Neurovirulence and immunogenicity of attenuated recombinant vesicular stomatitis viruses in nonhuman primates.

UNLABELLED: In previous work, a prototypic recombinant vesicular stomatitis virus Indiana serotype (rVSIV) vector expressing simian immunodeficiency virus (SIV) gag and human immunodeficiency virus type 1 (HIV-1) env antigens protected nonhuman primates (NHPs) from disease following challenge with an HIV-1/SIV recombinant (SHIV). However, when tested in a stringent NHP neurovirulence (NV) model, this vector was not adequately attenuated for clinical evaluation. For the work described here, the prototypic rVSIV vector was attenuated by combining specific G protein truncations with either N gene translocations or mutations (M33A and M51A) that ablate expression of subgenic M polypeptides, by incorporation of temperature-sensitive mutations in the N and L genes, and by deletion of the VSIV G gene to generate a replicon that is dependent on trans expression of G protein for in vitro propagation. When evaluated in a series of NHP NV studies, these attenuated rVSIV variants caused no clinical disease and demonstrated a very significant reduction in neuropathology compared to wild-type VSIV and the prototypic rVSIV vaccine vector. In spite of greatly increased in vivo attenuation, some of the rVSIV vectors elicited cell-mediated immune responses that were similar in magnitude to those induced by the much more virulent prototypic vector. These data demonstrate novel approaches to the rational attenuation of VSIV NV while retaining vector immunogenicity and have led to identification of an rVSIV N4CT1gag1 vaccine vector that has now successfully completed phase I clinical evaluation. IMPORTANCE: The work described in this article demonstrates a rational approach to the attenuation of vesicular stomatitis virus neurovirulence. The major attenuation strategy described here will be most likely applicable to other members of the Rhabdoviridae and possibly other families of nonsegmented negative-strand RNA viruses. These studies have also enabled the identification of an attenuated, replication-competent rVSIV vector that has successfully undergone its first clinical evaluation in humans. Therefore, these studies represent a major milestone in the development of attenuated rVSIV, and likely other vesiculoviruses, as a new vaccine platform(s) for use in humans.

Neurovirulence properties of recombinant vesicular stomatitis virus vectors in non-human primates.

Although vesicular stomatitis virus (VSV) neurovirulence and pathogenicity in rodents have been well studied, little is known about VSV pathogenicity in non-human primates. To address this question, we measured VSV viremia, shedding, and neurovirulence in macaques. Following intranasal inoculation, macaques shed minimal recombinant VSV (rVSV) in nasal washes for 1 day post-inoculation; viremia was not detected. Following intranasal inoculation of macaques, wild type (wt) VSV, rVSV, and two rVSV-HIV vectors showed no evidence of spread to CNS tissues. However, macaques inoculated intrathalamically with wt VSV developed severe neurological disease. One of four macaques receiving rVSV developed clinical and histological signs similar to the wt group, while the remaining three macaques in this group and all of the macaques in the rVSV-HIV vector groups showed no clinical signs of disease and reduced severity of histopathology compared to the wt group. The implications of these findings for rVSV vaccine development are discussed.

Ribavirin causes error catastrophe during Hantaan virus replication.

Except for ribavirin, no other antiviral drugs for treating hantaviral diseases have been identified. It is well established that ribavirin will inhibit the production of infectious Hantaan virus (HTNV); however, its mechanism of action is unknown. To characterize the inhibitory effect of ribavirin on HTNV, the levels of viral RNAs, proteins, and infectious particles were measured for 3 days posttreatment of HTNV-infected Vero E6 cells. HTNV-infected cells treated with ribavirin showed a slight reduction in the levels of cRNA, viral RNA, and mRNA populations on the first day postinfection. The amount of cRNA and viral RNA increased to that observed for untreated HTNV-infected cells on day 2, whereas mRNA levels were more greatly reduced on days 2 and 3. Despite the finding of S-segment mRNA, albeit low, three of the viral proteins-nucleocapsid (N) protein and glycoproteins G1 and G2-could not be detected by immunohistochemistry in ribavirin-treated cells. To test the hypothesis that these effects were caused by incorporation of ribavirin into nascent RNA and a resultant "error catastrophe" was occurring, we cloned and sequenced the S-segment cRNA/mRNA from ribavirin-treated or untreated cells from day 3. We found a high mutation frequency (9.5/1,000 nucleotides) in viral RNA synthesized in the presence of ribavirin. Hence, the transcripts produced in the presence of the drug were not functional. These results suggest that ribavirin's mechanism of action lies in challenging the fidelity of the hantavirus polymerase, which causes error catastrophe.