Early clinical trial experience with vaccine therapies in non-small-cell lung cancer

Cancer immunotherapy has made great progress because of advances in immunology and molecular biology. Increased understanding of mechanisms by which lung cancer cells escape the immune system and recognition of key tumor antigens and immune system components involved in tumor ignorance have led to the development of a variety of lung cancer vaccines. Immunotherapy has advanced from using nonspecific immunomodulatory agents to lung cancer-specific tumor antigens and tumor cell-derived vaccines. While understanding of immune processes and malignancy has improved, there is great opportunity for further research of vaccine therapies in non-small-cell lung cancer. Herein, we review the development and evolution of early lung cancer vaccine trials.

Virus purification, detection and removal

The biopharmaceutical industry has a growing demand and an increasing need to improve the current virus purification technologies, especially as more and more vaccines are produced from cell-culture derived virus particles. Downstream purification strategies can be expensive and account for 70% of the overall manufacturing costs. The economic pressure and purification processes can be particularly challenging when the virus to be purified is small, as in our model virus, porcine parvovirus (PPV). Our efforts are focused on designing an easy, economical, scalable and efficient system for virus purification, and we focused on aqueous two-phase systems. Industry acceptable standards for virus vaccine recovery can be as low as 30% due to demand of high final titer, virus transduction inhibitors and presence of empty or defective virus capsids as impurities. We have overcome these shortcomings by recovering a high 64% of infectious virus using an aqueous two-phase system. We used high molecular weight polymer and citrate salt to achieve a good yield and eliminated the major contaminant bovine serum albumin. Viruses are also studied for ensuring pure and safe drinking water. Low pressure microfiltrations are continuously being investigated for water filters as they allow high permeate flux and low fouling. Viruses such as PPV are small enough to pass through the microporous membranes. Control of viruses in water is crucial for public health and we have designed an affinity based membrane filter to capture virus. Nanofibers have a high surface to volume ratio providing a highly accessible surface area for virus adsorption. Chitosan an insoluble, biocompatible and biodegradable polymer was used for adsorbing trimer peptide WRW. About 0.2 μmoles of cysteine terminal WRW peptide was conjugated to amine terminal chitosan using maleimide conjugation chemistry. We achieved 90-99% virus removal from water adjusted to a neutral pH. The virus removal from affinity based chitosan was attributed to electrostatic and hydrophobic driven binding effect.

Virosomes can enter cells by non-phagocytic mechanisms

Phagocytosis of fine particles (1 mum) by macrophages is a ligand-receptor-mediated, actin-based process, whereas the entering of smaller particles (vaccines. We investigated their interactions with airway cells, in particular penetration into monocyte-derived macrophages. The microscopic analysis of phagocytic cells incubated with virosomes and polystyrene particles showed that virosomes and particles penetrated cells even in the presence of cytochalasin D, a drug inhibiting actin-based phagocytosis. The charge of the virosomes and particles did not influence their penetration. Also, different inhibitors of endocytotic pathways did not prevent the particles and virosomes from penetrating into the cells. Additionally, to study the ability of virosomes to overcome the epithelial airway barrier, a triple cell co-culture model composed of epithelial cells, monocyte-derived macrophages and dendritic cells of the respiratory tract was used. We found virosomes and polystyrene particles in both populations of antigen-presenting cells, monocyte-derived macrophages, and dendritic cells, in the latter even if they were not directly exposed. In conclusion, virosomes are readily taken up by monocyte-derived macrophages, both by conventional phagocytosis and by actin-independent mechanisms. Further, they can penetrate the airway barrier and reach resident dendritic cells. Therefore, virosomes are promising vaccine candidates.

