Long-term persistence of zoster vaccine efficacy.

BACKGROUND: The Shingles Prevention Study (SPS) demonstrated zoster vaccine efficacy through 4 years postvaccination. A Short-Term Persistence Substudy (STPS) demonstrated persistence of vaccine efficacy for at least 5 years. A Long-Term Persistence Substudy (LTPS) was undertaken to further assess vaccine efficacy in SPS vaccine recipients followed for up to 11 years postvaccination. Study outcomes were assessed for the entire LTPS period and for each year from 7 to 11 years postvaccination. METHODS: Surveillance, case determination, and follow-up were comparable to those in SPS and STPS. Because SPS placebo recipients were offered zoster vaccine before the LTPS began, there were no unvaccinated controls. Instead, SPS and STPS placebo results were used to model reference placebo groups. RESULTS: The LTPS enrolled 6867 SPS vaccine recipients. Compared to SPS, estimated vaccine efficacy in LTPS decreased from 61.1% to 37.3% for the herpes zoster (HZ) burden of illness (BOI), from 66.5% to 35.4% for incidence of postherpetic neuralgia, and from 51.3% to 21.1% for incidence of HZ, and declined for all 3 outcome measures from 7 through 11 years postvaccination. Vaccine efficacy for the HZ BOI was significantly greater than zero through year 10 postvaccination, whereas vaccine efficacy for incidence of HZ was significantly greater than zero only through year 8. CONCLUSIONS: Estimates of vaccine efficacy decreased over time in the LTPS population compared with modeled control estimates. Statistically significant vaccine efficacy for HZ BOI persisted into year 10 postvaccination, whereas statistically significant vaccine efficacy for incidence of HZ persisted only through year 8.

In vivo CD8+ T-cell suppression of siv viremia is not mediated by CTL clearance of productively infected cells.

The CD8+ T-cell is a key mediator of antiviral immunity, potentially contributing to control of pathogenic lentiviral infection through both innate and adaptive mechanisms. We studied viral dynamics during antiretroviral treatment of simian immunodeficiency virus (SIV) infected rhesus macaques following CD8+ T-cell depletion to test the importance of adaptive cytotoxic effects in clearance of cells productively infected with SIV. As previously described, plasma viral load (VL) increased following CD8+ T-cell depletion and was proportional to the magnitude of CD8+ T-cell depletion in the GALT, confirming a direct relationship between CD8+ T-cell loss and viral replication. Surprisingly, first phase plasma virus decay following administration of antiretroviral drugs was not slower in CD8+ T-cell depleted animals compared with controls indicating that the short lifespan of the average productively infected cell is not a reflection of cytotoxic T-lymphocyte (CTL) killing. Our findings support a dominant role for non-cytotoxic effects of CD8+ T-cells on control of pathogenic lentiviral infection and suggest that cytotoxic effects, if present, are limited to early, pre-productive stages of the viral life cycle. These observations have important implications for future strategies to augment immune control of HIV.

Evaluation of a highly sensitive qualitative human immunodeficiency virus type 1 (HIV-1) RNA assay for detection of HIV-1 suppression.

By using a mathematical model, the APTIMA human immunodeficiency virus type 1 (HIV-1) RNA qualitative assay was evaluated as a semiquantitative assay to distinguish HIV-1 patient samples needing quantitation from samples in which the virus was suppressed with antiretroviral therapy.

A Novel Shiga Toxin 1a-Converting Bacteriophage of Shigella sonnei With Close Relationship to Shiga Toxin 2-Converting Pages of Escherichia coli.

In recent studies, strains of non-dysenteriae 1 Shigella (NDS) expressing Shiga toxin have been reported. In this study, we report a novel stx1a-converting bacteriophage of Shigella sonnei associated with travel to Mexico. Phylogenetic comparison between this and other stx-converting phages suggests that toxigenic NDS strains have arisen through separate horizontal transfer events from toxigenic Escherichia coli.

Bacterial and viral co-infections complicating severe influenza: Incidence and impact among 507 U.S. patients, 2013-14.

