scholarly journals Replication and Propagation of Attenuated Vesicular Stomatitis Virus Vectors In Vivo: Vector Spread Correlates with Induction of Immune Responses and Persistence of Genomic RNA

2006 ◽  
Vol 81 (4) ◽  
pp. 2078-2082 ◽  
Author(s):  
Ian D. Simon ◽  
Jean Publicover ◽  
John K. Rose
Keyword(s):  
Immune Responses ◽  
Lymph Nodes ◽  
Vesicular Stomatitis Virus ◽  
Genomic Rna ◽  
Wild Type ◽  
Single Cycle ◽  
Pcr Assay ◽  
Vesicular Stomatitis

ABSTRACT Live-attenuated vesicular stomatitis virus (VSV) vectors expressing foreign antigens induce potent immune responses and protect against viral diseases in animal models. Highly attenuated (VSV-CT1) or single-cycle VSV (VSVΔG) vectors induce immune responses lower than those generated by attenuated wild-type VSV vectors when given intranasally. We show here that reduced spread of the more highly attenuated or single-cycle vectors to other organs, including lymph nodes, correlates with the reduction in the immune responses. A reverse transcription, real-time PCR assay for VSV genomic RNA (gRNA) sequences showed long-term persistence of gRNA from replicating vectors in lymph nodes, long after viral clearance. Such persistence may be important for induction of potent immune responses by VSV vectors.

scholarly journals Vesicular Stomatitis Virus Genomic RNA Persists In Vivo in the Absence of Viral Replication

2009 ◽  
Vol 84 (7) ◽  
pp. 3280-3286 ◽  
Author(s):  
Ian D. Simon ◽  
Nico van Rooijen ◽  
John K. Rose
Keyword(s):  
Lymph Node ◽  
Lymph Nodes ◽  
Viral Replication ◽  
Vesicular Stomatitis Virus ◽  
Genomic Rna ◽  
Intramuscular Inoculation ◽  
Vesicular Stomatitis ◽  
High Level

ABSTRACT Our previous studies using intranasal inoculation of mice with vesicular stomatitis virus (VSV) vaccine vectors showed persistence of vector genomic RNA (gRNA) for at least 60 days in lymph nodes in the absence of detectable infectious virus. Here we show high-level concentration of virus and gRNA in lymph nodes after intramuscular inoculation of mice with attenuated or single-cycle VSV vectors as well as long-term persistence of gRNA in the lymph nodes. To determine if the persistence of gRNA was due to ongoing viral replication, we developed a tagged-primer approach that was critical for detection of VSV mRNA specifically. Our results show that VSV gRNA persists long-term in the lymph nodes while VSV mRNA is present only transiently. Because VSV transcription is required for replication, our results indicate that persistence of gRNA does not result from continuing viral replication. We also performed macrophage depletion studies that are consistent with initial trapping of VSV gRNA largely in lymph node macrophages and subsequent persistence elsewhere in the lymph node.

scholarly journals A Single-Cycle Vaccine Vector Based on Vesicular Stomatitis Virus Can Induce Immune Responses Comparable to Those Generated by a Replication-Competent Vector

2005 ◽  
Vol 79 (21) ◽  
pp. 13231-13238 ◽  
Author(s):  
Jean Publicover ◽  
Elizabeth Ramsburg ◽  
John K. Rose
Keyword(s):  
Immune Responses ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Cytotoxic T Lymphocyte ◽  
Antibody Responses ◽  
Single Cycle ◽  
Live Attenuated Vaccine ◽  
Vesicular Stomatitis ◽  
Env Protein

ABSTRACT Live attenuated vaccine vectors based on recombinant vesicular stomatitis virus (VSV) are effective in several viral disease models. In this study, we asked if a VSV vector capable of only a single cycle of replication might be an effective alternative to replication-competent VSV vectors. We compared the cellular immune responses to human immunodeficiency virus (HIV) envelope protein (Env) expressed by replication-competent and single-cycle VSV vectors and also examined the antibody response to Env. The single-cycle vector was grown by complementation with VSV G protein and then tested initially for immunogenicity when given by four different routes. When given by the intramuscular route in mice, we found that the single-cycle vector was equivalent to the replication-competent VSV vector in generating high-level primary and memory CD8 T-cell responses as well as antibody responses to Env. Cellular responses were analyzed using major histocompatibility complex class I tetramers and direct measurement of cytotoxic T-lymphocyte activity in vivo. We also found that the recall responses after boosting were equivalent in animals vaccinated with replication-competent or single-cycle vectors. Additionally, we observed recall and heightened memory responses after boosting animals with a single-cycle vector complemented with G protein from a different vesiculovirus. Because expression of HIV Env by G-deleted VSV might allow replication in human cells expressing CD4, we generated a single-cycle VSV recombinant expressing a secreted form of the HIV Env protein. This virus was just as effective as the recombinant expressing the membrane-anchored Env protein at producing CD8 T cells and antibody responses.

