Limited Replication Research Articles

Cross-scale variation in top-down and bottom-up control of algal abundance

Previous studies of top-down and bottom-up effects in Oregon rocky intertidal communities suggested that, unlike sea star–mussel dominated food chains, grazer-alga food chains deviated from simple food web predictions. However, this conclusion lacked generality due to limited replication at the site level. We explored how grazing and algal colonization and growth in different zones and multiple sites in intertidal communities varied in relation to processes varying on local to mesoscales, including oceanographic variation. Our approach employed the comparative-experimental method, in which identically-designed and replicated experiments are performed at multiple sites spanning the environmental gradient of interest. In a limpet–algae–barnacle interaction web, we tested the hypothesis that top-down and bottom-up effects were inversely related along the Oregon coast. To distinguish between the alternatives of large-scale oceanographic forcing vs. local control, our experiments spanned scales of meters (between mid and low zones), kilometers (seven sites), 10's to 100's of km (three regions), and ∼ 260 km (central and southern coasts). Identically designed and executed experiments on effects and rates of grazing were conducted in a southern coast region of persistent upwelling and two central coast regions, one with intermittent upwelling in an area of weak offshore currents, and another with intermittent upwelling in an area of strong offshore currents. Grazer effects and grazing rates varied on all scales, indicating a complex interplay of processes operating at zone, site, regional, and coastal scales. Grazing was generally highest in all mid zones and in the low zone at the middle region (Cape Perpetua) on the central coast. Growth of early successional colonists tended to be higher at the northern and southern regions (Capes Foulweather and Blanco). Grazers had no effect on barnacle cover, but their foraging was inhibited by high densities of barnacles. Differences in grazing appear to be a complex consequence of direct and indirect effects of variable oceanographic conditions, limpet recruitment, and barnacle abundance. In contrast to positive associations between predation strength and the magnitude of prey subsidies, higher grazing strength was generally associated with lower magnitude of bottom-up effects and vice versa. Hence in this system predator–prey (sea star–filter feeder) and herbivore–plant (limpet–microalgae) interactions evidently respond differently to ecological subsidies.

A full Bayesian hierarchical mixture model for the variance of gene differential expression.

BackgroundIn many laboratory-based high throughput microarray experiments, there are very few replicates of gene expression levels. Thus, estimates of gene variances are inaccurate. Visual inspection of graphical summaries of these data usually reveals that heteroscedasticity is present, and the standard approach to address this is to take a log2 transformation. In such circumstances, it is then common to assume that gene variability is constant when an analysis of these data is undertaken. However, this is perhaps too stringent an assumption. More careful inspection reveals that the simple log2 transformation does not remove the problem of heteroscedasticity. An alternative strategy is to assume independent gene-specific variances; although again this is problematic as variance estimates based on few replications are highly unstable. More meaningful and reliable comparisons of gene expression might be achieved, for different conditions or different tissue samples, where the test statistics are based on accurate estimates of gene variability; a crucial step in the identification of differentially expressed genes.ResultsWe propose a Bayesian mixture model, which classifies genes according to similarity in their variance. The result is that genes in the same latent class share the similar variance, estimated from a larger number of replicates than purely those per gene, i.e. the total of all replicates of all genes in the same latent class. An example dataset, consisting of 9216 genes with four replicates per condition, resulted in four latent classes based on their similarity of the variance.ConclusionThe mixture variance model provides a realistic and flexible estimate for the variance of gene expression data under limited replicates. We believe that in using the latent class variances, estimated from a larger number of genes in each derived latent group, the p-values obtained are more robust than either using a constant gene or gene-specific variance estimate.

A less restrictive technique for the estimation of understory light under variable weather conditions

Many techniques have been developed to estimate mean available light in forest understories. The standard methods estimate average seasonal PAR by recording it in situ with quantum sensors or through simulation using hemispherical image analysis (HIA). Other techniques have been developed to overcome some of the drawbacks of the standard methods, such as high cost, cumbersome equipment, user bias, and limited replication. The “Overcast” method allows rapid evaluation of average seasonal PAR in most situations by taking instantaneous measurements, but is restricted by the overcast sky conditions required. The present study proposes a new, less restrictive method (BF2), that uses a sensor array under a hemispherical dome to overcome the sky condition restrictions of the Overcast method, and tests it against the Overcast and HIA techniques. The study also evaluates the method's ability to predict seedling growth and morphology. The BF2 method provided rapid and precise estimations of %PAR under variable sky conditions, as well as good estimations of seedling's responses, and is suitable for multiple site or highly replicated designs. The methods were used under deciduous stands with contrasting structures.