BACKGROUND: Influenza acts synergistically with bacterial co-pathogens. Few studies have described co-infection in a large cohort with severe influenza infection. OBJECTIVES: To describe the spectrum and clinical impact of co-infections. STUDY DESIGN: Retrospective cohort study of patients with severe influenza infection from September 2013 through April 2014 in intensive care units at 33 U.S. hospitals comparing characteristics of cases with and without co-infection in bivariable and multivariable analysis. RESULTS: Of 507 adult and pediatric patients, 114 (22.5%) developed bacterial co-infection and 23 (4.5%) developed viral co-infection. Staphylococcus aureus was the most common cause of co-infection, isolated in 47 (9.3%) patients. Characteristics independently associated with the development of bacterial co-infection of adult patients in a logistic regression model included the absence of cardiovascular disease (OR 0.41 [0.23-0.73], p=0.003), leukocytosis (>11K/µl, OR 3.7 [2.2-6.2], p<0.001; reference: normal WBC 3.5-11K/µl) at ICU admission and a higher ICU admission SOFA score (for each increase by 1 in SOFA score, OR 1.1 [1.0-1.2], p=0.001). Bacterial co-infections (OR 2.2 [1.4-3.6], p=0.001) and viral co-infections (OR 3.1 [1.3-7.4], p=0.010) were both associated with death in bivariable analysis. Patients with a bacterial co-infection had a longer hospital stay, a longer ICU stay and were likely to have had a greater delay in the initiation of antiviral administration than patients without co-infection (p<0.05) in bivariable analysis. CONCLUSIONS: Bacterial co-infections were common, resulted in delay of antiviral therapy and were associated with increased resource allocation and higher mortality.

Characterization of human immunodeficiency virus (HIV)-2 vector mobilization by HIV-1.

Conditionally replicating human immunodeficiency virus type 2 (crHIV-2) vectors can compete with HIV-1 for packaging in HIV-1-infected cells, indicating that the mobilization of vectors could selectively target as well as protect reservoirs susceptible to HIV-1 infection. The incorporation of HIV-1-specific antiviral transgenes in crHIV-2 vectors, although increasing the direct antiviral effect, may decrease mobilization and transmission to surrounding cells. To investigate how HIV-1-specific catalytic RNA cassettes (ribozymes) affect this balance between antiviral activity and mobilization, crHIV-2 vectors shown to display anti-HIV-1 activity were packaged by HIV-2 and used to transduce cells previously infected with HIV-1 or to transduce uninfected cells that were subsequently challenged with HIV-1. Vector mobilization was greater when HIV-1-infected cells were transduced with vector than when transduced cells were infected with HIV-1, and approximately 3-fold lower vector production was observed in cultures transduced with vectors expressing anti-HIV-1 ribozymes. Vector and antiviral effects could be transferred to new cultures by passaging supernatants to fresh cultures. No evidence of recombination with HIV-1 was observed. Vector mobilization and protection from HIV-1 infection were also demonstrated in human peripheral blood mononuclear cells. These data suggest that strategies employing vector mobilization for HIV-1 gene therapy should use vectors with maximal antiviral potency, despite resulting reductions in mobilization of the vector.

Inhibition of HIV-1 replication by siRNA targeting conserved regions of gag/pol.

Short interfering RNAs (siRNAs) targeting HIV-1gag, vif, tat, rev and host CD4 and CCR5 have been reported to inhibit HIV replication. However, the sequence divergence of HIV and the concentration dependence of siRNA activity represent significant challenges to RNAi mediated inhibition. To determine the parameters of RNAi in suppression of HIV-1 we screened seven siRNA candidates targeting highly conserved regions of gag/pol, based on target site GC content, for antiviral activity at varying concentrations. Only two of these inhibited CA-p24 production more than 50%, 2064 and 2161. Activity varied with concentration, with 100 nM producing optimal suppression. Requirements for target sequence conservation and activity over a range of concentrations may severely limit the number of siRNA candidates for therapeutic development.

Severe Influenza in 33 US Hospitals, 2013-2014: Complications and Risk Factors for Death in 507 Patients.

BACKGROUND: Influenza A (H1N1) pdm09 became the predominant circulating strain in the United States during the 2013-2014 influenza season. Little is known about the epidemiology of severe influenza during this season. METHODS: A retrospective cohort study of severely ill patients with influenza infection in intensive care units in 33 US hospitals from September 1, 2013, through April 1, 2014, was conducted to determine risk factors for mortality present on intensive care unit admission and to describe patient characteristics, spectrum of disease, management, and outcomes. RESULTS: A total of 444 adults and 63 children were admitted to an intensive care unit in a study hospital; 93 adults (20.9%) and 4 children (6.3%) died. By logistic regression analysis, the following factors were significantly associated with mortality among adult patients: older age (>65 years, odds ratio, 3.1 [95% CI, 1.4-6.9], P=.006 and 50-64 years, 2.5 [1.3-4.9], P=.007; reference age 18-49 years), male sex (1.9 [1.1-3.3], P=.031), history of malignant tumor with chemotherapy administered within the prior 6 months (12.1 [3.9-37.0], P<.001), and a higher Sequential Organ Failure Assessment score (for each increase by 1 in score, 1.3 [1.2-1.4], P<.001). CONCLUSION: Risk factors for death among US patients with severe influenza during the 2013-2014 season, when influenza A (H1N1) pdm09 was the predominant circulating strain type, shifted in the first postpandemic season in which it predominated toward those of a more typical epidemic influenza season.

