Development of a high-throughput RT-PCR based viral infectivity assay for monitoring the stability of a replicating recombinant Lymphocytic Choriomeningitis viral vector.

Traditional virus infectivity titration methods for lymphocytic choriomeningitis virus (LCMV) are laborious, time-consuming, and low-throughput (e.g., focus forming unit (FFA) assay). In this report, we developed a high-throughput reverse transcription quantitative PCR (RT-qPCR)-based virus infectivity assay for relative quantitation of a live, recombinant replicating LCMV -based viral vector (TT1). This in vitro infectivity assay demonstrated a 4-log linear range for TT1 titer quantitation. A high positive Pearson correlation coefficient value (≥ 0.80) was obtained between the RT-qPCR vs. the "gold-standard" FFU assay when comparing the stability profiles of stressed TT1 vector samples. In addition to the RT-qPCR infectivity assay, the stability of the TT1 vector upon freeze-thaw stress was investigated further with complementary viral particle characterization techniques (e.g., TEM, NTA, MFI). Correlations between viral infectivity and particle measurements during forced degradation studies were observed to be specific to the TT1 vector and its various formulations and such results facilitated the rank-ordering of formulation conditions. Overall, this infectivity RT-qPCR method showed increased sample throughput and improved assay flexibility compared to traditional viral infectivity assays. These results are discussed in the context of enabling future TT1 vector formulation development work, and potential utilization as an in-process monitoring tool during TT1 vector manufacturing.

Development of novel replication-defective lymphocytic choriomeningitis virus vectors expressing SIV antigens.

An important focus in vaccine research is the design of vaccine vectors with low seroprevalence and high immunogenicity. Replication-incompetent lymphocytic choriomeningitis virus (rLCMV) vectors do not elicit vector-neutralizing antibody responses, and homologous prime-boost regimens with rLCMV vectors induce boostable and protective T cell responses to model antigens in mice. However, cellular and humoral immune responses following homologous rLCMV vaccine regimens have not been rigorously evaluated in non-human primates (NHPs). To test whether rLCMV vectors constitute an effective vaccine platform in NHPs, we developed rLCMV vectors expressing SIVmac239 Env and Gag antigens and assessed their immunogenicity in mice and cynomolgus macaques. Immunization with rLCMV vaccine vectors expressing SIV Env and Gag was effective at generating SIV-specific T cell and antibody responses in both mice and NHPs. Epitope mapping using SIV Env in C57BL/6 mice demonstrated that rLCMV vectors induced sustained poly-functional responses to both dominant and subdominant epitopes. Our results suggest the potential of rLCMV vectors as vaccine candidates. Future SIV challenge experiments in rhesus macaques will be needed to assess immune protection by these vaccine vectors.

Replication-defective lymphocytic choriomeningitis virus vectors expressing guinea pig cytomegalovirus gB and pp65 homologs are protective against congenital guinea pig cytomegalovirus infection.

BACKGROUND: Congenital cytomegalovirus infection can be life-threatening and often results in significant developmental deficits and/or hearing loss. Thus, there is a critical need for an effective anti-CMV vaccine. OBJECTIVE: To determine the efficacy of replication-defective lymphocytic choriomeningitis virus (rLCMV) vectors expressing the guinea pig CMV (GPCMV) antigens, gB and pp65, in the guinea pig model of congenital CMV infection. METHODS: Female Hartley strain guinea pigs were divided into three groups: Buffer control group (n = 9), rLCMV-gB group (n = 11), and rLCMV-pp65 (n = 11). The vaccines were administered three times IM at 1.54 × 10(6)FFU per dose at 21-day intervals. At two weeks after vaccination, the female guinea pigs underwent breeding. Pregnant guinea pigs were challenged SQ at ∼ 45-55 days of gestation with 1 × 10(5)PFU of GPCMV. Viremia in the dams, pup survival, weights of pups at delivery, and viral load in both dam and pup tissues were determined. RESULTS: Pup survival was significantly increased in the LCMV-gB vaccine group. There was 23% pup mortality in the gB vaccine group (p = 0.044) and 26% pup mortality in the pp65 vaccine group (p = 0.054) compared to 49% control pup mortality. The gB vaccine induced high levels of gB binding and detectable neutralizing antibodies, reduced dam viremia, and significantly reduced viral load in dam tissues compared to control dams (p < 0.03). Reduced viral load and transmission in pups born to gB-vaccinated dams was observed compared to pups from pp65-vaccinated or control dams. CONCLUSIONS: The rLCMV-gB vaccine significantly improved pup survival and also increased pup weights and gestation time. The gB vaccine was also more effective at decreasing viral load in dams and pups and limiting congenital transmission. Thus, rLCMV vectors that express CMV antigens may be an effective vaccine strategy for congenital CMV infection.

The ability of E1A to rescue ras-induced premature senescence and confer transformation relies on inactivation of both p300/CBP and Rb family proteins.

In primary cells, oncogenic ras induces a stable growth arrest known as premature senescence. Ras-induced premature senescence is considered as a tumor-suppressing defense response that needs to be bypassed before oncogenic potential ras can be revealed. To gain insights into the mechanism of senescence bypass during oncogenic transformation, we dissected the activities of an adenoviral oncoprotein E1A, which is capable of overcoming ras-induced senescence. Our results have indicated that the senescence bypassing activity resides in the NH2 terminus and requires both Rb-binding and p300/CBP-binding functions of E1A. Although interference with the p16(INK4A)/Rb pathway or inactivation of p300/CBP alone did not lead to senescence bypass, these two types of genetic alterations complemented the Rb-binding defective and the p300/CBP-binding defective mutants of E1A, respectively, to rescue premature senescence. Therefore, genetic alterations disrupting the p16(INK4A)/Rb pathway or the p300/CBP functions both contribute to the bypass of senescence. We further showed that p300/CBP were essential for ras-induced p53 activity, providing a potential mechanism underlying the important role of p300/CBP in senescence. Furthermore, p300/CBP inactivation led to cellular transformation in cooperation with the p300/CBP-binding defective E1A mutants, MDM2 and Ha-RasV12. These results have shown that p300 and CBP are integral components of the pathway that mediates ras-induced senescence. The critical role of p300 and CBP in the senescence response that limits the oncogenic potential of ras has provided a mechanistic basis for the tumor-suppressing function of these proteins.

Transferrin ensures survival of ovarian carcinoma cells when apoptosis is induced by TNFalpha, FasL, TRAIL, or Myc.

The activation of Myc induces apoptosis of human ovarian adenocarcinoma N.1 cells when serum factors are limited. However, the downstream mechanism that is triggered by Myc is unknown. Myc-activation and treatment with the proapoptotic ligands TNFalpha, FasL, and TRAIL induced H-ferritin expression under serum-deprived conditions. H-ferritin chelates intracellular iron and also intracellular iron sequestration by deferoxamine-induced apoptosis of N.1 cells. Supplementation of serum-free medium with holo-transferrin blocked apoptosis of N.1 cells that was induced by Myc-activation or by treatment with TNFalpha, FasL, and TRAIL, whereas apotransferrin did not prevent apoptosis. This suggests that intracellular iron depletion was a trigger for apoptosis and that transferrin-bound iron rescued N.1 cells. Furthermore, apoptosis of primary human ovarian carcinoma cells, which was induced by TNFalpha, FasL, and TRAIL, was also inhibited by holo-transferrin. The data suggest that Myc-activation, FasL, TNFalpha, and TRAIL disturbed cellular iron homeostasis, which triggered apoptosis of ovarian carcinoma cells and that transferrin iron ensured survival by re-establishing this homeostasis.