Early restriction of alphavirus replication and dissemination contributes to age-dependent attenuation of systemic hyperinflammatory disease

Severity of alphavirus infection in humans tends to be strongly age-dependent and several studies using laboratory-adapted Sindbis virus (SB) AR339 strains have indicated that SB-induced disease in mice is similarly contingent upon host developmental status. In the current studies, the consensus wild-type SB, TR339, and in vivo imaging technology have been utilized to examine virus replication and disease manifestations in mice infected subcutaneously at 5 days of age (5D) vs 11D. Initial virulence studies with TR339 indicated that this age range is coincident with rapid transition from fatal to non-fatal outcome. Fatal infection of 5D mice is characterized by high-titre serum viraemia, extensive virus replication in skin, fibroblast connective tissue, muscle and brain, and hyperinflammatory cytokine induction. In contrast, 11D-infected mice experience more limited virus replication and tissue damage and develop mild, immune-mediated pathologies including encephalitis. These results further establish the linkage between hyperinflammatory cytokine induction and fatal outcome of infection. In vivo imaging using luciferase-expressing viruses and non-propagative replicons revealed that host development results in a restriction of virus replication within individual infected cells that is manifested as a delay in reduction of virus replication in the younger mice. Thus, an important contributing factor in age-dependent resistance to alphavirus infection is restriction of replication within first infected cells in peripheral tissues, which may augment other developmentally regulated attenuating effects, such as increasing neuronal resistance to virus infection and apoptotic death.

Meta-analysis of genome-wide scans provides evidence for sex- and site-specific regulation of bone mass.

Several genome-wide scans have been performed to detect loci that regulate BMD, but these have yielded inconsistent results, with limited replication of linkage peaks in different studies. In an effort to improve statistical power for detection of these loci, we performed a meta-analysis of genome-wide scans in which spine or hip BMD were studied. Evidence was gained to suggest that several chromosomal loci regulate BMD in a site-specific and sex-specific manner. BMD is a heritable trait and an important predictor of osteoporotic fracture risk. Several genome-wide scans have been performed in an attempt to detect loci that regulate BMD, but there has been limited replication of linkage peaks between studies. In an attempt to resolve these inconsistencies, we conducted a collaborative meta-analysis of genome-wide linkage scans in which femoral neck BMD (FN-BMD) or lumbar spine BMD (LS-BMD) had been studied. Data were accumulated from nine genome-wide scans involving 11,842 subjects. Data were analyzed separately for LS-BMD and FN-BMD and by sex. For each study, genomic bins of 30 cM were defined and ranked according to the maximum LOD score they contained. While various densitometers were used in different studies, the ranking approach that we used means that the results are not confounded by the fact that different measurement devices were used. Significance for high average rank and heterogeneity was obtained through Monte Carlo testing. For LS-BMD, the quantitative trait locus (QTL) with greatest significance was on chromosome 1p13.3-q23.3 (p = 0.004), but this exhibited high heterogeneity and the effect was specific for women. Other significant LS-BMD QTLs were on chromosomes 12q24.31-qter, 3p25.3-p22.1, 11p12-q13.3, and 1q32-q42.3, including one on 18p11-q12.3 that had not been detected by individual studies. For FN-BMD, the strongest QTL was on chromosome 9q31.1-q33.3 (p = 0.002). Other significant QTLs were identified on chromosomes 17p12-q21.33, 14q13.1-q24.1, 9q21.32-q31.1, and 5q14.3-q23.2. There was no correlation in average ranks of bins between men and women and the loci that regulated BMD in men and women and at different sites were largely distinct. This large-scale meta-analysis provided evidence for replication of several QTLs identified in previous studies and also identified a QTL on chromosome 18p11-q12.3, which had not been detected by individual studies. However, despite the large sample size, none of the individual loci identified reached genome-wide significance.