Prospective surveillance of varicella-zoster virus infections in an out-patient setting in Switzerland

Limited data are available on the clinical impact of varicella in the ambulatory setting. Our goal was to determine real-life data on the clinical outcomes, medical management, and resource utilization in patients with varicella in Switzerland, a country without a universal immunization program against varicella. A total of 236 patients (222 = 94% primarily healthy individuals) with a clinical diagnosis of varicella were recruited by pediatricians and general practitioners. Age range of patients was 0-47 years with a median of 5 years. The great majority of patients (179 = 76%) were

Economic evaluation of varicella vaccination in Swiss children and adolescents

This study explores the effects of three different 2-dose varicella zoster virus (VZV) vaccination strategies in Switzerland. The EVITA model was used to assess clinical benefits and costs of strategies (1) vaccination of 11-15 year old adolescents with a negative or uncertain history for chickenpox, (2) universal vaccination of toddlers at age 1 to 2 years, and (3) strategy 2 plus catch-up vaccination of 11-15 year old susceptible adolescents. The cost-effectiveness analysis compares strategies 2 and 3 versus strategy 1 (current vaccination policy in Switzerland). Probabilities for clinical outcomes and medical resource utilization were derived from a real-world survey among Swiss pediatricians and general practitioners including 236 individuals with VZV infection, published information on varicella complications, and expert opinion. Costs of medical resource utilization represent official Swiss medical tariffs. The model predicts both universal childhood vaccination strategies to be more effective in reducing varicella disease burden compared to strategy 1. Economically, both universal childhood vaccination strategies with or without catch-up result in net savings from the societal perspective reflected by a benefit cost ratio (BCR) of 1.22 or 1.29, respectively. In contrast, the model predicts net costs from the payer perspective (BCR of 0.27 and 0.30, respectively). These economic findings are comparable to those reported from other similar evaluations. However, due to the recent recommendation for using a 2-dose varicella vaccination schedule, our economic results for Switzerland are somewhat less favorable than those for other country analyses in which a less expensive 1-dose vaccination regimen for toddlers has been studied.

Gene therapy for immunodeficiency due to adenosine deaminase deficiency

BACKGROUND: We investigated the long-term outcome of gene therapy for severe combined immunodeficiency (SCID) due to the lack of adenosine deaminase (ADA), a fatal disorder of purine metabolism and immunodeficiency. METHODS: We infused autologous CD34+ bone marrow cells transduced with a retroviral vector containing the ADA gene into 10 children with SCID due to ADA deficiency who lacked an HLA-identical sibling donor, after nonmyeloablative conditioning with busulfan. Enzyme-replacement therapy was not given after infusion of the cells. RESULTS: All patients are alive after a median follow-up of 4.0 years (range, 1.8 to 8.0). Transduced hematopoietic stem cells have stably engrafted and differentiated into myeloid cells containing ADA (mean range at 1 year in bone marrow lineages, 3.5 to 8.9%) and lymphoid cells (mean range in peripheral blood, 52.4 to 88.0%). Eight patients do not require enzyme-replacement therapy, their blood cells continue to express ADA, and they have no signs of defective detoxification of purine metabolites. Nine patients had immune reconstitution with increases in T-cell counts (median count at 3 years, 1.07x10(9) per liter) and normalization of T-cell function. In the five patients in whom intravenous immune globulin replacement was discontinued, antigen-specific antibody responses were elicited after exposure to vaccines or viral antigens. Effective protection against infections and improvement in physical development made a normal lifestyle possible. Serious adverse events included prolonged neutropenia (in two patients), hypertension (in one), central-venous-catheter-related infections (in two), Epstein-Barr virus reactivation (in one), and autoimmune hepatitis (in one). CONCLUSIONS: Gene therapy, combined with reduced-intensity conditioning, is a safe and effective treatment for SCID in patients with ADA deficiency. (ClinicalTrials.gov numbers, NCT00598481 and NCT00599781.)

An outbreak of mumps in a population partially vaccinated with the Rubini strain

Since 1991, 6 years after the recommendation of universal childhood triple vaccination against measles, mumps and rubella (M + M + R), Switzerland has been confronted with an increasing number of mumps cases affecting both vaccinated and unvaccinated children. The M + M + R vaccine mainly used in the Swiss population after 1986 contains the highly attenuated Rubini strain of mumps virus. We analysed an outbreak of 102 suspected mumps cases by virus isolation, determination of IgM antibodies to mumps virus in 27 acute phase sera, and verification of vaccination histories. Mumps was confirmed by virus isolation in 88 patients, of whom 72 had previously received the Rubini vaccine strain. IgM antibodies to mumps virus were detected in 24/27 acute phase serum samples. A group of 92 subjects from the same geographic area without signs of mumps virus infection served as controls. IgG antibodies to mumps virus and vaccination status were assessed in these children. The vaccination rate in these controls was 61%, with equal seropositivity for unvaccinated and Rubini-vaccinated subjects. These data support other recent reports which indicate an insufficient protective efficacy of current mumps vaccines.

