Designing phase III or IV trials for vaccines: choosing between individual or cluster randomised trial designs.

The choice of design between individual randomisation, cluster or pseudo-cluster randomisation is often made difficult. Clear methodological guidelines have been given for trials in general practice, but not for vaccine trials. This article proposes a decisional flow-chart to choose the most adapted design for evaluating the effectiveness of a vaccine in large-scale studies. Six criteria have been identified: importance of herd immunity or herd protection, ability to delimit epidemiological units, homogeneity of transmission probability across sub-populations, population's acceptability of randomisation, availability of logistical resources, and estimated sample size. This easy to use decisional method could help sponsors, trial steering committees and ethical committees adopt the most suitable design.

Meningococcal disease in a kidney transplant recipient with mannose-binding lectin (MBL) deficiency

Background: There is an increasing amount of data associating MBL deficiency with a higher susceptibility to meningococca[ disease. In addition, meningococca[ disease has been reported in patients with various immunosuppressive conditions. However, to our knowledge, only three cases of meningococca[ disease have been reported in solid organ recipients (SOT). Methods & Results: A 32 year-old male patient underwent cadaveric kidney transplantation for endstage renal disease of unknown origin. On day 71 post-transplantation he developed fever (39.6°C), shaking chilis, and tachycardia without hypotension. At this time, immunosuppression consisted of tacro[imus, prednisone 10mg daily and mycopheno[ ate mofeti[ 2 g daily. Physical examination on admission was normal, except for two small petechia[ lesions on the forearm. No meningeal signs were present. Three sets of blood cultures grew Neisseria meningitidis group C susceptible to ceftriaxone (MIC=0.003mg/[). Antibiotic therapy consisted in intravenous ceftriaxone 2 g per day for a total duration of 7 days. Serum immunog[obu[in levels, C3, C4 and CHS0 were normal However, using a method to screen for the functional activity of a[[ three pathways of complement (Wies[ab, Lund, Sweden), no activation via the MBL pathway could be detected (0%). A subsequent quantification of MBL pathway components revealed normal levels of MASP 2 but undetectab[e amounts of MBL (below 10 ng/m[, normal range: >500 ng/m[). Conclusion: Since the exact incidence and the possible relationship between meningococca[ disease and organ transplantation is not we[[ understood, we strongly encourage transplantation centers to report additional cases. The potential clinical usefu[ ness of screening SOT candidates for MBL deficiency in relation to infectious complications after transplantation remains to be determined.

The effect of vaccines based on ovalbumin coupled to gas-filled microbubbles for reducing infection by ovalbumin-expressing Listeria monocytogenes.

Gas-filled microbubbles (MB) are a very promising alternative to the currently evaluated lipid- or polymer-based particulate Ag delivery systems. We recently demonstrated the ability of MB to deliver associated Ag to DC, to activate them and thereby induce both humoral and cellular immune responses. We now extended the characterization of MB as antigen-delivery system by appraising the efficiency of MB-associated ovalbumin (OVA-MB) at protecting mice against pathogen infection. Ultrasound-mediated imaging demonstrated that the administration of OVA via MB generates a depot at the injection site that lasts for several hours. We found that OVA-MB injected subcutaneously is far more effective at inducing specific Ab and T cell immunity than immunization with free OVA. Moreover, a covalent link between MB and OVA causes a stronger bias towards a Th1-type of immune response than adsorption of the Ag or its covalent link to liposomes of the same lipid composition. Finally, vaccination of mice with OVA-MB partially protects against a systemic infection with OVA-expressing Listeria monocytogenes. The vaccine induces specific effector CD8 T cell responses capable of decreasing more than 100 fold the bacterial load. MB thus represent a potent Ag delivery system for vaccination against intracellular infectious agents.

Molecularly defined vaccines for cancer immunotherapy, and protective T cell immunity.

Malignant cells are frequently recognized and destroyed by T cells, hence the development of T cell vaccines against established tumors. The challenge is to induce protective type 1 immune responses, with efficient Th1 and CTL activation, and long-term immunological memory. These goals are similar as in many infectious diseases, where successful immune protection is ideally induced with live vaccines. However, large-scale development of live vaccines is prevented by their very limited availability and vector immunogenicity. Synthetic vaccines have multiple advantages. Each of their components (antigens, adjuvants, delivery systems) contributes specifically to induction and maintenance of T cell responses. Here we summarize current experience with vaccines based on proteins and peptide antigens, and discuss approaches for the molecular characterization of clonotypic T cell responses. With carefully designed step-by-step modifications of innovative vaccine formulations, T cell vaccination can be optimized towards the goal of inducing therapeutic immune responses in humans.