Restricted replication and lysosomal trafficking of yellow fever 17D vaccine virus in human dendritic cells

The yellow fever virus attenuated 17D vaccine strain is a safe and effective vaccine and a valuable model system for evaluating immune responses against attenuated viral variants. This study compared the in vitro interactions of the commercially available yellow fever vaccine (YF-VAX), Dengue virus and the live-attenuated dengue vaccine PDK50 with dendritic cells (DCs), the main antigen-presenting cells at the initiation of immune responses. Similar to PDK50, infection with YF-VAX generated activated DCs; however, for YF-VAX, activation occurred with limited intracellular virus replication. The majority of internalized virus co-localized with endolysosomal markers within 90 min, suggesting that YF-VAX is processed rapidly in DCs. These results indicate that restricted virus replication and lysosomal compartmentalization may be important contributing factors to the success of the YF-VAX vaccine.

Characterization of the protective T‐cell response generated in CD4‐deficient mice by a live attenuated Mycobacterium tuberculosis vaccine

The global epidemic of tuberculosis, fuelled by acquired immune-deficiency syndrome, necessitates the development of a safe and effective vaccine. We have constructed a DeltaRD1DeltapanCD mutant of Mycobacterium tuberculosis (mc(2)6030) that undergoes limited replication and is severely attenuated in immunocompromised mice, yet induces significant protection against tuberculosis in wild-type mice and even in mice that completely lack CD4(+) T cells as a result of targeted disruption of their CD4 genes (CD4(-/-) mice). Ex vivo studies of T cells from mc(2)6030-immunized mice showed that these immune cells responded to protein antigens of M. tuberculosis in a major histocompatibility complex (MHC) class II-restricted manner. Antibody depletion experiments showed that antituberculosis protective responses in the lung were not diminished by removal of CD8(+), T-cell receptor gammadelta (TCR-gammadelta(+)) and NK1.1(+) T cells from vaccinated CD4(-/-) mice before challenge, implying that the observed recall and immune effector functions resulting from vaccination of CD4(-/-) mice with mc(2)6030 were attributable to a population of CD4(-) CD8(-) (double-negative) TCR-alphabeta(+), TCR-gammadelta(-), NK1.1(-) T cells. Transfer of highly enriched double-negative TCR-alphabeta(+) T cells from mc(2)6030-immunized CD4(-/-) mice into naive CD4(-/-) mice resulted in significant protection against an aerosol tuberculosis challenge. Enriched pulmonary double-negative T cells transcribed significantly more interferon-gamma and interleukin-2 mRNA than double-negative T cells from naive mice after a tuberculous challenge. These results confirmed previous findings on the potential for a subset of MHC class II-restricted T cells to develop and function without expression of CD4 and suggest novel vaccination strategies to assist in the control of tuberculosis in human immunodeficiency virus-infected humans who have chronic depletion of their CD4(+) T cells.

Caliciviruses Differ in Their Functional Requirements for eIF4F Components

Two classes of viruses, namely members of the Potyviridae and Caliciviridae, use a novel mechanism for the initiation of protein synthesis that involves the interaction of translation initiation factors with a viral protein covalently linked to the viral RNA, known as VPg. The calicivirus VPg proteins can interact directly with the initiation factors eIF4E and eIF3. Translation initiation on feline calicivirus (FCV) RNA requires eIF4E because it is inhibited by recombinant 4E-BP1. However, to date, there have been no functional studies carried out with respect to norovirus translation initiation, because of a lack of a suitable source of VPg-linked viral RNA. We have now used the recently identified murine norovirus (MNV) as a model system for norovirus translation and have extended our previous studies with FCV RNA to examine the role of the other eIF4F components in translation initiation. We now demonstrate that, as with FCV, MNV VPg interacts directly with eIF4E, although, unlike FCV RNA, translation of MNV RNA is not sensitive to 4E-BP1, eIF4E depletion, or foot-and-mouth disease virus Lb protease-mediated cleavage of eIF4G. We also demonstrate that both FCV and MNV RNA translation require the RNA helicase component of the eIF4F complex, namely eIF4A, because translation was sensitive (albeit to different degrees) to a dominant negative form and to a small molecule inhibitor of eIF4A (hippuristanol). These results suggest that calicivirus RNAs differ with respect to their requirements for the components of the eIF4F translation initiation complex.

