J. Erik Johnson

Construction of a Novel Virus That Targets HIV-1-Infected Cells and Controls HIV-1 Infection

We describe a recombinant vesicular stomatitis virus lacking its glycoprotein gene and expressing instead the HIV-1 receptor CD4 and a coreceptor, CXCR4. This virus was unable to infect normal cells but did infect, propagate on, and kill cells that were first infected with HIV-1 and therefore had the HIV membrane fusion protein on their surface. Killing of HIV-1-infected cells controlled HIV infection in a T cell line and reduced titers of infectious HIV-1 in the culture by as much as 10(4)-fold. Such a targeted virus could have therapeutic value in reducing HIV viral load. Our results also demonstrate a general strategy of targeting one virus to the envelope protein of another virus to control infection.

Attenuation of Recombinant Vesicular Stomatitis Virus-Human Immunodeficiency Virus Type 1 Vaccine Vectors by Gene Translocations and G Gene Truncation Reduces Neurovirulence and Enhances Immunogenicity in Mice

ABSTRACT Recombinant vesicular stomatitis virus (rVSV) has shown great potential as a new viral vector for vaccination. However, the prototypic rVSV vector described previously was found to be insufficiently attenuated for clinical evaluation when assessed for neurovirulence in nonhuman primates. Here, we describe the attenuation, neurovirulence, and immunogenicity of rVSV vectors expressing human immunodeficiency virus type 1 Gag. These rVSV vectors were attenuated by combinations of the following manipulations: N gene translocations (N4), G gene truncations (CT1 or CT9), noncytopathic M gene mutations (Mncp), and positioning of the gag gene into the first position of the viral genome (gag1). The resulting N4CT1-gag1, N4CT9-gag1, and MncpCT1-gag1 vectors demonstrated dramatically reduced neurovirulence in mice following direct intracranial inoculation. Surprisingly, in spite of a very high level of attenuation, the N4CT1-gag1 and N4CT9-gag1 vectors generated robust Gag-specific immune responses following intramuscular immunization that were equivalent to or greater than immune responses generated by the more virulent prototypic vectors. MncpCT1-gag1 also induced Gag-specific immune responses following intramuscular immunization that were equivalent to immune responses generated by the prototypic rVSV vector. Placement of the gag gene in the first position of the VSV genome was associated with increased in vitro expression of Gag protein, in vivo expression of Gag mRNA, and enhanced immunogenicity of the vector. These findings demonstrate that through directed manipulation of the rVSV genome, vectors that have reduced neurovirulence and enhanced immunogenicity can be made.

Synergistic Attenuation of Vesicular Stomatitis Virus by Combination of Specific G Gene Truncations and N Gene Translocations

ABSTRACT A variety of rational approaches to attenuate growth and virulence of vesicular stomatitis virus (VSV) have been described previously. These include gene shuffling, truncation of the cytoplasmic tail of the G protein, and generation of noncytopathic M gene mutants. When separately introduced into recombinant VSV (rVSV), these mutations gave rise to viruses distinguished from their “wild-type” progenitor by diminished reproductive capacity in cell culture and/or reduced cytopathology and decreased pathogenicity in vivo. However, histopathology data from an exploratory nonhuman primate neurovirulence study indicated that some of these attenuated viruses could still cause significant levels of neurological injury. In this study, additional attenuated rVSV variants were generated by combination of the above-named three distinct classes of mutation. The resulting combination mutants were characterized by plaque size and growth kinetics in cell culture, and virulence was assessed by determination of the intracranial (IC) 50% lethal dose (LD50) in mice. Compared to virus having only one type of attenuating mutation, all of the mutation combinations examined gave rise to virus with smaller plaque phenotypes, delayed growth kinetics, and 10- to 500-fold-lower peak titers in cell culture. A similar pattern of attenuation was also observed following IC inoculation of mice, where differences in LD50 of many orders of magnitude between viruses containing one and two types of attenuating mutation were sometimes seen. The results show synergistic rather than cumulative increases in attenuation and demonstrate a new approach to the attenuation of VSV and possibly other viruses.

Neurovirulence and Immunogenicity of Attenuated Recombinant Vesicular Stomatitis Viruses in Nonhuman Primates

ABSTRACT 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.

Simultaneous Quantification of Four RNA Targets by Multiplex, Real-Time RT-PCR without Optimization

Researchers carrying out quantitative, multiplex, realtime PCR and RT-PCR require master mixes that provide accurate, simultaneous quantification of multiple targets in the same reaction without time-consuming optimization. Our results at Wyeth Research show that QuantiTect® Multiplex Kits from QIAGEN are well suited for multiplex analysis of up to four RNA targets in two-step and onestep real-time RT-PCR, requiring no PCR optimization and providing the same efficiency as single-target amplification (singleplex) reactions. Multiplex, real-time PCR refers to the use of multiple fluorescently-labeled oligonucleotide probes for the quantification of amplicons produced by different primer pairs in the same reaction. This method is becoming more common with the introduction of cyclers capable of spectrally resolving different fluorophores (e.g., Applied Biosystems® 7500, Mx3000P®, LightCycler® 480, and iCycler iQ®), and with the development of master mixes that facilitate the quantification of multiple targets in the same reaction tube. Some master mixes intended for singleplex analysis have been adapted for multiplex analysis1, 2, but require time-consuming optimization of primer–probe sets. Therefore, evaluating and selecting a suitable master mix is critical when establishing multiplex assays.