Improved NYVAC-based vaccine vectors.

While as yet there is no vaccine against HIV/AIDS, the results of the phase III Thai trial (RV144) have been encouraging and suggest that further improvements of the prime/boost vaccine combination of a poxvirus and protein are needed. With this aim, in this investigation we have generated derivatives of the candidate vaccinia virus vaccine vector NYVAC with potentially improved functions. This has been achieved by the re-incorporation into the virus genome of two host range genes, K1L and C7L, in conjunction with the removal of the immunomodulatory viral molecule B19, an antagonist of type I interferon action. These novel virus vectors, referred to as NYVAC-C-KC and NYVAC-C-KC-ΔB19R, have acquired relevant biological characteristics, giving higher levels of antigen expression in infected cells, replication-competency in human keratinocytes and dermal fibroblasts, activation of selective host cell signal transduction pathways, and limited virus spread in tissues. Importantly, these replication-competent viruses have been demonstrated to maintain a highly attenuated phenotype.

Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI).

The Cancer Vaccine Consortium of the Sabin Vaccine Institute (CVC/SVI) is conducting an ongoing large-scale immune monitoring harmonization program through its members and affiliated associations. This effort was brought to life as an external validation program by conducting an international Elispot proficiency panel with 36 laboratories in 2005, and was followed by a second panel with 29 participating laboratories in 2006 allowing for application of learnings from the first panel. Critical protocol choices, as well as standardization and validation practices among laboratories were assessed through detailed surveys. Although panel participants had to follow general guidelines in order to allow comparison of results, each laboratory was able to use its own protocols, materials and reagents. The second panel recorded an overall significantly improved performance, as measured by the ability to detect all predefined responses correctly. Protocol choices and laboratory practices, which can have a dramatic effect on the overall assay outcome, were identified and lead to the following recommendations: (A) Establish a laboratory SOP for Elispot testing procedures including (A1) a counting method for apoptotic cells for determining adequate cell dilution for plating, and (A2) overnight rest of cells prior to plating and incubation, (B) Use only pre-tested serum optimized for low background: high signal ratio, (C) Establish a laboratory SOP for plate reading including (C1) human auditing during the reading process and (C2) adequate adjustments for technical artifacts, and (D) Only allow trained personnel, which is certified per laboratory SOPs to conduct assays. Recommendations described under (A) were found to make a statistically significant difference in assay performance, while the remaining recommendations are based on practical experiences confirmed by the panel results, which could not be statistically tested. These results provide initial harmonization guidelines to optimize Elispot assay performance to the immunotherapy community. Further optimization is in process with ongoing panels.

Clinical studies of experimental vaccines.

PURPOSE OF REVIEW: The scope of this review is to provide the current status of HIV vaccine clinical development. A series of issues regarding the type of immune response stimulated by the candidate vaccines in the pipeline, the advances in the immune correlates of protection, the need for an effective decision-making process for selection of candidate vaccines into further clinical development and the rationale for clinical trials will also be discussed. RECENT FINDINGS: Efforts in the development of HIV vaccines inducing broad neutralizing antibodies have failed so far. The current pipeline is predominantly composed of candidate vaccines designed to induce cellular immunity and particularly T-cell response. For these reasons, these candidate vaccines have been termed 'T-cell vaccines'. A large number of candidate vaccines or vaccine combinations have entered phase I-II clinical trials in 2005. Furthermore, an adenovirus vector-based vaccine has entered proof-of-concept efficacy trial and a canarypox vector in combination with a protein-based vaccine is currently being evaluated in phase III clinical trials. T-cell vaccines have been shown to be safe and the most recent generation of these vaccines also has substantial immunogenicity. SUMMARY: Only clinical trials can provide the definitive answer to immune correlates of protection and vaccine efficacy.

Vaccines safety; effect of supervision or SMS on reporting rates of adverse events following immunization (AEFI) with meningitis vaccine (MenAfriVac?): a randomized controlled trial.