The efficiency of a novel delivery system (PEI600-Tat) in development of potent DNA vaccine using HPV16 E7 as a model antigen.

DNA vaccination is a promising approach for inducing both humoral and cellular immune responses. The mode of plasmid DNA delivery is critical to make progress in DNA vaccination. Using human papillomavirus type 16 E7 as a model antigen, this study evaluated the effect of peptide-polymer hybrid including PEI600-Tat conjugate as a novel gene delivery system on the potency of antigen-specific immunity in mice model. At ratio of 10:50 PEI-Tat/E7DNA (w/w), both humoral and cellular immune responses were significantly enhanced as compared with E7DNA construct and induced Th1 response. Therefore, this new delivery system could have promising applications in gene therapy.

Use of recombinant tissue plasminogen activator in children with meningococcal purpura fulminans: a retrospective study.

OBJECTIVE: Meningococcal disease causes septic shock with associated disseminated intravascular coagulation and hemorrhagic skin necrosis. In severe cases, widespread vascular thrombosis leads to gangrene of limbs and digits and contributes to morbidity and mortality. Uncontrolled case reports have suggested that thrombolytic therapy may prevent some complications, and the use of tissue plasminogen activator (t-PA) has been widespread. Our aim was to summarize the clinical outcome and adverse effects where systemic t-PA has been used to treat children with fulminant meningococcemia. DESIGN: International, multiple-center, retrospective, observational case note study between January 1992 and June 2000. SETTING: Twenty-four different hospitals in seven European countries and Australia. PATIENTS: A total of 62 consecutive infants and children with severe meningococcal sepsis in whom t-PA was used for the treatment of predicted amputations and/or refractory shock (40 to treat severe ischemia, 12 to treat shock, and ten to treat both). INTERVENTIONS: t-PA was administered with a median dose of 0.3 mg.kg(-1).hr(-1) (range, 0.008-1.13) and a median duration of 9 hrs (range, 1.2-83). MAIN RESULTS: Twenty-nine of 62 patients died (47%; 95% confidence interval, 28-65). Seventeen of 33 survivors had amputations (11 below knee/elbow or greater loss; six less severe). In 12 of 50 patients to whom t-PA was given for imminent amputation, no amputations were observed. Five developed intracerebral hemorrhages (five of 62, 8%; 95% confidence interval, 0.5-16). Of these five, three died, one developed a persistent hemiparesis, and one recovered completely. CONCLUSIONS: The high incidence of intracerebral hemorrhage in our study raises concerns about the safety of t-PA in children with fulminant meningococcemia. However, due to the absence of a control group in such a severe subset of patients, whether t-PA is beneficial or harmful cannot be answered from the unrestricted use of the drug that is described in this report. Our experience highlights the need to avoid strategies that use experimental drugs in an uncontrolled fashion and to participate in multiple-center trials, which are inevitably required to study rare diseases.

Saponins from the Spanish saffron Crocus sativus are efficient adjuvants for protein-based vaccines.

Protein and peptide-based vaccines provide rigorously formulated antigens. However, these purified products are only weakly immunogenic by themselves and therefore require the addition of immunostimulatory components or adjuvants in the vaccine formulation. Various compounds derived from pathogens, minerals or plants, possess pro-inflammatory properties which allow them to act as adjuvants and contribute to the induction of an effective immune response. The results presented here demonstrate the adjuvant properties of novel saponins derived from the Spanish saffron Crocus sativus. In vivo immunization studies and tumor protection experiments unambiguously establish the value of saffron saponins as candidate adjuvants. These saponins were indeed able to increase both humoral and cellular immune responses to protein-based vaccines, ultimately providing a significant degree of protection against tumor challenge when administered in combination with a tumor antigen. This preclinical study provides an in depth immunological characterization of a new saponin as a vaccine adjuvant, and encourages its further development for use in vaccine formulations.

Therapeutic cancer vaccines based on molecularly defined human tumor antigens.

The results of numerous phases I and II clinical trials testing the safety and immunogenicity of various cancer vaccine formulations based on cytolytic T lymphocytes (CTLs)-defined tumor antigens have been reported recently. Specific T cell responses can be detected in only a fraction of immunized patients. A smaller but significant fraction of these patients have objective tumor responses. Efficient therapeutic vaccination should aim at boosting naturally occurring anti-tumor responses and at sustaining a large contingent of tumor antigen-specific and fully functional effector T cells at tumor sites.

Nicotine vaccines for smoking cessation.