Efficacy pneumococcal vaccination in adults: a meta-analysis

Background: Clinical trials and meta-analyses have produced conflicting results of the efficacy of unconjugated pneumococcal polysaccharide vaccine in adults. We sought to evaluate the vaccine’s efficacy on clinical outcomes as well as the methodologic quality of the trials. Methods: We searched several databases and all bibliographies of reviews and meta-analyses for clinical trials that compared pneumococcal polysaccharide vaccine with a control. We examined rates of pneumonia and death, taking the methodologic quality of the trials into consideration. Results: We included 22 trials involving 101 507 participants: 11 trials reported on presumptive pneumococcal pneumonia, 19 on all-cause pneumonia and 12 on allcause mortality. The current 23-valent vaccine was used in 8 trials. The relative risk (RR) was 0.64 (95% confidence interval [CI] 0.43–0.96) for presumptive pneumococcal pneumonia and 0.73 (95% CI 0.56–0.94) for all-cause pneumonia. There was significant heterogeneity between the trials reporting on presumptive pneumonia (I2 = 74%, p < 0.001) and between those reporting on all-cause pneumonia (I2 = 90%, p < 0.001). The RR for all-cause mortality was 0.97 (95% CI 0.87–1.09), with moderate heterogeneity between trials (I2 = 44%, p = 0.053). Trial quality, especially regarding double blinding, explained a substantial proportion of the heterogeneity in the trials reporting on presumptive pneumonia and all-cause pneumonia. There was little evidence of vaccine protection in trials of higher methodologic quality (RR 1.20, 95% CI 0.75–1.92, for presumptive pneumonia; and 1.19, 95% CI 0.95–1.49, for allcause pneumonia in double-blind trials; p for heterogeneity > 0.05). The results for all-cause mortality in double-blind trials were similar to those in all trials combined. There was little evidence of vaccine protection among elderly patients or adults with chronic illness in analyses of all trials (RR 1.04, 95% CI 0.78–1.38, for presumptive pneumococcal pneumonia; 0.89, 95% CI 0.69–1.14, for all-cause pneumonia; and 1.00, 95% CI 0.87–1.14, for all-cause mortality). Interpretation: Pneumococcal vaccination does not appear to be effective in preventing pneumonia, even in populations for whom the vaccine is currently recommended.

Detection of Streptococcus pneumoniae strain cocolonization in the nasopharynx

Colonization with more than one distinct strain of the same species, also termed cocolonization, is a prerequisite for horizontal gene transfer between pneumococcal strains that may lead to change of the capsular serotype. Capsule switch has become an important issue since the introduction of conjugated pneumococcal polysaccharide vaccines. There is, however, a lack of techniques to detect multiple colonization by S. pneumoniae strains directly in nasopharyngeal samples. Two hundred eighty-seven nasopharyngeal swabs collected during the prevaccine era within a nationwide surveillance program were analyzed by a novel technique for the detection of cocolonization, based on PCR amplification of a noncoding region adjacent to the pneumolysin gene (plyNCR) and restriction fragment length polymorphism (RFLP) analysis. The numbers of strains and their relative abundance in cocolonized samples were determined by terminal RFLP. The pneumococcal carriage rate found by PCR was 51.6%, compared to 40.0% found by culture. Cocolonization was present in 9.5% (10/105) of samples, most (9/10) of which contained two strains in a ratio of between 1:1 and 17:1. Five of the 10 cocolonized samples showed combinations of vaccine types only (n = 2) or combinations of nonvaccine types only (n = 3). Carriers of multiple pneumococcal strains had received recent antibiotic treatment more often than those colonized with a single strain (33% versus 9%, P = 0.025). This new technique allows for the rapid and economical study of pneumococcal cocolonization in nasopharyngeal swabs. It will be valuable for the surveillance of S. pneumoniae epidemiology under vaccine selection pressure.