CIMMYT-selected derived synthetic bread wheats for rainfed environments: Yield evaluation in Mexico and Australia

Synthetic backcrossed-derived bread wheats (SBWs) from CIMMYT were grown in the Northwest of Mexico at Centro de Investigaciones Agrícolas del Noroeste (CIANO) and sites across Australia during three seasons. During three consecutive years Australia received “shipments” of different SBWs from CIMMYT for evaluation. A different set of lines was evaluated each season, as new materials became available from the CIMMYT crop enhancement program. These consisted of approximately 100 advanced lines (F7) per year. SBWs had been top and backcrossed to CIMMYT cultivars in the first two shipments and to Australian wheat cultivars in the third one. At CIANO, the SBWs were trialled under receding soil moisture conditions. We evaluated both the performance of each line across all environments and the genotype-by-environment interaction using an analysis that fits a multiplicative mixed model, adjusted for spatial field trends. Data were organised in three groups of multienvironment trials (MET) containing germplasm from shipment 1 (METShip1), 2 (METShip2), and 3 (METShip3), respectively. Large components of variance for the genotype × environment interaction were found for each MET analysis, due to the diversity of environments included and the limited replication over years (only in METShip2, lines were tested over 2 years). The average percentage of genetic variance explained by the factor analytic models with two factors was 50.3% for METShip1, 46.7% for METShip2, and 48.7% for METShip3. Yield comparison focused only on lines that were present in all locations within a METShip, or “core” SBWs. A number of core SBWs, crossed to both Australian and CIMMYT backgrounds, outperformed the local benchmark checks at sites from the northern end of the Australian wheat belt, with reduced success at more southern locations. In general, lines that succeeded in the north were different from those in the south. The moderate positive genetic correlation between CIANO and locations in the northern wheat growing region likely reflects similarities in average temperature during flowering, high evaporative demand, and a short flowering interval. We are currently studying attributes of this germplasm that may contribute to adaptation, with the aim of improving the selection process in both Mexico and Australia.

Resveratrol inhibition of varicella-zoster virus replication in vitro

Resveratrol was found to inhibit varicella-zoster virus (VZV) replication in a dose-dependent and reversible manner. This decrease in virus production in the presence of resveratrol was not caused by direct inactivation of VZV or inhibition of virus attachment to MRC-5 cells. The drug effectively limited VZV replication if added during the first 30 h of infection. Western blot analysis and real-time RT-PCR studies demonstrated that protein and mRNA levels of IE62, an essential immediate early viral protein, were reduced when compared to controls. These results demonstrate that VZV replication is adversely affected by resveratrol which is negatively impacting IE62 synthesis.

Mycobacterium tuberculosis Δ RD1 Δ panCD: A safe and limited replicating mutant strain that protects immunocompetent and immunocompromised mice against experimental tuberculosis

The global epidemic of tuberculosis (TB), fueled by the growing HIV pandemic, warrants the development of a safe and effective vaccine against TB. We report the construction and characterization of an unlinked double deletion mutant of Mycobacterium tuberculosis H37Rv that deletes both the primary attenuating mutation of BCG (Δ RD1) and two genes required for the synthesis of pantothenate (Δ panCD). The M. tuberculosis Δ RD1 Δ panCD (mc 26030) mutant undergoes limited replication in mice, and yet is both significantly safer than BCG in immunocompromised mice and also safe in guinea pigs. Additionally, the mc 26030 strain does not reactivate in a mouse chemo-immunosuppression model. Importantly, long-lived protective immune responses following immunization with the mc 26030 strain prolong the survival of wild type mice, and CD4-deficient mice against an aerosol challenge with virulent M. tuberculosis. Given its overall safety and effectiveness, the mc 26030 live attenuated strain should be considered as a human vaccine candidate for protecting both healthy and HIV-infected individuals against TB.