Feline immunodeficiency virus: a concise review.

Among non-primate vertebrates, feline immunodeficiency virus (FIV) infection in the cat may be the closest model of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS). Clinical evolution and immunological and virological relationships between human HIV/AIDS and disease produced by FIV infection in cats are very close. These similarities should facilitate progress in the understanding of mechanisms of viral infection and immunopathology, and make this model potentially very valuable in evaluation of experimental therapeutic approaches to AIDS in man. Development of feline immunodeficiency virus vectors bearing therapeutic genes targeting different human diseases is a promising strategy for gene therapy, despite some recent studies which suggest that despite lack of evidence of infection of man by FIV, additional epidemiological surveillance may be indicated to determine if transmission can occur from this close companion to humans in some circumstances.

A cysteine-rich LIM-only protein mediates regulation of smooth muscle-specific gene expression by cGMP-dependent protein kinase.

Vascular smooth muscle cells (VSMCs) undergo phenotypic modulation, changing from a differentiated, contractile to a de-differentiated, synthetic phenotype; the change is associated with decreased expression of smooth muscle (SM)-specific genes and loss of cGMP-dependent protein kinase (PKG), but transfection of PKG into de-differentiated VSMCs restores SM-specific gene expression. We show that small interference RNA-mediated down-regulation or pharmacologic inhibition of PKG reduced SM-specific gene expression in differentiated VSMCs and provide a mechanism for cGMP/PKG regulation of SM-specific genes involving the cysteine-rich LIM-only protein CRP4. PKG associated with CRP4 and phosphorylated the protein in intact cells. CRP4 had no intrinsic transcriptional activity, but exhibited adaptor function, because it acted synergistically with serum response factor (SRF) and GATA6 to activate the SM-alpha-actin promoter. cGMP stimulation of the promoter required PKG and CRP4 co-expression with SRF and GATA6. A phosphorylation-deficient mutant CRP4 and a CRP4 deletion mutant deficient in PKG binding did not support cGMP/PKG stimulation of the SM-alpha-actin promoter. In the presence of wild-type but not mutant CRP4, cGMP/PKG enhanced SRF binding to a probe encoding the distal SM-alpha-actin promoter CArG (CC(AT)(6)GG) element. CRP4 and SRF associated with CArG elements of endogenous SM-specific genes in intact chromatin. Small interference RNA-mediated down-regulation of CRP4 prevented the positive effects of cGMP/PKG on SM-specific gene expression. In the presence of CRP4, cGMP/PKG increased SRF- and GATA6-dependent expression of endogenous SM-specific genes in pluripotent 10T1/2 cells. Thus, CRP4 mediates cGMP/PKG stimulation of SM-specific gene expression, and PKG plays an important role in regulating the phenotype of VSMCs.

Efficient gene transfer to retinal pigment epithelium cells with long-term expression.

PURPOSE: To evaluate the safety and efficiency of feline immunodeficiency virus (FIV) vectors for gene delivery into the mammalian retina. METHODS: A first-generation FIV vector was constructed and administered into rabbit eyes at two different concentrations by intravitreal or subretinal routes. A second-generation FIV vector was also constructed and administered subretinally into both rabbit and rat eyes at the same concentration. After vector administration, eyes were monitored using slit-lamp biomicroscopy, indirect ophthalmoscopy, fundus photography, and electroretinogram. After the rabbits were killed, eye tissues were processed for light microscopy and immunohistochemical analysis. RESULTS: Administration of both first- and second-generation FIV vectors produced transient vitritis and/or papillitis in rabbits, without other pathologic abnormalities. Retinal pigment epithelium (RPE) cells were the predominant cell type transduced in rabbit eyes, but ganglion cells and Muller cells were also transduced. Transduction was confined to the retinal bleb area. The second-generation FIV vector transduced RPE cells much more efficiently than the first-generation vector (95% vs. 4.5%, respectively; P = 0.0015) in rabbit eyes. In contrast, no toxicity was evident over a 24- to 25-month follow-up period after injection of the second-generation FIV vector into rat eyes. Tropism in the rat eye was similar, including RPE and ganglion cells, and the RPE transduction rate was also high (50%). Transgene expression was persistent in both species over the duration of the experiment. CONCLUSION: Second-generation FIV vectors can efficiently transfer genes into RPE cells with resulting long-term expression, properties potentially valuable to gene therapy approaches to some retinal diseases.