BACKGROUND: To ensure vaccines safety, given the weaknesses of the national pharmacovigilance system in Cameroon, there is a need to identify effective interventions that can contribute to improving AEFI reporting. OBJECTIVE: To assess the effect of: (i) sending weekly SMS, or (ii) weekly supervisory visits on AEFI reporting rate during a meningitis immunization campaign conducted in Cameroon in 2012 using the meningitis A conjugate vaccine (MenAfriVac?). METHODS: Health facilities that met the inclusion criteria were randomly assigned to receive: (i) a weekly standardized SMS, (ii) a weekly standardized supervisory visits or (iii) no intervention. The primary outcome was the reported AEFI incidence rate from week 5 to 8 after the immunization campaign. Poisson regression model was used to estimate the effect of interventions after adjusting for health region, type of health facility, type and position of health workers as well as the cumulative number of AEFI reported from weeks 1 to 4. RESULTS: A total of 348 (77.2%) of 451 health facility were included, and 116 assigned to each of three groups. The incidence rate of reported AEFI per 100 health facility per week was 20.0 (15.9-24.1) in the SMS group, 40.2 (34.4-46.0) in supervision group and 13.6 (10.1-16.9) in the control group. Supervision led to a significant increase of AEFI reporting rate compared to SMS [adjusted RR=2.1 (1.6-2.7); p<0.001] and control [RR=2.8(2.1-3.7); p<0.001)] groups. The effect of SMS led to some increase in AEFI reporting rate compared to the control group, but the difference was not statistically significant [RR=1.4(0.8-1.6); p=0.07)]. CONCLUSION: Supervision was more effective than SMS or routine surveillance in improving AEFI reporting rate. It should be part of any AEFI surveillance system. SMS could be useful in improving AEFI reporting rates but strategies need to be found to improve its effectiveness, and thus maximize its benefits.

Capsid modified replicating adenovirus to selectively target colon cancer

Résumé: Pratiquement tous les cancers du colon contiennent des mutations dans la voie de signalisation de Wnt qui active constitutivement cette voie. Cette activation mène à la stabilisation de la β-catenine. La β-catenin est transportée dans le noyau ou elle active des gènes cible en interagissant avec le facteur de transcription de TCF/LEF. Des adénovirus qui peuvent sélectivement se répliquer dans les cellules tumorales sont les agents qui peuvent permettre la déstruction de la tumeur mais pas le tissu normal. In vitro, les adénovirus avec des sites d'attachement du facteur de transcription TCF dans les promoteurs de l'adénovirus montrent une sélectivité et une activité dans une large sélection de lignées cellulaires de cancer du colon. Au contraire, in vivo, quand les adénovirus modifiés sont injectés dans la circulation, ils sont moins efficaces à cause de leur fixation par le foie et à cause de l'absence d'expression du récepteur du Coxsackie-Adénovirus (CAR). Le but de ma thèse était de modifier la protéine principale de capside de l'adénovirus, fibre, pour augmenter l'infection des tumeurs du cancer du colon. La fibre de l'adénovirus est responsable de l'attachement aux cellules et de l'entrée virale. J'ai inséré un peptide RGD dans la boucle HI de la fibre qui dirige sélectivement le virus aux récepteurs des integrines. Les integrines sont surexprimées par les cellules du cancer du colon et l'endothélium des vesseaux de la tumeur. Le virus re-ciblé, vKH6, a montré une activité accrue dans toutes les lignées cellulaires de cancer du colon, tandis que la sélectivité était maintenue. In vivo, vKH6 était supérieur au virus avec une capside de type sauvage en retardant la croissance de la tumeur. Le virus s'est répliqué plus vite et dispersé graduellement dans la tumeur. Cet effet a été montré par hybridation in situ et par PCR quantitative. Cependant, la monothérapie avec le virus n'a pu retarder la croissance des cellules tumorales SW620 greffées que de 2 semaines, mais à cause des régions non infectées la tumeur n'a pas pu être éliminée. Bien que la combinaison avec les chimiothérapies conventionnelles soit d'intérêt potentiel, presque toutes interfèrent avec la réplication virale. Les drogues antiangiogéniques sont des agents anti-tumoraux efficaces et prometteurs. Ces drogues n'interfèrent pas avec le cycle de vie de l'adénovirus. RAD001 est un dérivé de la rapamycine et il inhibe mTOR, une protéine kinase de la voie de PI3K. RAD001 empêche la croissance des cellules et il a aussi des effets anti-angiogénique et immunosuppressifs. RAD001 in vitro n'affecte pas l'expression des gènes viraux et la production virale. La combinaison de VKH6 et RAD001 in vivo a un effet additif en retardant la croissance de la tumeur. Des nouveaux peptides plus efficaces dans le ciblage de l'adénovirus sont nécessaires pour augmenter l'infection des tumeurs. J'ai créé un système de recombinaison qui permettra la sélection de nouveaux peptides dans le contexte du génome de l'adénovirus. Summary Virtually all colon cancers have mutations in the Wnt signalling pathway which result in the constitutive activation of the pathway. This activation leads to stabilization of β-catenin. β-catenin enters the nucleus and activates its target genes through interaction with the TCF transcription factor. Selectively replicating adenoviruses are promising novel agents that can destroy the tumour but not the surrounding normal tissue. In vitro, adenoviruses with TCF binding sites in the early viral promoters show selectivity and activity in a broad panel of viruses but in vivo they are less effective due to the lack of expression of the Coxsackie-Adenovirus receptor (CAR). The aim of my thesis was to modify the major capsid protein of the adenovirus, fibre, to increase the infection of colon tumours. Fibre of adenovirus is responsible for the binding to cells and for the viral uptake. I inserted an RGD binding peptide into the HI loop of fibre that selectively targets the virus to integrins that are overexpressed on tumour cells and on tumour endothelium. The retargeted virus, vKH6, showed increased activity in all colon cancer cell lines while selectivity was maintained. In vivo, vKH6 is superior to a matched virus with a wild type capsid in delaying tumour growth. vKH6 replicates and gradually spreads within the tumour as shown by in situ hybridization and Q-PCR. The virus alone can delay the growth of SW620 xenografts by 2 weeks but due to uninfected tumour regions the tumour cannot be cured. Although combination with conventional chemotherapeutics is of potential interest, almost all of them interfere with the viral replication. Growing evidence supports that anti-angiogenic drugs are effective and promising anti-tumour agents. These drugs interfere less with the viral life cycle. RAD001 is a rapamycin derivative and it blocks mTOR, a protein kinase in the PI3K pathway. RAD001 inhibits cell growth and has strong anti-angiogenic and immunosuppressive effects. RAD001 in vitro does not affect viral gene expression and viral burst size. In vivo vKH6 and RAD001 have an additive effect in delaying tumour growth, but tumour growth is still not completely inhibited. To further increase tumour infection new tumour specific targeting peptides are needed. I created an adenovirus display library that will allow the selection of targeting peptides. This system may also facilitate the production of fibre modified viruses.