scholarly journals Recombinant Vesicular Stomatitis Virus Vectors Expressing Herpes Simplex Virus Type 2 gD Elicit Robust CD4+ Th1 Immune Responses and Are Protective in Mouse and Guinea Pig Models of Vaginal Challenge

2006 ◽  
Vol 80 (9) ◽  
pp. 4447-4457 ◽  
Author(s):  
Robert J. Natuk ◽  
David Cooper ◽  
Min Guo ◽  
Priscilla Calderon ◽  
Kevin J. Wright ◽  
...  
Keyword(s):  
Herpes Simplex ◽  
Immune Responses ◽  
Vesicular Stomatitis Virus ◽  
Guinea Pigs ◽  
Wild Type ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Vesicular Stomatitis ◽  
Simplex Virus

ABSTRACT Recombinant vesicular stomatitis virus (rVSV) vectors offer an attractive approach for the induction of robust cellular and humoral immune responses directed against human pathogen target antigens. We evaluated rVSV vectors expressing full-length glycoprotein D (gD) from herpes simplex virus type 2 (HSV-2) in mice and guinea pigs for immunogenicity and protective efficacy against genital challenge with wild-type HSV-2. Robust Th1-polarized anti-gD immune responses were demonstrated in the murine model as measured by induction of gD-specific cytotoxic T lymphocytes and increased gamma interferon expression. The isotype makeup of the serum anti-gD immunoglobulin G (IgG) response was consistent with the presence of a Th1-CD4+ anti-gD response, characterized by a high IgG2a/IgG1 IgG subclass ratio. Functional anti-HSV-2 neutralizing serum antibody responses were readily demonstrated in both guinea pigs and mice that had been immunized with rVSV-gD vaccines. Furthermore, guinea pigs and mice were prophylactically protected from genital challenge with high doses of wild-type HSV-2. In addition, guinea pigs were highly protected against the establishment of latent infection as evidenced by low or absent HSV-2 genome copies in dorsal root ganglia after virus challenge. In summary, rVSV-gD vectors were successfully used to elicit potent anti-gD Th1-like cellular and humoral immune responses that were protective against HSV-2 disease in guinea pigs and mice.

scholarly journals The 5′ Terminal Trailer Region of Vesicular Stomatitis Virus Contains a Position-Dependent cis-Acting Signal for Assembly of RNA into Infectious Particles

1999 ◽  
Vol 73 (1) ◽  
pp. 307-315 ◽  
Author(s):  
Sean P. J. Whelan ◽  
Gail W. Wertz
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Genomic Rna ◽  
Wild Type ◽  
Rna Sequences ◽  
Defective Interfering Particles ◽  
Cis Acting ◽  
Efficient Replication ◽  
Vesicular Stomatitis

