A novel Sin Nombre virus DNA vaccine and its inclusion in a candidate pan-hantavirus vaccine against hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS)

Abstract

Sin Nombre virus (SNV; family Bunyaviridae, genus Hantavirus) causes a hemorrhagic fever known as hantavirus pulmonary syndrome (HPS) in North America. There have been approximately 200 fatal cases of HPS in the United States since 1993, predominantly in healthy working-age males (case fatality rate 35%). There are no FDA-approved vaccines or drugs to prevent or treat HPS. Previously, we reported that hantavirus vaccines based on the full-length M gene segment of Andes virus (ANDV) for HPS in South America, and Hantaan virus (HTNV) and Puumala virus (PUUV) for hemorrhagic fever with renal syndrome (HFRS) in Eurasia, all elicited high-titer neutralizing antibodies in animal models. HFRS is more prevalent than HPS (>20,000 cases per year) but less pathogenic (case fatality rate 1-15%). Here, we report the construction and testing of a SNV full-length M gene-based DNA vaccine to prevent HPS. Rabbits vaccinated with the SNV DNA vaccine by muscle electroporation (mEP) developed high titers of neutralizing antibodies. Furthermore, hamsters vaccinated three times with the SNV DNA vaccine using a gene gun were completely protected against SNV infection. This is the first vaccine of any kind that specifically elicits high-titer neutralizing antibodies against SNV. To test the possibility of producing a pan-hantavirus vaccine, rabbits were vaccinated by mEP with an HPS mix (ANDV and SNV plasmids), or HFRS mix (HTNV and PUUV plasmids), or HPS/HFRS mix (all four plasmids). The HPS mix and HFRS mix elicited neutralizing antibodies predominantly against ANDV/SNV and HTNV/PUUV, respectively. Furthermore, the HPS/HFRS mix elicited neutralizing antibodies against all four viruses. These findings demonstrate a pan-hantavirus vaccine using a mixed-plasmid DNA vaccine approach is feasible and warrants further development.

Keywords: DNA vaccine; HFRS; HPS; Hantavirus; Sin Nombre virus; mEP; muscle electroporation.

Fig. 1

Hantavirus neutralizing antibodies produced in rabbits vaccinated with full-length hantavirus M gene-based DNA vaccines using mEP. Groups of 3 rabbits were vaccinated with DNA vaccines (A) pWRG/HTN-M(x) and (B) pWRG/PUU-M(s2) on days noted by black arrows by mEP (Inovio Elgen device, dose 0.4 mg DNA per injection). Sera collected were tested in homotypic PRNT. Symbols represent the mean of two separate PRNT₅₀ ± SE. (C) The same data from (A) and (B) were combined to show mean titers for the groups. Previously published mean titers from rabbits vaccinated with pWRG/AND-M were shown for comparison. Note the vaccination days were different for the Andes DNA vaccine (shown in gray arrows). Groups of 3 or 4 rabbits were vaccinated with (D) the first generation SNV M gene-based DNA vaccine, pWRG/SN-M(2a) or (E) the optimized SNV M gene-based DNA vaccine, pWRG/SN-M(opt), on days noted by black arrows. Sera collected were tested in homotypic PRNT. (F) The same data from (D) and (E) were combined to show mean titers for the groups. Black arrows indicate vaccination days for pWRG/SN-M(2a) and gray arrows indicate vaccination days for pWRG/SN-M(opt). The PRNT limit of detection was a titer of 20 (dashed lines).

Fig. 2

Single-injection multiagent hantavirus DNA vaccines are feasible by mEP. Three mixtures of hantavirus DNA vaccine plasmids were delivered to rabbits by mEP (Ichor Tri-grid). Groups of 3 rabbits were vaccinated at 3-week intervals and sera were collected for PRNT analysis. The HFRS mixture was comprised of equal volumes of pWRG/HTN-M(x) and pWRG/PUU-M(s2), (2 mg DNA total, 1 mg/plasmid/injection, 1 injection/vaccination). The HPS mixture was comprised of equal volumes of pWRG/ANDM and pWRG/SN-M(opt) (2 mg DNA total, 1 mg/plasmid/injection, 1 injection/vaccination). The HFRS/HPS mixture was comprised of equal volumes of HTNV, PUUV, ANDV, and SNV DNA vaccine plasmids (4 mg DNA total, 1 mg/plasmid/injection, 2 injections/vaccination). (A) Neutralizing antibody titers for individual rabbits are shown. The virus used in the neutralization test is shown on the y-axis. (B) Mean neutralization titers for each group ± SE. The PRNT limit of detection was a titer of 20 (dashed lines).

Fig. 3

PRNT₈₀ GMT against HTNV, PUUV, ANDV, and SNV for each DNA vaccine formulation after 1, 2, or 3 vaccinations. These data are from the same experiment shown in Fig. 2; however, PRNT₈₀ GMT are presented (A) after 1 vaccination, (B)after 2 vaccinations, and (C) after 3 vaccinations. The PRNT limit of detection was a titer of 20 (dashed lines). ns indicates a lack of statistical significance when titers were compared from HFRS or HPS mix to HFRS/HPS mix vaccine. Significance lines pertain to (A), (B), and (C).

Fig. 4

pWRG/SN-M(opt) DNA vaccine (gene gun) is immunogenic and protective in hamsters. Groups of 7–8 hamsters received 2 or 3 vaccinations with the pWRG/SN-M(opt) SNV DNA vaccine, 3 vaccinations with a negative control DNA vaccine, or no vaccine. (A) Sera collected were tested for SNV neutralizing antibodies by PRNT. Mean titers ± SE are shown. (B) Individual PRNT₅₀ titers from sera collected on week 9 are presented with the GMT and 95% confidence interval depicted. The PRNT limit of detection was a titer of 20 (dashed lines). (C) Sera collected on week 16 (5 weeks postchallenge) were tested by ELISA for evidence of SNV infection. All prechallenge sera samples were negative by ELISA (data not shown).<indicates titer was below level of detection for the assay. *indicates antibody responses were statistically significant when compared to negative DNA vaccination controls.

Fig. 5

pWRG/SN-M(opt) DNA vaccine (gene gun) does not protect hamsters from ANDV challenge. A group of 8 hamsters received 3 vaccinations with pWRG/SNM(opt). (A) Vaccinated and unvaccinated hamsters were challenged with 200 PFU ANDV i.m. and observed for survival. (B) Sera collected prechallenge were tested by SNV PRNT for homotypic neutralizing antibodies. The PRNT limit of detection was a titer of 20 (dashed lines). *indicate antibody response was statistically significant when compared to no vaccine controls. s indicates the PRNT titer of a surviving hamster.