Can we translate vitamin D immunomodulating effect on innate and adaptive immunity to vaccine response?

Vitamin D (VitD), which is well known for its classic role in the maintenance of bone mineral density, has now become increasingly studied for its extra-skeletal roles. It has an important influence on the body's immune system and modulates both innate and adaptive immunity and regulates the inflammatory cascade. In this review our aim was to describe how VitD might influence immune responsiveness and its potential modulating role in vaccine immunogenicity. In the first instance, we consider the literature that may provide molecular and genetic support to the idea that VitD status may be related to innate and/or adaptive immune response with a particular focus on vaccine immunogenicity and then discuss observational studies and controlled trials of VitD supplementation conducted in humans. Finally, we conclude with some knowledge gaps surrounding VitD and vaccine response, and that it is still premature to recommend "booster" of VitD at vaccination time to enhance vaccine response.

Local Salmonella immunostimulation recruits vaccine-specific CD8 T cells and increases regression of bladder tumor.

NlmCategory="UNASSIGNED">The efficacy of antitumoral responses can be increased using combinatorial vaccine strategies. We recently showed that vaccination could be optimized by local administration of diverse molecular or bacterial agents to target and augment antitumoral CD8 T cells in the genital mucosa (GM) and increase regression of cervical cancer in an animal model. Non muscle-invasive bladder cancer is another disease that is easily amenable to local therapies. In contrast to data obtained in the GM, in this study we show that intravesical (IVES) instillation of synthetic toll-like receptor (TLR) agonists only modestly induced recruitment of CD8 T cells to the bladder. However, IVES administration of Ty21a, a live bacterial vaccine against typhoid fever, was much more effective and increased the number of total and vaccine-specific CD8 T cells in the bladder approximately 10 fold. Comparison of chemokines induced in the bladder by either CpG (a TLR-9 agonist) or Ty21a highlighted the preferential increase in complement component 5a, CXCL5, CXCL2, CCL8, and CCL5 by Ty21a, suggesting their involvement in the attraction of T cells to the bladder. IVES treatment with Ty21a after vaccination also significantly increased tumor regression compared to vaccination alone, resulting in 90% survival in an orthotopic murine model of bladder cancer expressing a prototype tumor antigen. Our data demonstrate that combining vaccination with local immunostimulation may be an effective treatment strategy for different types of cancer and also highlight the great potential of the Ty21a vaccine, which is routinely used worldwide, in such combinatorial therapies.