ABSTRACT The cis-acting genomic RNA requirements for the assembly of vesicular stomatitis virus (VSV) ribonucleocapsids into infectious particles were investigated. Using a biological assay based on particle infectivity, we demonstrated that subgenomic replicons that contained all four possible combinations of the natural genomic termini, the 3′ leader (Le) and 5′ trailer (Tr) regions, were replication competent; however, a 3′ copyback replicon (3′CB), containing the natural 3′ terminus but having the 5′ Tr replaced by a sequence complementary to the 3′ Le for 46 nucleotides, was unable to assemble infectious particles, despite efficient replication. When a copy of Tr was inserted 51 nucleotides from the 5′ end of 3′CB, infectious particles were produced. However, analysis of the replication products of these particles showed that the 51 nucleotides which corresponded to the Le complement sequences at the 5′ terminus were removed during RNA replication, thus restoring the wild-type 5′ Tr to the exact 5′ terminus. These data showed that acis-acting signal was necessary for assembly of VSV RNAs into infectious particles and that this signal was supplied by Tr when located at the 5′ end. The regions within Tr required for assembly were analyzed by a series of deletions and exchanges for Le complement sequences, which demonstrated that the 5′ terminal 29 nucleotides of Tr allowed assembly of infectious particles but that the 5′ terminal 22 nucleotides functioned poorly. Deletions in Tr also altered the balance between negative- and positive-strand genomic RNA and affected levels of replication. RNAs that retained fewer than 45 but at least 22 nucleotides of the 5′ terminus could replicate but were impaired in RNA replication, and RNAs that retained only 14 nucleotides of the 5′ terminus were severely reduced in ability to replicate. These data define the VSV Tr as a position-dependent, cis-acting element for the assembly of RNAs into infectious particles, and they delineate RNA sequences that are essential for negative-strand RNA synthesis. These observations are consistent with, and offer an explanation for, the absence of 3′ copyback defective interfering particles in nature.

scholarly journals Identification of a Novel Tripartite Complex Involved in Replication of Vesicular Stomatitis Virus Genome RNA

2003 ◽  
Vol 77 (1) ◽  
pp. 732-738 ◽  
Author(s):  
Ashim K. Gupta ◽  
Daniel Shaji ◽  
Amiya K. Banerjee
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Reverse Genetics ◽  
Insect Cells ◽  
Recombinant Baculovirus ◽  
Wild Type ◽  
Bhk Cells ◽  
P Protein ◽  
Vesicular Stomatitis

ABSTRACT Our laboratory's recent observations that transcriptionally inactive phosphoprotein (P) mutants can efficiently function in replicating vesicular stomatitis virus (VSV) defective interfering particle in a three-plasmid-based (L, P, and N) reverse genetics system in vivo (A. K. Pattnaik, L. Hwang, T. Li, N. Englund, M. Mathur, T. Das, and A. K. Banerjee, J. Virol. 71:8167-8175, 1997) led us to propose that a tripartite complex consisting of L-(N-P) protein may represent the putative replicase for synthesis of the full-length genome RNA. In this communication we demonstrate that such a complex is indeed detectable in VSV-infected BHK cells. Furthermore, coexpression of L, N, and P proteins in Sf21 insect cells by recombinant baculovirus containing the respective genes also resulted in the formation of a tripartite complex, as shown by immunoprecipitation with specific antibodies. A basic amino acid mutant of P protein, P260A, previously shown to be inactive in transcription but active in replication (T. Das, A. K. Pattnaik, A. M. Takacs, T. Li, L. N. Hwang, and A. K. Banerjee, Virology 238:103-114, 1997) was also capable of forming the mutant [L-(N-Pmut)] complex in both insect cells and BHK cells. Sf21 extract containing either the wild-type P protein or the mutant P protein along with the L and N proteins was capable of synthesizing 42S genome-sense RNA in an in vitro replication reconstitution reaction. Addition of N-Pmut or wild-type N-P complex further stimulated the synthesis of the genome-length RNA. These results indicate that the transcriptase and replicase complexes of VSV are possibly two distinct entities involved in carrying out capped mRNAs and uncapped genome and antigenome RNAs, respectively.

scholarly journals Immune Response in the Absence of Neurovirulence in Mice Infected with M Protein Mutant Vesicular Stomatitis Virus

2008 ◽  
Vol 82 (18) ◽  
pp. 9273-9277 ◽  
Author(s):  
Maryam Ahmed ◽  
Tracie R. Marino ◽  
Shelby Puckett ◽  
Nancy D. Kock ◽  
Douglas S. Lyles
Keyword(s):  
Central Nervous System ◽  
Immune Response ◽  
Nervous System ◽  
Vesicular Stomatitis Virus ◽  
M Protein ◽  
Wild Type ◽  
Protein Mutants ◽  
The Central Nervous System ◽  
Vesicular Stomatitis

ABSTRACT Matrix (M) protein mutants of vesicular stomatitis virus (VSV), such as rM51R-M virus, are less virulent than wild-type (wt) VSV strains due to their inability to suppress innate immunity. Studies presented here show that when inoculated intranasally into mice, rM51R-M virus was cleared from nasal mucosa by day 2 postinfection and was attenuated for spread to the central nervous system, in contrast to wt VSV, thus accounting for its reduced virulence. However, it stimulated an antibody response similar to that in mice infected with the wt virus, indicating that it has the ability to induce adaptive immunity in vivo without causing disease. These results support the use of M protein mutants of VSV as vaccine vectors.

