Abstract We used a replication-incompetent, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the extracellular domain of human cytomegalovirus ...(CMV) glycoprotein B or a pp65/IE1 fusion protein. Efficient production methods were scaled to produce pilot lots and clinical lots of each alphavirus replicon vaccine component. The vaccine induced high-titered antibody responses in mice and rabbits, as measured by ELISA and CMV neutralization assays, and robust T-cell responses in mice, as measured by IFN-γ ELISPOT assay. A toxicity study in rabbits showed no adverse effects in any toxicology parameter. These studies support clinical testing of this novel CMV alphavirus replicon vaccine in humans.
Alphavirus vectors and vaccination Rayner, Jonathan O.; Dryga, Sergey A.; Kamrud, Kurt I.
Reviews in medical virology,
September/October 2002, Letnik:
12, Številka:
5
Journal Article
HIV-1 genetic diversity among circulating strains presents a major challenge for HIV-1 vaccine development, particularly for developing countries where less sequence information is available. To ...identify representative viruses for inclusion in candidate vaccines targeted for South Africa, we applied an efficient sequence survey strategy to samples from recently and chronically infected persons residing in potential vaccine trial sites. All 111 sequences were subtype C, including 30 partial gag, 26 partial pol, 27 V2-V3 env, and 28 V5-partial gp41 sequences. Of the 10 viruses cultured from recently infected individuals, 9 were R5 and 1 was R5X4. Two isolates, Du151 and Du422, collected within 2 months of infection, were selected as vaccine strains on the basis of their amino acid similarity to a derived South African consensus sequence The selection of recently transmitted R5 isolates for vaccine design may provide an advantage in a subtype C R5-dominant epidemic. The full-length Du422 gag and Du151 pol and env genes were cloned into the Venezuelan equine encephalitis (VEE) replicon particle (VRP) expression system. Du422 Gag protein expressed from the VRP accumulated to a high level and was immunogenic as demonstrated by cytotoxic T lymphocyte responses in mice vaccinated with gag-VRPs. Optimization of codon use for VRP expression in human cells did not enhance expression of the gag gene. The cloned Du151 env gene encoded a functional protein as demonstrated by fusion of VRP-infected cells with cells expressing CD4 and CCR5. Genes identified in this study have been incorporated into the VEE VRP candidate vaccines targeted for clinical trial in South Africa.
Alphaviruses are positive-strand RNA viruses that can mediate efficent cytoplasmic gene expression in insect and vertebrate cells. Through recombinant DNA technology, the alphavirus RNA replication ...machinery has been engineered for high-level expression of heterologous RNAs and proteins. Amplification of replication-competent alpha-virus RNAs (replicons) can be initiated by RNA or DNA transfection and a variety of packaging systems have been developed for producing high titers of infectious viral particles. Although normally cytocidal for vertebrate cells, variants with adaptive mutations allowing noncytopathic replication have been isolated from persistently infected cultures or selected using a dominant selectable marker. Such mutations have been mapped and used to create new alphavirus vectors for noncytopathic gene expression in mammalian cells. These vectors allow long-term expression at moderate levels and complement previous vectors designed for short-term highlevel expression. Besides their use for a growing number of basic research applications, recombinant alphavirus RNA replicons may also facilitate genetic vaccination and transient gene therapy.
Replicon particles based on Venezuelan equine encephalitis virus (VEE) contain a self‐replicating RNA encoding the VEE replicase proteins and expressing a gene of interest in place of the viral ...structural protein genes. Structural proteins for packaging of replicon RNA into VEE replicon particles (VRPs) are expressed from separate helper RNAs. Aspects of the biology of VEE that are exploited in VRP vaccines include 1) expression of very high levels of immunogen, 2) expression of immunizing proteins in cells in the draining lymph node, and 3) the ability to induce mucosal immunity from a parental inoculation. Results of experiments with VRPs expressing green fluorescent protein or influenza virus hemagglutinin (HA) demonstrated that specific mutations in the VRP envelope glycoproteins affect both targeting in the draining lymph node and efficiency of the immune response in mice. VRPs expressing either the matrix‐capsid portion of Gag, the full‐length envelope gp160, or the secreted gp140 of cloned SIVsm H‐4i were mixed in a cocktail and used to immunize macaques at 0, 1, and 4 months. Neutralizing antibodies against SIVsm H‐4 were induced in 6 of 6 vaccinates and CTL in 4 of 6. An intrarectal challenge with the highly pathogenic SIVsm E660 was given at 5 months. A vaccine effect was seen in reduced peak virus loads, reduced virus loads both at set point and at 41 weeks postchallenge, and preserved or increased CD4 counts compared to controls. A candidate VRP HIV vaccine expressing Clade C Gag contains a sequence that is very close to the South African Clade C consensus and was selected from a recent seroconverter in the Durban cohort to represent currently circulating genotypes in South Africa. A GMP lot of this vaccine has been manufactured and tested for a phase I trial in the first months of 2002.