BACKGROUND: By reducing the amount of nicotine that reaches the brain when a person smokes a cigarette, nicotine vaccines may help people to stop smoking or to prevent recent quitters from relapsing. OBJECTIVES: The aims of this review are to assess the efficacy of nicotine vaccines for smoking cessation and for relapse prevention, and to assess the frequency and type of adverse events associated with the use of nicotine vaccines. SEARCH METHODS: We searched the Cochrane Tobacco Addiction Review Group specialised register for trials, using the term 'vaccine' in the title or abstract, or in a keyword (date of most recent search April 2012). To identify any other material including reviews and papers potentially relevant to the background or discussion sections, we also searched MEDLINE, EMBASE, and PsycINFO, combining terms for nicotine vaccines with terms for smoking and tobacco use, without design limits or limits for human subjects. We searched the Annual Meeting abstracts of the Society for Research on Nicotine and Tobacco up to 2012, using the search string 'vaccin'. We searched Google Scholar for 'nicotine vaccine'. We also searched company websites and Google for information related to specific vaccines. We searched clinicaltrials.gov in March 2012 for 'nicotine vaccine' and for the trade names of known vaccine candidates. SELECTION CRITERIA: We included randomized controlled trials of nicotine vaccines, at Phase II and Phase III trial stage and beyond, in adult smokers or recent ex-smokers. We included studies of nicotine vaccines used as part of smoking cessation or relapse prevention interventions. DATA COLLECTION AND ANALYSIS: We extracted data on the type of participants, the dose and duration of treatment, the outcome measures, the randomization procedure, concealment of allocation, blinding of participants and personnel, reporting of outcomes, and completeness of follow-up.Our primary outcome measure was a minimum of six months abstinence from smoking. We used the most rigorous definition of abstinence, and preferred cessation rates at 12 months and biochemically validated rates where available. We have used the risk ratio (RR) to summarize individual trial outcomes. We have not pooled the current group of included studies as they cover different vaccines and variable regimens. MAIN RESULTS: There are no nicotine vaccines currently licensed for public use, but there are a number in development. We found four trials which met our inclusion criteria, three comparing NicVAX to placebo and one comparing NIC002 (formerly NicQbeta) to placebo. All were smoking cessation trials conducted by pharmaceutical companies as part of the drug development process, and all trials were judged to be at high or unclear risk of bias in at least one domain. Overall, 2642 smokers participated in the included studies in this review. None of the four included studies detected a statistically significant difference in long-term cessation between participants receiving vaccine and those receiving placebo. The RR for 12 month cessation in active and placebo groups was 1.35 (95% Confidence Interval (CI) 0.82 to 2.22) in the trial of NIC002 and 1.74 (95% CI 0.73 to 4.18) in one NicVAX trial. Two Phase III NicVAX trials, for which full results were not available, reported similar quit rates of approximately 11% in both groups. In the two studies with full results available, post hoc analyses detected higher cessation rates in participants with higher levels of nicotine antibodies, but these findings are not readily generalisable. The two studies with full results showed nicotine vaccines to be well tolerated, with the majority of adverse events classified as mild or moderate. In the study of NIC002, participants receiving the vaccine were more likely to report mild to moderate adverse events, most commonly flu-like symptoms, whereas in the study of NicVAX there was no significant difference between the two arms. Information on adverse events was not available for the large Phase III trials of NicVAX.Vaccine candidates are likely to undergo significant changes before becoming available to the general public, and those included in this review may not be the first to reach market; this limits the external validity of the results reported in this review in terms of both effectiveness and tolerability. AUTHORS' CONCLUSIONS: There is currently no evidence that nicotine vaccines enhance long-term smoking cessation. Rates of serious adverse events recorded in the two trials with full data available were low, and the majority of adverse events reported were at mild to moderate levels. The evidence available suggests nicotine vaccines do not induce compensatory smoking or affect withdrawal symptoms. No nicotine vaccines are currently licensed for use in any country but a number are under development.Further trials of nicotine vaccines are needed, comparing vaccines with placebo for smoking cessation. Further trials are also needed to explore the potential of nicotine vaccines to prevent relapse. Results from past, current and future research should be reported in full. Adverse events and serious adverse events should continue to be carefully monitored and thoroughly reported.

Single cell gene expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines

The CD8 T cell response generatedby gene-based vaccines is importantfor protective immunity againstmany infectious diseases but its complexityis incompletely understood.Here, we report that different vaccinesencoding HIV Env elicit qualitativelydistinct CD8 T cells that wereidentified by patterns of gene expressionin individual cells. Three alternativeprime-boost vector combinationsstimulated antigen-specific CD8 Tcell populations of similar magnitudeand function by intracellular cytokinestaining; however, single cell geneexpression profiling enabled the discriminationof distinct CM and EMCD8 cells elicited by the three vaccines.Two previously unrecognizedCD8 T cell subsets have been definedby their coexpression of Eomes,Cxcr3 and Ccr7; or Klrk1, Klrg1 andCcr5 in CM and EM cells respectively.