The atypical chemokine receptor CCX-CKR scavenges homeostatic chemokines in circulation and tissues and suppresses Th17 responses

Blood ◽  
2010 ◽  
Vol 116 (20) ◽  
pp. 4130-4140 ◽  
Author(s):  
Iain Comerford ◽  
Robert J. B. Nibbs ◽  
Wendel Litchfield ◽  
Mark Bunting ◽  
Yuka Harata-Lee ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Lymph Nodes ◽  
Chemokine Receptor ◽  
Fold Increase ◽  
Wild Type ◽  
Peripheral Lymph ◽  
The Central Nervous System

Abstract Our previous in vitro studies led to proposals that the atypical chemokine receptor CCX-CKR is a scavenger of CCR7 ligand homeostatic chemokines. In the present study, we generated CCX-CKR−/− mice and confirm this scavenger function in vivo. Compared with wild-type mice, CCX-CKR−/− have a 5-fold increase in the level of CCL21 protein in blood, and 2- to 3-fold increases in CCL19 and CCL21 in peripheral lymph nodes. The effect of these protein increases on immunity was investigated after immunization with MOG35-55 peptide emulsified in complete Freund adjuvant (CFA). The subsequent characteristic paralysis develops with enhanced kinetics and severity in CCX-CKR−/− versus wild-type mice. Despite this effect, antigen-specific immune responses in the draining lymph nodes are diminished in CCX-CKR−/− mice. Instead, the earlier onset of disease is associated with enhanced T-cell priming in the CCX-CKR−/− spleen and a skewing of CD4+ T-cell responses toward Th17 rather than Th1. This observation correlates with increased expression of IL-23 in the CCX-CKR−/− spleen and increased CCL21 levels in the central nervous system postimmunization. The early onset of disease in CCX-CKR−/− mice is reversed by systemic administration of neutralizing anti-CCL21 antibodies. Thus, by regulating homeostatic chemokine bioavailability, CCX-CKR influences the localization, kinetics, and nature of adaptive immune responses in vivo.

scholarly journals Overlapping Signals for Transcription and Replication at the 3′ Terminus of the Vesicular Stomatitis Virus Genome

1999 ◽  
Vol 73 (1) ◽  
pp. 444-452 ◽  
Author(s):  
Tong Li ◽  
Asit K. Pattnaik
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Virus Genome ◽  
Gene Analysis ◽  
Genomic Rna ◽  
Wild Type ◽  
Transcription Activity ◽  
Transcription Reinitiation ◽  
Vesicular Stomatitis ◽  
Negative Sense ◽  
Transcription And Replication

ABSTRACT Transcription and replication signals within the negative-sense genomic RNA of vesicular stomatitis virus (VSV) are located at the 3′ terminus. To identify these signals, we have used a transcription- and replication-competent minigenome of VSV to generate a series of deletions spanning the first 47 nucleotides at the 3′ terminus of the VSV genome corresponding to the leader gene. Analysis of these mutants for their ability to replicate showed that deletion of sequences within the first 24 nucleotides abrogated or greatly reduced the level of replication. Deletion of downstream sequences from nucleotides 25 to 47 reduced the level of replication only to 55 to 70% of that of the parental template. When transcription activity of these templates was measured, the first 24 nucleotides were also found to be required for transcription, since deletion of these sequences blocked or significantly reduced transcription. Downstream sequences from nucleotides 25 to 47 were necessary for optimal levels of transcription. Furthermore, replacement of sequences within the 25 to 47 nucleotides with random heterologous nonviral sequences generated mutant templates that replicated well (65 to 70% of the wild-type levels) but were transcribed poorly (10 to 15% of the wild-type levels). These results suggest that the minimal promoter for transcription and replication could be as small as the first 19 nucleotides and is contained within the 3′-terminal 24 nucleotides of the VSV genome. The sequences from nucleotides 25 to 47 may play a more important role in optimal transcription than in replication. Our results also show that deletion of sequences within the leader gene does not influence the site of transcription reinitiation of the downstream gene.