Comparative evaluation of two severe acute respiratory syndrome (SARS) vaccine candidates in mice challenged with SARS coronavirus

Two different severe acute respiratory syndrome (SARS) vaccine strategies were evaluated for their ability to protect against live SARS coronavirus (CoV) challenge in a murine model of infection. A whole killed (inactivated by beta-propiolactone) SARS-CoV vaccine and a combination of two adenovirus-based vectors, one expressing the nucleocapsid (N) and the other expressing the spike (S) protein (collectively designated Ad S/N), were evaluated for the induction of serum neutralizing antibodies and cellular immune responses and their ability to protect against pulmonary SARS-CoV replication. The whole killed virus (WKV) vaccine given subcutaneously to 129S6/SvEv mice was more effective than the Ad S/N vaccine administered either intranasally or intramuscularly in inhibiting SARS-CoV replication in the murine respiratory tract. This protective ability of the WKV vaccine correlated with the induction of high serum neutralizing-antibody titres, but not with cellular immune responses as measured by gamma interferon secretion by mouse splenocytes. Titres of serum neutralizing antibodies induced by the Ad S/N vaccine administered intranasally or intramuscularly were significantly lower than those induced by the WKV vaccine. However, Ad S/N administered intranasally, but not intramuscularly, significantly limited SARS-CoV replication in the lungs. Among the vaccine groups, SARS-CoV-specific IgA was found only in the sera of mice immunized intranasally with Ad S/N, suggesting that mucosal immunity may play a role in protection for the intranasal Ad S/N delivery system. Finally, the sera of vaccinated mice contained antibodies to S, further suggesting a role for this protein in conferring protective immunity against SARS-CoV infection.

OII-A-3PK/PD in silico and in vitro simulations of linezolid (LZD) activity against methicillin resistant staphylococcus aureus (MRSA)

BACKGROUND To predict the activity of various LZD dosage regimens for MRSA. The MIC90 of LZD against MRSA is ∼4 mg/L. METHODS For 2 clinical MRSA isolates (MIC2, MIC4), 24h kill curve experiments were fit by a PD model: capacity limited replication inhibited by LZD via a Hill function, & 1st order elimination. A mixture model described both strains as 2 populations: a predominant ‘sensitive’ population (EC50 = 0.4 & 0.6xMIC) & a ‘resistant’ subpopulation (EC50 of 3 & 5xMIC). LZD PK parameters derived from our previous population PK analysis were used to predict PD responses to 4 LZD regimens: 600mg PO q12h w/o (BID) or with (LBID) a loading dose (LD) of 900mg; 600mg PO q8h w/o (TID) or with (LTID) a LD of 1200mg. For verification, these regimens were studied against these MRSA isolates in an in vitro PK/PD infection model, over 96h. RESULTS All 4 regimens were effective against MIC2 (LBID≈ BID). In silico & in vitro, BID & LBID were ineffective, while TID & LTID were effective (with similar activity) against MIC4. For eg, in vitro at 48h, the log10(bacterial CFU) were decreased by 3.6 & 3.2, for MIC2, & by 1 & 2.6, for MIC4, with BID & TID regimens. CONCLUSIONS The BID regimen might not provide adequate activity at MIC ≥ 4 mg/L (though these studies did not include any effect due to the immune system). At MIC=4, a TID regimen may offer a substantial benefit over BID. With its ‘slow’ rate of kill & its shallow concentration-effect curve, LZD appears to show only modest benefit for the evaluated loading doses. Clinical Pharmacology & Therapeutics (2005) 79, P4–P4; doi: 10.1016/j.clpt.2005.12.012

Employment of liver tissue slice analysis to assay hepatotoxicity linked to replicative and nonreplicative adenoviral agents