Sindbis virus is a positive strand RNA virus that has provided a valuable model for studying virus structure and replication. It is also being developed as a vector for the expression of heterologous ...proteins. Many studies with this virus are carried out in cultured BHK cells where infection is usually highly cytopathic and within 1 or 2 days after infection all of the cells are dead. Weisset al.had established a persistently infected culture of BHK cells by infecting the cells with a virus preparation highly enriched in defective interfering (DI) particles and had isolated an attenuated virus, SIN-1 virus, from the culture Weisset al.(1980)J. Virol.33, 463–474. SIN-1 virus, free of DI particles, was able to establish a persistent infection in BHK cells. We initiated studies to determine what changes in the genome of the virus were responsible for this phenotype. We describe here the cDNA cloning and sequencing of the 5′ terminus and the four nonstructural protein genes from SIN-1 virus. A single coding mutation in the nsP2 gene (a predicted change of Pro-726 → Ser) produced a virus that was able to establish persistent infection in BHK cells. Additional mutations in the other genes were required to decrease the synthesis of viral RNA to a level similar to that found in cells infected with SIN-1 virus. Incorporation of the nsP2 mutation into a Sindbis virus expression vector led to a higher level of synthesis of the reporter protein, β-galactosidase, than that obtained with the original Sindbis virus replicon.
Abstract
ELISpot assays measure antigen specific cell mediated immunity, and are useful indicators of vaccine and therapeutic efficacy. Methods that yield consistent and reproducible results are ...required to compare data in longitudinal studies or among laboratories. Here, we describe the performance of a validated ELISpot assay using PBMC controls and standardized antigenic stimulants among laboratories participating in an External Quality Assurance (EQA) Program. Responses from four PBMC controls (high, medium, low and negative responders) were compared in an IFN-γ ELISpot assay using SeraCare (SC) kits and individual laboratory specific assay protocol and kits in an ELISpot EQA. SC kit performance was also compared to competitor kits in previous rounds of an EQA, each with 13 participants. SC kits showed high sensitivity in detection of low responders (Spot forming cells SFC range 10.9 - 49.2/well) compared with in house kit results (SFC range 0.4 - 67.2). Competitor kits yielded larger numbers of Too Numerous To Count results with high responders and greater variability in comparison to SC kits. SC kits yielded a broad linear dose response curve (10-1000 SFC/well) as a result of smaller spot size and improved resolution. SC kits and standardized assay protocols used by multiple laboratories allowed improved sensitivity of detection of low level antigenic responses, higher consistency and a broad linear range.
Abstract only
INTRODUCTION: Cellular immune response is often evaluated on frozen Peripheral Blood Mononuclear Cells (PBMC). Here, we evaluate the effect of shipping and storage conditions on PBMC ...viability and functional response using ELISpot assay.
METHODS: Cryopreserved human PBMC were evaluated after overnight placement on dry ice and subsequent storage at −80
o
C, −20°C, in LN
2
and on dry ice for up to 6 months. Cell count and viability was measured at time of thaw and after 24 hour rest. ELISpot assays were performed using huIFN‐γ ELISpot Kit (SeraCare) after stimulation with PHA, CMV and CEF peptide pools.
RESULTS: Cells stored overnight on dry ice showed no significant decrease in viability at thaw and post rest. Cells stored at −20°C were not suitable for ELISpot assay. Cells stored on dry ice, at −80°C or in LN
2
for 6 month demonstrated 92–93% viability upon thaw and 80–86% after 24 hr rest. The control cells remained 90% viable both post thaw and post rest. ELISpot responses of cells stored at −80°C or in LN
2
for 6 month, or cells stored on dry ice for 4 weeks were comparable to control.
CONCLUSIONS: The PBMC shipped in dry ice can be stored at −80°C for up to 6 months or on dry ice for 4 weeks with no appreciable loss of viability or functionality. Therefore, the use of PBMC for ELISpot proficiency testing is possible even in laboratories which lack ultra‐cold storage facilities.
SUPPORT: This work was supported by NIAID contract NO1‐AI‐85341.
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