scholarly journals Protection of Cattle against Rinderpest by Vaccination with Wild-Type but Not Attenuated Strains of Peste des Petits Ruminants Virus

2016 ◽  
Vol 90 (10) ◽  
pp. 5152-5162 ◽  
Author(s):  
Barbara Holzer ◽  
Sophia Hodgson ◽  
Nicola Logan ◽  
Brian Willett ◽  
Michael D. Baron
Keyword(s):  
Immune Responses ◽  
Peste Des Petits Ruminants ◽  
Wild Type ◽  
Rinderpest Virus ◽  
Live Virus ◽  
In The Wild ◽  
Deliberate Release ◽  
T Cell Immune Responses ◽  
Vesicular Stomatitis

ABSTRACTAlthough rinderpest virus (RPV) has been eradicated in the wild, efforts are still continuing to restrict the extent to which live virus is distributed in facilities around the world and to prepare for any reappearance of the disease, whether through deliberate or accidental release. In an effort to find an alternative vaccine which could be used in place of the traditional live attenuated RPV strains, we have determined whether cattle can be protected from rinderpest by inoculation with vaccine strains of the related morbillivirus, peste des petits ruminants virus (PPRV). Cattle were vaccinated with wild-type PPRV or either of two established PPRV vaccine strains, Nigeria/75/1 or Sungri/96. All animals developed antibody and T cell immune responses to the inoculated PPRV. However, only the animals given wild-type PPRV were protected from RPV challenge. Animals given PPRV/Sungri/96 were only partially protected, and animals given PPRV/Nigeria/75/1 showed no protection against RPV challenge. While sera from animals vaccinated with the vaccine strain of RPV showed cross-neutralizing ability against PPRV, none of the sera from animals vaccinated with any strain of PPRV was able to neutralize RPV although sera from animals inoculated with wild-type PPRV were able to neutralize RPV-pseudotyped vesicular stomatitis virus.IMPORTANCERinderpest virus has been eradicated, and it is only the second virus for which this is so. Significant efforts are still required to ensure preparedness for a possible escape of RPV from a laboratory or its deliberate release. Since RPV vaccine protects sheep and goats from PPRV, it is important to determine if the reverse is true as this would provide a non-RPV vaccine for dealing with suspected RPV outbreaks. This is probably the lastin vivostudy with live RPV that will be approved.

Neurovirulence Mutant of Vesicular Stomatitis Virus with an Altered Target Cell Tropism In Vivo

1980 ◽  
Vol 29 (2) ◽  
pp. 744-757
Author(s):  
Olivia T. Preble ◽  
Lauren E. Costello ◽  
Diana D. Huang ◽  
Mamdouha A. Barmada
Keyword(s):  
Spinal Cord ◽  
Vesicular Stomatitis Virus ◽  
Target Cell ◽  
Ependymal Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Neuronal Necrosis ◽  
Lateral Ventricles ◽  
Vesicular Stomatitis

Intracerebral infection of weanling Swiss mice with a temperature-sensitive ( ts ) mutant of vesicular stomatitis virus (VSV), ts pi364, resulted in a unique neuropathological syndrome not previously described with other VSV mutants. Mice infected with wild-type VSV died from an acute encephalitis characterized by neuronal necrosis and efficient virus replication in both brain and spinal cord. In contrast, with VSV ts pi364, the most prominent histopathological feature was destruction of the ependyma of the lateral ventricles. Virus antigen was also limited to the leptomeninges and the lateral ventricles. Infected mice survived and developed hydrocephalus. Replication of ts pi364 in the brain was 10- to 100- fold less than that of wild-type VSV, and appearance of virus in the spinal cord was delayed. VSV ts pi364 was isolated from mouse cells persistently infected with VSV. Another VSV ts pi mutant, isolated from the same persistent infection, behaved in vivo like wild-type VSV, even though both mutants were very similar in plaque size, reversion frequency, cut-off temperature, and synthesis of virus-specific proteins at semipermissive temperature. These results strongly suggest that VSV ts pi364 has a second, non- ts mutation which results in a restricted target cell range in vivo; wild-type VSV can infect both neurons and ependymal cells, whereas ts pi364 does not replicate in neurons.

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