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. 2009 Jul;83(14):7285-95.
doi: 10.1128/JVI.00373-09. Epub 2009 Apr 29.

Adenovirus vectors expressing hantavirus proteins protect hamsters against lethal challenge with andes virus

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Adenovirus vectors expressing hantavirus proteins protect hamsters against lethal challenge with andes virus

David Safronetz et al. J Virol. 2009 Jul.

Abstract

Hantaviruses infect humans following aerosolization from rodent feces and urine, producing hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Due to the high rates of mortality and lack of therapies, vaccines are urgently needed. Nonreplicating adenovirus (Ad) vectors that express Andes hantavirus (ANDV) nucleocapsid protein (AdN) or glycoproteins (AdG(N) and AdG(C)) were constructed. Ad vectors were tested for their ability to protect Syrian hamsters from a lethal ANDV infection that mimics the pulmonary disease seen in humans. When administered once, all three Ad vectors, individually or in combination, elicited a robust immune response that protected hamsters. No vaccinated animal died, and there were no obvious clinical signs of disease. Further, hantavirus RNA was not detected by sensitive reverse transcription-PCR in tissues and blood of hamsters immunized with both AdG(N) and AdG(C). Cellular immunity appeared to be important for protection because the AdN vector completely protected animals. All three Ad vectors produced strong cytotoxic T-lymphocyte responses directed to hantavirus proteins in mice. Moreover, hamsters vaccinated with AdN, AdG(N), or AdG(C) produced no detectable neutralizing antibodies yet were protected. These Ad vectors represent the first vaccines that prevent lethal hantavirus disease and, in some instances (AdG(N) and AdG(C)), provide sterile immunity. These observations set the stage for a more detailed characterization of the types of immunity required to protect humans from hantavirus infections.

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Figures

FIG. 1.
FIG. 1.
Construction of recombinant Ad vectors expressing ANDV proteins. (A) The ANDV S, M, and L RNA segments encoding the N protein, the GPC protein that gives rise to GN and GC, and polymerase, respectively. (B) N-, GPC-, GN-, and GC-coding regions were amplified by PCR from plasmids and inserted into a shuttle plasmid, pDC316(io), for rescue into E1− E3− Ad vectors. This placed hantavirus coding sequences downstream of an MCMV immediate-early promoter sequence that was separated from the hantavirus genes by a LacZ repressor binding site and an intron, all inserted with Ad5 E1 sequences. These pDC316(io) plasmids were cotransfected with plasmid pBHGloxDE1,3Cre, which contains other Ad sequences and lacks E3 sequences, into 293 IQ cells. Recombination between loxP sites in pDC316(io) and pBHGloxDE1,3Cre produced Ad vectors. 293 IQ cells express a LacZ repressor protein that reduces expression of hantavirus proteins during Ad vector propagation.
FIG. 2.
FIG. 2.
Expression of ANDV proteins by Ad vectors. (A) Human U373 cells were infected with Ad vector AdGN, AdGC, AdGPC, or AdN using 50 (293 cell) PFU/cell for 24 h, and then the cells were radiolabeled with [35S]methionine-cysteine for 3 h. (B) U373 cells were infected with AdGN, AdGC, or both AdGN and AdGC using 50 PFU/cell for 22 h, and then cells were radiolabeled with [35S]methionine-cysteine for 3 h. Detergent extracts of cells were mixed with GN-, GC-, or N-specific antibodies, and various proteins were immunoprecipitated and analyzed by gel electrophoresis. Molecular mass markers are at the right, and the positions of GN, GC, and N proteins indicated on the left and right.
FIG. 3.
FIG. 3.
Mouse CTL lysis of mouse cells transduced with hantavirus proteins. Groups of five mice were vaccinated with 1 ×ばつ 108 (293 cell) PFU per animal of AdN (A), AdGN (B), AdGC (C), or both AdGN and AdGC (D). Splenocytes were removed after 6 to 8 days, pooled for all five animals, and restimulated with interleukin-2 and gamma-irradiated syngeneic SVBalb cells infected with the relevant Ad vectors for 7 to 8 days. The splenocytes (effectors) were mixed with 51Cr-labeled target cells consisting of SVBalb cells that were left uninfected (UI) or infected with Ad vectors (including AdEmpty, which expresses no hantavirus proteins) using various effector-to-target cell ratios in triplicate wells. After 4 to 5 h the release of 51Cr into cell culture supernatants was measured, as well as spontaneous release and maximum release. The percent specific release was calculated as described in the Materials and Methods.
FIG. 4.
FIG. 4.
Protection of hamsters from lethal ANDV disease following vaccination with Ad vectors. Groups of 12 or 13 hamsters were immunized once using 108 PFU/animal with various Ad vectors by the intramuscular route. Twenty-eight days later, the animals were challenged using 100 LD50s of ANDV and monitored for 45 days for signs of disease and survival.
FIG. 5.
FIG. 5.
Detection of ANDV RNA in hamsters vaccinated with Ad vectors and then challenged with ANDV. Three hamsters from each group (AdEmpty, AdN, AdGN, AdGC, or both AdGN and AdGC) were euthanized at 6 (upper panel) and 9 (lower panel) days postchallenge with ANDV. Lung, liver, and blood samples were collected and RNA extracted and analyzed for the presence of ANDV RNA using real-time quantitative RT-PCR. The data represents the average values for triplicate analyses of tissues from three hamsters. Numbers above bars indicate the number of hamsters that were positive for RNA. Asterisks indicate statistically significant differences compared with AdEmpty-immunized hamsters (*, P < 0.0001; **, P < 0.0096). Error bars represent 2 times the standard error of the mean.
FIG. 6.
FIG. 6.
N protein-specific and neutralizing antibody responses in hamsters vaccinated with Ad vectors before and after ANDV challenge. Sera from vaccinated hamsters were obtained 28 days after vaccination with Ad vectors and prior to ANDV challenge (pre) or 45 days after ANDV challenge (post). Sera were tested for N-specific antibodies using recombinant SNV N protein in ELISA (upper panel). Neutralizing antibodies were detected using VSV pseudotypes bearing the ANDV glycoproteins (lower panel). Each symbol represents the titer for a single hamster. †, hamster died and serum was not collected.

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