Live-attenuated vesicular stomatitis virus (VSV) vectors expressing foreign antigens induce potent

Live-attenuated vesicular stomatitis virus (VSV) vectors expressing foreign antigens induce potent immune responses and protect against viral diseases in animal Rabbit polyclonal to ABCA13. models. virus (VSV) a nonsegmented negative-strand RNA virus infects cattle horses and pigs causing vesicular lesions. Other mammals including humans and mice are also infected in areas of enzootic outbreaks (16 22 Live-attenuated VSV-based vectors expressing specific foreign proteins produce strong immune responses and protect against viral SL251188 diseases in many animal models (4-6 12 14 15 18 19 including a monkey model for AIDS (3 11 17 A live-attenuated VSV vector expressing human immunodeficiency virus proteins is moving into clinical trials as an AIDS vaccine. Given the importance of the VSV vector system we wanted to determine the extent of vector replication SL251188 and persistence in vivo. A recombinant wild-type VSV (rwt) derived from DNA (7) is already attenuated for pathogenesis in mice compared to the wild-type VSV (14). We have also characterized a highly attenuated VSV mutant with a truncation of the VSV G cytoplasmic tail to 1 1 amino acid (20). This CT1 mutation eliminates all vector-associated pathogenesis after intranasal (i.n.) inoculation of mice (9 13 Previous studies showed that this VSV-CT1 vector induces humoral and cellular immune responses but these responses are four- to fivefold lower than those generated by rwtVSV when given i.n. (9). In contrast the highly attenuated CT1 vector or a single-cycle vector lacking the VSV G gene (VSVΔG) induced immune SL251188 responses comparable to rwtVSV when given intramuscularly (10). Spread of VSV vectors in vivo. We hypothesized that after i.n. immunization the rwt vector might need to replicate extensively and spread to other organs to induce strong immune responses. To examine the extent of replication of rwtVSV and VSV-CT1 in detail during an in vivo contamination groups of four to seven 8 BALB/c mice were infected i.n. with 5 × 105 PFU of each virus. We harvested lungs liver spleen plasma and lymph nodes from mice at various times after contamination. We determined virus titers from snap-frozen homogenized tissue and expressed them as PFU per gram or as PFU/ml in the case of plasma titers (Fig. ?(Fig.1).1). The brain was omitted from these experiments because a previous study from our lab focusing on neurotropism of our attenuated rwtVSV virus showed that it spread only to the olfactory bulb and no farther into the brain after i.n. administration in young mice (24). FIG. 1. Replication and spread of recombinant VSV vectors following intranasal inoculation. Eight-week-old BALB/c mice were inoculated with 5 × 105 PFU of rwtVSV (solid squares) and VSV-CT1 (open triangles). Lungs (A) lymph nodes (B) spleen (C) liver … Within the lungs and lymph nodes we observed the highest titers for rwtVSV at the first time point 12 h postinfection (hpi) (Fig. 1A and B). In contrast the peak VSV-CT1 titers in all organs occurred at 24 hpi. At 12 hpi we recovered a total of 9.5 × 105 PFU rwtVSV from the organs tested. This amount was twice the input virus amount (5 × 105 PFU) a clear indication that this virus was replicating. In contrast the total amount of VSV-CT1 recovered was less than the input amount. However the increase in titers from 12 to 24 hpi suggested that VSV-CT1 was replicating after i.n. inoculation but that replication of VSV-CT1 was less efficient than that of rwtVSV in vivo. Our data indicate that VSV-CT1 and rwtVSV replicate and spread in a similar pattern by initially replicating within the lungs and then likely traveling to peripheral organs via the blood. In addition VSV-CT1 was cleared faster than SL251188 rwtVSV from the lungs and lymph nodes SL251188 (Fig. 1A and B). VSV-CT1 reached peak titers nearly as high as rwtVSV in lungs and lymph nodes (Fig. ?(Fig.1) 1 while it grew to titers 10- to 30-fold lower than rwtVSV in tissue culture (20). We also performed a control experiment to ensure that the CT1 mutant was not reverting in vivo because the spread in vivo seemed greater than expected based on the 30-fold reduced growth in tissue culture. The deletion of 28 amino acids from the cytoplasmic tail of G in the CT1 mutant is usually easily detected from the more rapid mobility of the mutant G protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To examine this issue we.