals.

Fig. 5

Rabies virus-neutralizing antibody (RVNA) titers in mice vaccinated with various RV-G plasmid DNA constructs were determined. The bars represent the geometric mean of the RVNA titers obtained with individual serum samples (represented by various symbols) in a group of vaccinated mice. RVNA titer equivalent to 0.5 IU/ml is the minimum adequate titer against rabies as recommended by WHO. The figure represents RVNA titers on day 62.

Fig. 6

Concentrations of cytokines in cell-culture supernatants of BALB/c mouse splenocytes. Splenocytes (5 × 10⁵ cells/ml) were stimulated with 5 μg/ml of BPL-inactivated PV-11 virus. 24, 48 and 72 h later, culture supernatants were collected and analyzed by a capture ELISA for IL-2, IL-4, IL-12 and IFN-γ. Splenocytes from two mice immunized with DNA vaccine constructs were included in each experiment. Data are expressed as mean values ± S.D. of triplicates.

Fig. 7

Survival percentage of mice immunized with Rabies DNA vaccine. Mice were immunized with the various constructs or empty vector control. All mice were challenged intracerebrally with 20 LD₅₀ of CVS strain of rabies virus on day 21 post-immunization and observed for 18 days for rabies specific symptoms or death.