"To date, a number of different viral vectors have been extensively characterized and studied. A significant vaccine trial using human adenovirus-5 failed to result in protection of high-risk individuals from HIV infection . One of the major drawbacks of this system was thought to be the presence of pre-existing antibodies against the viral vector , necessitating the characterization of viral vectors with potential for use in HIV vaccine development. "
[Show abstract][Hide abstract] ABSTRACT: This study analyzed a heterologous prime-boost vaccine approach against HIV-1 using three different antigenically unrelated negative-stranded viruses (NSV) expressing HIV-1 Gag as vaccine vectors: rabies virus (RABV), vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV). We hypothesized that this approach would result in more robust cellular immune responses than those achieved with the use of any of the vaccines alone in a homologous prime-boost regimen. To this end, we primed BALB/c mice with each of the NSV-based vectors. Primed mice were rested for thirty-five days after which we administered a second immunization with the same or heterologous NSV-Gag viruses. The magnitude and quality of the Gag-specific CD8(+) T cells in response to these vectors post boost were measured. In addition, we performed challenge experiments using vaccinia virus expressing HIV-1 Gag (VV-Gag) thirty-three days after the boost inoculation. Our results showed that the choice of the vaccine used for priming was important for the detected Gag-specific CD8(+) T cell recall responses post boost and that NDV-Gag appeared to result in a more robust recall of CD8(+) T cell responses independent of the prime vaccine used. However, the different prime-boost strategies were not distinct for the parameters studied in the challenge experiments using VV-Gag but did indicate some benefits compared to single immunizations. Taken together, our data show that NSV vectors can individually stimulate HIV-Gag specific CD8(+) T cells that are effectively recalled by other NSV vectors in a heterologous prime-boost approach. These results provide evidence that RABV, VSV and NDV can be used in combination to develop vaccines needing prime-boost regimens to stimulate effective immune responses.
PLoS ONE 06/2013; 8(6):e67123. DOI:10.1371/journal.pone.0067123 · 3.23 Impact Factor
"Vaccinees and controls who became infected got similar viral loads, but vaccination was associated with a modestly increased risk of infection [2,23]. Intriguingly, the increased infection risk correlated weakly with pre-existing Ab titers to the adenovirus vector among uncircumcised male vaccinees, while such titers inversely correlated with the risk of infection in the control group . A partly analogous effect was later observed in a macaque experiment: monkeys that were chronically infected with a host-range mutant of adenovirus type 5 (Ad5) and then immunized with a replication-defective Ad5 SIVmac239 Gag-Pol-Nef vaccine showed increased susceptibility to a sub-preputial penile inoculation with low-dose SIVmac251. "
[Show abstract][Hide abstract] ABSTRACT: Correlates of protection (CoPs) against infection by primate lentiviruses remain undefined. Modest protection against HIV-1 was observed in one human vaccine trial, whereas previous trials and vaccine-challenge experiments in non-human primates have yielded inconsistent but intriguing results. Although high levels of neutralizing antibodies are known to protect macaques from mucosal and intravenous viral challenges, antibody or other adaptive immune responses associated with protection might also be mere markers of innate immunity or susceptibility. Specific strategies for augmenting the design of both human trials and animal experiments could help to identify mechanistic correlates of protection and clarify the influences of confounding factors. Robust protection may, however, require the combined actions of immune responses and other host factors, thereby limiting what inferences can be drawn from statistical associations. Here, we discuss how to analyze immune protection against primate lentiviruses, and how host factors could influence both the elicitation and effectiveness of vaccine-induced responses.
"Interest in the field has also been stimulated by the approval of several veterinary gene-based therapeutics and on-going clinical trials of plasmid-based human therapeutics . In addition, a recent Phase 2 trial of an adenoviral-vectored DNA vaccine yielded disappointing results , fueling safety and efficacy concerns surrounding adenoviral vaccines. Finally, recent advances in delivery vehicles and adjuvants for use in concert with naked plasmid DNA have helped increase the efficacy of these therapies . "
[Show abstract][Hide abstract] ABSTRACT: Background:
There has been renewed interest in biopharmaceuticals based on plasmid DNA (pDNA) in recent years due to the approval of several veterinary DNA vaccines, on-going clinical trials of human pDNA-based therapies, and significant advances in adjuvants and delivery vehicles that have helped overcome earlier efficacy deficits. With this interest comes the need for high-yield, cost-effective manufacturing processes. To this end, vector engineering is one promising strategy to improve plasmid production.
In this work, we have constructed a new DNA vaccine vector, pDMB02-GFP, containing the runaway R1 origin of replication. The runaway replication phenotype should result in plasmid copy number amplification after a temperature shift from 30°C to 42°C. However, using Escherichia coli DH5α as a host, we observed that the highest yields of pDMB02-GFP were achieved during constant-temperature culture at 30°C, with a maximum yield of approximately 19 mg pDNA/g DCW being observed. By measuring mRNA and protein levels of the R1 replication initiator protein, RepA, we determined that RepA may be limiting pDMB02-GFP yield at 42°C. A mutant plasmid, pDMB-ATG, was constructed by changing the repA start codon from the sub-optimal GTG to ATG. In cultures of DH5α[pDMB-ATG], temperature-induced plasmid amplification was more dramatic than that observed with pDMB02-GFP, and RepA protein was detectable for several hours longer than in cultures of pDMB02-GFP at 42°C.
Overall, we have demonstrated that R1-based plasmids can produce high yields of high-quality pDNA without the need for a temperature shift, and have laid the groundwork for further investigation of this class of vectors in the context of plasmid DNA production.
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