Global phylogenomic analysis of nonencapsulated Streptococcus pneumoniae reveals a deep-branching classic lineage that is distinct from multiple sporadic lineages.

The surrounding capsule of Streptococcus pneumoniae has been identified as a major virulence factor and is targeted by pneumococcal conjugate vaccines (PCV). However, nonencapsulated S. pneumoniae (non-Ec-Sp) have also been isolated globally, mainly in carriage studies. It is unknown if non-Ec-Sp evolve sporadically, if they have high antibiotic nonsusceptiblity rates and a unique, specific gene content. Here, whole-genome sequencing of 131 non-Ec-Sp isolates sourced from 17 different locations around the world was performed. Results revealed a deep-branching classic lineage that is distinct from multiple sporadic lineages. The sporadic lineages clustered with a previously sequenced, global collection of encapsulated S. pneumoniae (Ec-Sp) isolates while the classic lineage is comprised mainly of the frequently identified multilocus sequences types (STs) ST344 (n = 39) and ST448 (n = 40). All ST344 and nine ST448 isolates had high nonsusceptiblity rates to β-lactams and other antimicrobials. Analysis of the accessory genome reveals that the classic non-Ec-Sp contained an increased number of mobile elements, than Ec-Sp and sporadic non-Ec-Sp. Performing adherence assays to human epithelial cells for selected classic and sporadic non-Ec-Sp revealed that the presence of a integrative conjugative element (ICE) results in increased adherence to human epithelial cells (P = 0.005). In contrast, sporadic non-Ec-Sp lacking the ICE had greater growth in vitro possibly resulting in improved fitness. In conclusion, non-Ec-Sp isolates from the classic lineage have evolved separately. They have spread globally, are well adapted to nasopharyngeal carriage and are able to coexist with Ec-Sp. Due to continued use of PCV, non-Ec-Sp may become more prevalent.

In vivo activation of melanoma-specific CD8(+) T cells by endogenous tumor antigen and peptide vaccines. A comparison to virus-specific T cells.

Many new types of vaccines against infectious or malignant diseases are currently being proposed. Careful characterization of the induced immune response is required in assessing their efficiency. While in most studies human tumor antigen-specific T cells are analyzed after in vitro re-stimulation, we investigated these T cells directly ex vivo using fluorescent tetramers. In peripheral blood lymphocytes from untreated melanoma patients with advanced disease, a fraction of tumor antigen (Melan-A/MART-1)-specific T cells were non-naive, thus revealing tumor-driven immune activation. After immunotherapy with synthetic peptides plus adjuvant, we detected tumor antigen-specific T cells that proliferated and differentiated to memory cells in vivo in some melanoma patients. However, these cells did not present the features of effector cells as found in cytomegalovirus specific T cells analyzed in parallel. Thus, peptide plus adjuvant vaccines can lead to activation and expansion of antigen specific CD8(+) T cells in PBL. Differentiation to protective CD8(+) effector cells may, however, require additional vaccine components that stimulate T cells more efficiently, a major challenge for the development of future immunotherapy.

Aerosol immunization with NYVAC and MVA vectored vaccines is safe, simple, and immunogenic.

Each year, approximately five million people die worldwide from putatively vaccine-preventable mucosally transmitted diseases. With respect to mass vaccination campaigns, one strategy to cope with this formidable challenge is aerosol vaccine delivery, which offers potential safety, logistical, and cost-saving advantages over traditional vaccination routes. Additionally, aerosol vaccination may elicit pivotal mucosal immune responses that could contain or eliminate mucosally transmitted pathogens in a preventative or therapeutic vaccine context. In this current preclinical non-human primate investigation, we demonstrate the feasibility of aerosol vaccination with the recombinant poxvirus-based vaccine vectors NYVAC and MVA. Real-time in vivo scintigraphy experiments with radiolabeled, aerosol-administered NYVAC-C (Clade C, HIV-1 vaccine) and MVA-HPV vaccines revealed consistent mucosal delivery to the respiratory tract. Furthermore, aerosol delivery of the vaccines was safe, inducing no vaccine-associated pathology, in particular in the brain and lungs, and was immunogenic. Administration of a DNA-C/NYVAC-C prime/boost regime resulted in both systemic and anal-genital HIV-specific immune responses that were still detectable 5 months after immunization. Thus, aerosol vaccination with NYVAC and MVA vectored vaccines constitutes a tool for large-scale vaccine efforts against mucosally transmitted pathogens.