Small interfering RNA-induced transcriptional gene silencing in human cells.

Small interfering RNA (siRNA) and microRNA silence genes at the transcriptional, posttranscriptional, and/or translational level. Using human tissue culture cells, we show that promoter-directed siRNA inhibits transcription of an integrated, proviral, elongation factor 1alpha (EF1A) promoter-green fluorescent protein reporter gene and of endogenous EF1A. Silencing was associated with DNA methylation of the targeted sequence, and it required either active transport of siRNA into the nucleus or permeabilization of the nuclear envelope by lentiviral transduction. These results demonstrate that siRNA-directed transcriptional silencing is conserved in mammals, providing a means to inhibit mammalian gene function.

Transduction of cell lines and primary cells by FIV-packaged HIV vectors.

Human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus, and feline immunodeficiency virus (FIV) are capable of packaging viral RNA derived from heterologous as well as homologous lentiviruses, a phenomenon referred to as "cross packaging." To remove the possibility of seroconversion to HIV proteins, and to avoid potential problems arising due to targeting of vector or packaging construct by antiviral genes, we investigated the feasibility of using an FIV-based packaging system to deliver human immunodeficiency virus type 2 (HIV-2)-based vectors bearing anti-HIV-1 RNA expression cassettes to target cells. In the absence of FIV rev, FIV was packaged by HIV-2 at only 3% the efficiency of FIV packaging by FIV, but this was increased to 39% of homologous controls by supplying FIV rev in trans. HIV-2 vectors were packaged by FIV at levels equal to or exceeding the homologous HIV-2 packaging system in the absence of HIV-1 tat and rev, and levels increased approximately four- to fivefold with the addition of tat and rev in trans. HIV-2 vectors bearing a polyribozyme cassette targeting multiple regions of HIV RNA were efficiently packaged by FIV and transferred to target cells. Upon challenge with cell-free HIV-1 (m.o.i. = 0.1) a significant reduction in replication was observed. These findings demonstrate that packaging HIV vectors with FIV is a viable alternative, which avoids use of HIV structural proteins.

Characterization of a mobilization-competent simian immunodeficiency virus (SIV) vector containing a ribozyme against SIV polymerase.

Exploitation of the intracellular virus machinery within infected cells to drive an anti-viral gene therapy vector may prove to be a feasible alternative to reducing viral loads or overall virus infectivity while propagating the spread of a therapeutic vector. Using a simian immunodeficiency virus (SIV)-based system, it was shown that the pre-existing retroviral biological machinery within SIV-infected cells can drive the expression of an anti-SIV pol ribozyme and mobilize the vector to transduce neighbouring cells. The anti-SIV pol ribozyme vector was derived from the SIV backbone and contained the 5'- and 3'LTR including transactivation-response, Psi and Rev-responsive elements, thus requiring Tat and Rev and therefore limiting expression to SIV-infected cells. The data presented here show an early reduction in SIV p27 levels in the presence of the anti-SIV pol ribozyme, as well as successful mobilization (vector RNA constituted approximately 17 % of the total virus pool) and spread of the vector containing this ribozyme. These findings provide direct evidence that mobilization of an anti-retroviral SIV gene therapy vector is feasible in the SIV/macaque model.

Lentiviral vectors pseudotyped with minimal filovirus envelopes increased gene transfer in murine lung.

A human immunodeficiency virus (HIV)-based vector pseudotyped with the Ebola Zaire (EboZ) viral envelope glycoprotein (GP) was recently shown to transduce murine airway epithelia cells in vivo. In this study, the vector was further redesigned to improve gene transfer and also to increase safety. We used mutant EboZ envelopes for pseudotyping, which resulted in higher titers and increased transduction of airway cells in vivo compared to vectors pseudotyped with wild-type EboZ GP. As these envelopes lack regions associated with toxicity of the wild-type EboZ GP, they should also be safer to use for pseudotyping of lentiviral vectors. In addition, lentiviral vectors were created based on feline immunodeficiency virus and shown to have similar efficiency of transduction compared to HIV-based vectors. The creation of lentiviral vectors with highly engineered EboZ envelopes improved the performance of the system and should also increase its safety since only minimal regions of the EboZ envelope, which lack the toxic domain, are used.