Paired frozen tumor and adjacent non-tumor lung from 30 NSCLC patients were obtained at the Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori IRST, S.r.l, IRCCS, in Meldola (FC), Italy. Written informed consent was obtained from all patients before sample analyses.
The cases from TCGA analysis consisted of 210 Lung squamous cell carcinoma (LUSC) samples with overall survival information and uc.339 expression computed. Clinical data were retrieved from cbio portal ( ). We downloaded RNA-seq BAM files from UCSC Cancer Genomics Hub (CGHub, ). TCGA BAM files were generated based on Mapsplice2 algorithm57 for alignment against the hg19 reference genome using default parameters. We quantified the expression of uc.339 as RPKM (reads per kilobase per million mapped reads)58. The data can be retrieved from TANRIC _design/basic/index.html. We downloaded patient clinical information for the TCGA patients with LUSC from cbioPortal ( ).
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I.V. designed the experiments, conducted the experiments, analyzed the data, and wrote the manuscript; P.M.W., K.B.C., M.P., M.R., E.B., F.F., G.P., M.C., Z.G, M.A.C., P.W., S.C., S.S., L.F., H.L., M.M. and P.N. conducted experiments; L.C. and S.Z. performed the secondary structure analysis of the uc.339 transcript, identified TRE sequences and wrote the manuscript; M. Ferracin, C.I., M.N. and C.F.-C. analyzed the data and wrote the manuscript; R.V.D. performed TP53 binding site analysis; X.Z. performed in situ hybridization experiments; L.H. and H.L. performed bioinformatics analysis on TCGA data and wrote the manuscript; B.J.G. and I.A.L.-O. analyzed the data and wrote the manuscript; P.N.-S., D.A. and A.C. designed the experiments and wrote the manuscript; and G.A.C. and M. Fabbri designed the experiments, analyzed the data, and wrote the manuscript.
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Subject matter experts provided oral presentations on various aspects of smallpox and smallpox vaccines, including a review of the smallpox eradication campaign, U.S. government vaccine response strategies, surveillance and research data from the DoD smallpox vaccination program, regulatory considerations for the use of smallpox vaccines, and incidence, severity, and treatment of adverse events. Vaccine manufacturers presented data and answered questions from the expert panel members, but did not observe or participate in any other aspect of the meeting to avoid introducing bias because of potential competing interests. The primary evidence base for the development of clinical guidance included relevant studies that were identified by literature searches of the PubMed database, manual searches of subject matter experts' personal libraries, and communication with vaccine manufacturers and researchers. Nonsystematic literature searches were performed using standard term indices to cover the terms "smallpox vaccine," "ACAM2000," "Imvamune," "Modified Vaccinia Ankara," "MVA," "Aventis Pasteur Smallpox Vaccine," and "APSV."
The primary strategy for controlling the spread of disease after confirmation of one or more human smallpox cases involves the use of smallpox vaccine in combination with other surveillance and containment activities. As demonstrated during the eradication campaign, the immune response generated by smallpox vaccination is one of the most effective tools for halting the transmission of smallpox (1). Smallpox vaccines are made from live vaccinia viruses that protect against smallpox disease (1). They do not contain variola virus, the causative agent of smallpox (1). The U.S. government has three different smallpox vaccines available in the U.S. SNS: ACAM2000, Imvamune, and Aventis Pasteur Smallpox Vaccine (APSV). ACAM2000 is licensed by FDA whereas Imvamune and APSV are expected to be used under Investigational New Drug (IND) or Emergency Use Authorization (EUA) regulatory mechanisms. Although an EUA cannot be issued until an emergency determination and declaration are in place, FDA can review submitted product data as a pre-EUA before a formal EUA request (38).