Whereas virotherapy has emerged as a novel and promising approach for neoplastic diseases, appropriate model systems have hampered preclinical evaluation of candidate conditionally replicative adenovirus agents (CRAds) with respect to liver toxicity. This is due to the inability of human viral agents to cross species. We have recently shown the human liver tissue slice model to be a facile means to validate adenoviral replication. On this basis, we sought to determine whether our ex vivo liver tissue slice model could be used to assess CRAd-mediated liver toxicity. We analyzed and compared the toxicity of a conditionally replicative adenovirus (AdDelta24) to that of a replication incompetent adenovirus (Adnull [E1-]) in mouse and human liver tissue slices. To accomplish this, we examined the hepatic apoptosis expression profile by DNA microarray analyses, and compared these results to extracellular release of aminotransferase enzymes, along with direct evidence of apoptosis by caspase-3 immunhistochemical staining and TUNEL assays. Human and mouse liver tissue slices demonstrated a marked increase in extracellular release of aminotransferase enzymes on infection with AdDelta24 compared to Adnull. AdDelta24-mediated liver toxicity was further demonstrated by apoptosis induction, as detected by caspase-3 immunohistochemical staining, TUNEL assay and microarray analysis. In conclusion, concordance of CRAd-mediated apoptosis in both the human and the mouse liver tissue slice models was demonstrated, despite the limited replication ability of CRAds in mouse liver slices. The results of this study, defining the CRAd-mediated apoptosis gene expression profiles in human and mouse liver, may lay a foundation for preclinical liver toxicity analysis of CRAd agents.

132. Increased GFP Gene Delivery to Human Prostate Cancer Cells Using Trancomplementing Adenoviral Vectors

Adenoviral vectors are commonly used in cancer research for transgene delivery and expression because of their rapid and significant transgene expression as well as facile production of high viral titers. Unfortunately, relatively inefficient in vivo transgene expression is a characteristic of first generation recombinant adenoviral vectors. Balancing between safety and effectiveness of transgene delivery, many approaches have been used including development of conditionally or limited replicating vectors and utilization of complementing adenoviruses. In this study we exploited two replication defective adenoviral vectors that are able to transcomplement each other to yield limited replication and greater gene delivery.

CD4+ immune escape and subsequent T-cell failure following chimpanzee immunization against hepatitis C virus

Hepatitis C is a major cause of chronic liver disease, with 170 million individuals infected worldwide and no available vaccine. We analyzed the effects of an induced T-cell response in 3 chimpanzees, targeting nonstructural proteins in the absence of neutralizing antibodies. In all animals the specific T-cell response modified the outcome of infection, producing a 10- to 1,000-fold reduction in peak virus titers. The challenge of 2 immunized animals that had been previously exposed to hepatitis C virus resulted in subclinical infections. Immune responses in the third animal, naive prior to immunization, limited viral replication immediately, evidenced by a 30-fold reduction in virus titer by week 2, declining to a nonquantifiable level by week 6. After 10 weeks of immunological control, we observed a resurgence of virus, followed by progression to a persistent infection. Comparing virus evolution with T-cell recognition, we demonstrated that: (i) resurgence was concomitant with the emergence of new dominant viral populations bearing single amino acid changes in the NS3 and NS5A regions, (ii) these mutations resulted in a loss of CD4+ T-cell recognition, and (iii) subsequent to viral resurgence and immune escape a large fraction of NS3-specific T cells became impaired in their ability to secrete IFN-gamma and proliferate. In contrast, NS3-specific responses were sustained in the recovered/immunized animals presenting with subclinical infections. In conclusion, viral escape from CD4+ T cells can result in the eventual failure of an induced T-cell response that initially controls infection. Vaccines that can induce strong T-cell responses prior to challenge will not necessarily prevent persistent HCV infection.

Improving recombinant MVA immune responses: Potentiation of the immune responses to HIV-1 with MVA and DNA vectors expressing Env and the cytokines IL-12 and IFN-gamma

Recombinants based on vaccinia virus vectors, especially on the highly attenuated modified vaccinia virus Ankara (MVA) strain, are now being tested in clinical trials for safety and immunogenicity, using prime/boost heterologous regimes of vaccination. Due to the limited replication capacity of MVA, it is necessary to develop procedures that can enhance the specific cellular immune responses to the recombinant antigen delivered by the MVA vector. In this investigation, we have characterized the systemic immune responses in BALB/c mice using interferon-gamma (IFN-γ) or interleukin-12 (IL-12) in an adjuvant-like manner elicited by MVA recombinants or naked DNA vectors expressing one of those cytokines in combination with the human immunodeficiency virus type 1 (HIV-1) envelope (Env) as antigen. In infected mice, virus gene expression in splenocytes and levels of cytokines IFN-γ and IL-12 in serum were maximal by 6 h post-infection (hpi) with MVA recombinants expressing IFN-γ (MVAIFN-γ) or IL-12 (MVAIL-12). In the infected animals, co-expression of HIV-1 env (MVAENV) and either IFN-γ or IL-12 from MVA recombinants produced a two and three-fold increase of anti-env CD8+ T cell response, respectively. When priming was carried out with DNA vectors expressing HIV-1 env and either IFN-γ or IL-12, the magnitude of the specific anti-env CD8+ T cell stimulation after MVAENV booster was further enhanced. Our findings revealed that IFN-γ or IL-12 can be used to potentiate the cellular immune response to HIV-1 env, when delivered either from a single MVA recombinant or from a DNA vector. The increment of the CD8+ T cell response was higher in a DNA/MVA prime/boost protocol. Thus, the immune response of MVA vectors can be improved with the co-delivery of the cytokines IFN-γ or IL-12.