The Ovarian Cancer Chemokine Landscape Is Conducive to Homing of Vaccine-Primed and CD3/CD28-Costimulated T Cells Prepared for Adoptive Therapy.

PURPOSE: Chemokines are implicated in T-cell trafficking. We mapped the chemokine landscape in advanced stage ovarian cancer and characterized the expression of cognate receptors in autologous dendritic cell (DC)-vaccine primed T cells in the context of cell-based immunotherapy. EXPERIMENTAL DESIGN: The expression of all known human chemokines in patients with primary ovarian cancer was analyzed on two independent microarray datasets and validated on tissue microarray. Peripheral blood T cells from five HLA-A2 patients with recurrent ovarian cancer, who previously received autologous tumor DC vaccine, underwent CD3/CD28 costimulation and expansion ex vivo. Tumor-specific T cells were identified by HER2/neu pentamer staining and were evaluated for the expression and functionality of chemokine receptors important for homing to ovarian cancer. RESULTS: The chemokine landscape of ovarian cancer is heterogeneous with high expression of known lymphocyte-recruiting chemokines (CCL2, CCL4, and CCL5) in tumors with intraepithelial T cells, whereas CXCL10, CXCL12, and CXCL16 are expressed quasi-universally, including in tumors lacking tumor-infiltrating T cells. DC-vaccine primed T cells were found to express the cognate receptors for the above chemokines. Ex vivo CD3/CD28 costimulation and expansion of vaccine-primed Tcells upregulated CXCR3 and CXCR4, and enhanced their migration toward universally expressed chemokines in ovarian cancer. CONCLUSIONS: DC-primed tumor-specific T cells are armed with the appropriate receptors to migrate toward universal ovarian cancer chemokines, and these receptors are further upregulated by ex vivo CD3/CD28 costimulation, which render T cells more fit for migrating toward these chemokines. Clin Cancer Res; 21(12); 2840-50. ©2015 AACR.

Vaccine-Induced Linear Epitope-Specific Antibodies to Simian Immunodeficiency Virus SIVmac239 Envelope Are Distinct from Those Induced to the Human Immunodeficiency Virus Type 1 Envelope in Nonhuman Primates.

To evaluate antibody specificities induced by simian immunodeficiency virus (SIV) versus human immunodeficiency virus type 1 (HIV-1) envelope antigens in nonhuman primate (NHP), we profiled binding antibody responses to linear epitopes in NHP studies with HIV-1 or SIV immunogens. We found that, overall, HIV-1 Env IgG responses were dominated by V3, with the notable exception of the responses to the vaccine strain A244 Env that were dominated by V2, whereas the anti-SIVmac239 Env responses were dominated by V2 regardless of the vaccine regimen.

Synergy of Radiotherapy and a Cancer Vaccine for the Treatment of HPV-Associated Head and Neck Cancer.

There is growing interest in the association of radiotherapy and immunotherapy for the treatment of solid tumors. Here, we report an extremely effective combination of local irradiation (IR) and Shiga Toxin B (STxB)-based human papillomavirus (HPV) vaccination for the treatment of HPV-associated head and neck squamous cell carcinoma (HNSCC). The efficacy of the irradiation and vaccine association was tested using a model of HNSCC obtained by grafting TC-1/luciferase cells at a submucosal site of the inner lip of immunocompetent mice. Irradiation and the STxB-E7 vaccine acted synergistically with both single and fractionated irradiation schemes, resulting in complete tumor clearance in the majority of the treated mice. A dose threshold of 7.5 Gy was required to elicit the dramatic antitumor response. The combined treatment induced high levels of tumor-infiltrating, antigen-specific CD8(+) T cells, which were required to trigger the antitumor activity. Treatment with STxB-E7 and irradiation induced CD8(+) T-cell memory, which was sufficient to exert complete antitumor responses in both local recurrences and distant metastases. We also report for the first time that a combination therapy based on local irradiation and vaccination induces an increased pericyte coverage (as shown by αSMA and NG2 staining) and ICAM-1 expression on vessels. This was associated with enhanced intratumor vascular permeability that correlated with the antitumor response, suggesting that the combination therapy could also act through an increased accessibility for immune cells. The combination strategy proposed here offers a promising approach that could potentially be transferred into early-phase clinical trials.