The clinical effectiveness of ACAM2000 is based on two pivotal clinical trials that demonstrated noninferiority to Dryvax (the smallpox vaccine licensed at the time of the trial) (47) (Table 4). The surrogate endpoints evaluated in these trials included major cutaneous reactions (i.e., "take" rates) and serum-neutralizing antibodies. ACAM2000 met two of the four primary endpoint criteria established in these trials. Among vaccinia virus-naïve subjects, ACAM2000 was determined to be noninferior to Dryvax in eliciting a major cutaneous reaction. Although ACAM2000 and Dryvax demonstrated similar trends in generating a serum-neutralizing antibody response (GMTs of 166 and 255 respectively on day 30), ACAM2000 did not meet the predefined criterion for noninferiority to Dryvax for this outcome. Among previously vaccinated subjects, ACAM2000 was determined to be noninferior to Dryvax in generating a serum neutralizing antibody response but did not meet the criterion for noninferiority in eliciting a major cutaneous reaction. The major cutaneous reaction is considered the primary determinant for an effective immune response in vaccinia virus-naïve subjects (47). However, the serum neutralizing antibody response might be a more informative measure of immune response in previously vaccinated subjects because pre-existing immunity can modify the cutaneous response to vaccination and add difficulty in evaluating for a major cutaneous reaction among revaccinees (47). Therefore, ACAM2000 was noninferior to Dryvax in the two most relevant surrogate endpoints (47). A summary of these clinical trials is provided (Table 4).
ACAM2000 has been used only in patients who have undergone intensive screening to exclude persons with known risk factors for adverse events. Because ACAM2000 is derived from the same NYCBOH strain that was used to manufacture Dryvax vaccine, it is anticipated that the safety profile of ACAM2000 is probably similar, if not identical, to that of Dryvax. Serious adverse events, including encephalitis, encephalomyelitis, encephalopathy, progressive vaccinia, generalized vaccinia, severe vaccinial skin infections, erythema multiforme major (including Stevens-Johnson syndrome), myocarditis/pericarditis, and eczema vaccinatum (severe and destructive infection of skin affected by eczema or other chronic skin disorder caused by spread of vaccinia virus) resulting in permanent sequelae or death, ocular complications, blindness, and fetal death have occurred following either primary vaccination or revaccination with replication-competent smallpox vaccines including Dryvax (9,43,46). Two studies conducted by CDC in 1968 are among the most comprehensive to evaluate the incidence of adverse events following routine smallpox vaccination with Dryvax. The first study relied on passive reporting of patients with suspected complications of smallpox vaccination to seven separate national surveillance systems (60). The second study implemented an active surveillance system whereby all physicians in 10 states were prospectively requested to record all smallpox vaccine adverse events they saw in practice (61). The physicians were then surveyed to collect the reports of these vaccine adverse events. The adverse event rates observed using the active surveillance from the 10 statewide surveys (Tables 5 and 6) were overall higher than those calculated from the passive national surveillance. The rates reported from the 10 statewide surveys often are considered to be more accurate on the basis of the methodology of the active surveillance employed in contrast to the passive data collection of the national surveillance systems (1,61). Rates of adverse events following revaccination were much lower than those for primary vaccination (with the exception of those in immunocompromised patients).
APSV, also known as "WetVax," is a liquid formulation of calf-lymph-origin vaccinia virus vaccine that has been maintained at -4F (-20C) since it was manufactured in 1956 and 1957 (62). APSV was produced from a vaccinia virus seed stock derived from the NYCBOH strain (62). The original seed #17633 was received from the Michigan Department of Health in 1947. The bulk material was manufactured under license to Aventis Pasteur and was released previously under then acceptable release criteria to the DoD vaccine reserves. The formulation contains live vaccinia virus in 50% glycerol, 0.4% phenol, and 0.00017% Brilliant Green. No antibiotics or other additives are present. Fourteen bottles tested for bioburden met specifications of
APSV is administered in a single dose (2.5 uL) by the percutaneous route (scarification) using 15 jabs of a stainless steel bifurcated needle that has been dipped into the vaccine. The site of vaccination is the upper arm over the deltoid muscle (62). Studies of undiluted vaccine potency found a titer of 10 x 107.6 plaque-forming units (PFU)/mL and there was no difference in vaccine success rates when comparing diluted (1:5) and undiluted vaccine (62). Yearly monitoring of APSV potency remains ongoing.
Imvamune is an investigational product that is stored in the U.S. SNS. CDC has submitted a pre-EUA submission to FDA for its potential use during a declared public health emergency involving smallpox. On the basis of the clinical trial information available to date, the pre-EUA submitted to FDA for review and authorization during an emergency would allow the use of Imvamune for persons of all ages with a diagnosis of HIV or atopic dermatitis during a postevent, pre-exposure setting. 2ff7e9595c
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