INTERPLAY BETWEEN DNA REPLICATION AND RECOMBINATION IN PROKARYOTES

The processes of DNA replication and recombination are intertwined at many different levels. In diverse systems, extensive DNA replication can be triggered by genetic recombination, with assembly of a replication complex onto a D-loop recombination intermediate. This and related pathways of replisome assembly allow the completion of DNA replication when forks initiated at a conventional replication origin fail before completing replication of the genome. In addition, the repair of double-strand breaks or gaps by homologous recombination requires at least limited DNA replication to replace the missing information. An intricate interplay between replication and recombination is also evident during the termination of bacterial DNA replication and during the induction of the bacterial SOS response to DNA damage.

Two Doses of Parenterally Administered Split Influenza Virus Vaccine Elicited High Serum IgG Concentrations which Effectively Limited Viral Shedding upon Challenge in Mice

We have previously found that whole influenza virus vaccine induced a more rapid and stronger humoral response, particularly after the first dose of vaccine, than split virus vaccine in mice. In this study, we have evaluated the protective efficacy of whole and split influenza virus vaccines in mice using a nonlethal upper respiratory tract challenge model. We have also investigated the immunological correlates associated with no or very little viral shedding after viral challenge. Vaccination resulted in reduced viral shedding and shortened the duration of infection by at least 2 days. After one dose of vaccine, whole virus vaccine generally resulted in less viral shedding than split virus vaccine. In contrast, two doses of split virus vaccine, particularly the highest vaccine strengths of 15 and 30 microg HA, most effectively limited viral replication and these mice had high concentrations of prechallenge influenza-specific serum IgG. The vaccine formulation influenced the IgG2a/IgG1 ratio, and this IgG subclass profile was maintained upon challenge to some extent, although it did not influence the level of viral shedding. The concentration of postvaccination serum IgG showed an inverse relationship with the level of viral shedding after viral challenge. Therefore, serum IgG is an important factor in limiting viral replication in the upper respiratory tract upon challenge of an antigenically similar virus.

Oncolytic activity of vesicular stomatitis virus in primary adult T-cell leukemia

Treatments for hematological malignancies have improved considerably over the past decade, but the growing therapeutic arsenal has not benefited adult T-cell leukemia (ATL) patients. Oncolytic viruses such as vesicular stomatitis virus (VSV) have recently emerged as a potential treatment of solid tumors and leukemias in vitro and in vivo. In the current study, we investigated the ability of VSV to lyse primary human T-lymphotropic virus type 1 (HTLV-1)-infected T-lymphocytes from patients with ATL. Ex vivo primary ATL cells were permissive for VSV and underwent rapid oncolysis in a time-dependent manner. Importantly, VSV infection showed neither viral replication nor oncolysis in HTLV-1-infected, nonleukemic cells from patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and in naive CD4(+) T-lymphocytes from normal individuals or in ex vivo cell samples from patients with chronic lymphocytic leukemia (CLL). Interestingly, activation of primary CD4(+) T-lymphocytes with anti-CD3/CD28 monoclonal antibody, and specifically with anti-CD3, was sufficient to induce limited viral replication and oncolysis. However, at a similar level of T-cell activation, VSV replication was increased fourfold in ATL cells compared to activated CD4(+) T-lymphocytes, emphasizing the concept that VSV targets genetic defects unique to tumor cells to facilitate its replication. In conclusion, our findings provide the first essential information for the development of a VSV-based treatment for ATL.