Vaccination with recombinant NcROP2 combined with recombinant NcMIC1 and NcMIC3 reduces cerebral infection and vertical transmission in mice experimentally infected with Neospora caninum tachyzoites

We investigated the protective potential of recombinant his-tagged antigens recNcMIC1, recNcMIC3 and recNcROP2, applied either as single vaccines or as vaccine combinations, in BALB/c mouse models for cerebral and fetal infection. Subsequently, mice were mated and challenged by i.p. inoculation of 2 x 10(6)Neospora caninum tachyzoites at day 7 of pregnancy. The mortality and morbidity of adult mice (non-pregnant and dams) and of the newborn pups was studied for a period of 40 days following birth. Vaccination of non-pregnant mice with recNcROP2 or combinations of recNcROP2 with recNcMIC antigens significantly reduced the numbers of mice suffering from clinical signs, and morbidity was completely prevented with the combination of all three antigens. Of the dams, the groups receiving either recNcROP2 alone or the combination of all three antigens did not exhibit any morbidity, the groups receiving ROP2 mixed with either MIC1 or MIC3 exhibited reduced numbers of deaths, and in the infection control group and the adjuvant group 50% and 43% of mice, respectively, succumbed to disease. For pups, the highest survival rates were noted for the groups receiving recNcROP2 (50%) and recNcROP2/NcMIC1/NcMIC3 (35%), while in the infection- and adjuvant- control groups all pups died, the latest at days 25 and 30, respectively. Quantification of parasite DNA by N. caninum-specific real-time PCR revealed consistently lower parasite burdens in brain tissue of pups from vaccinated groups compared with the controls. However, dense granule antigen 2 (GRA2) real-time reverse transcriptase-PCR on brain tissue of surviving pups (applied here to detect viable parasites) demonstrated that only the pups from the group vaccinated with all three antigens in combination appeared free of viable tachyzoites, while in all other groups viable parasites were still present. Serological analysis of humoral (total IgG, IgG1 and IgG2a) and serum cytokine (IL-4 and IFN-gamma) responses showed that this effect was associated with a Th-2-biased immune response, with a clearly elevated IL-4/IFN-gamma ratio in the mice receiving all three antigens in combination. In conclusion, a mixture of recombinant antigens representing important secretory micronemal and rhoptry proteins leads to a significant protection against vertical transmission of N. caninum in mice.

A 4-year study on clinical characteristics of children hospitalized with rotavirus gastroenteritis

Rotavirus (RV) is a frequent cause of severe gastroenteritis (GE) in children. With the licensure of new RV vaccines, data on the burden of disease are important regarding immunization strategies. We reviewed the medical records of children hospitalized with RV infection in our institution between July 2002 and March 2006. Relevant data were extracted in a standardized fashion from records of hospitalized children with a positive RV antigen test in a stool sample. Severity of disease was graded by the 20-point Vesikari score. Population data were obtained from the Federal Office of Statistics. Six hundred eighty-six RVGE were identified and records of 608 hospitalizations (in 607 children) were available. In 539 (89%) cases, RVGE was the primary reason for hospitalization and 69 (11%) were nosocomial infections; yearly peaks occurred between February and May. Cumulative incidence of RVGE was 26.7/1,000 children <3 years of age. Median age of 539 children (55.6% male) with primary RVGE was 1.4 years and median stay in the hospital for both community acquired and nosocomial RVGE was 4 days (interquartile range 3-5). Thirtypercent and 94% of RV hospitalizations were in children <1 and <3 years of age, respectively. Mean Vesikari score was 15 (range 6-20; 96% >11). Intravenous fluids were administered in 378 (70%) patients, 130 (24%) patients were rehydrated via nasogastral tube, and 31 (5.7%) received rehydration by mouth. RVGE causes a substantial burden in children with an estimated risk for hospitalization due to RVGE of one in 37 children <3 years of age.

Morphological and immunocytochemical analysis of Escherichia coli-specific surface antigens in wildtype strains and in recombinant Vibrio cholerae

Adhesion is the first step in the pathogenesis of enterotoxigenic Escherichia coli infections. The genes encoding the most prevalent adhesion factors CFA/I, CS3 and CS6 were cloned into Vibrio cholerae strain CVD 103-HgR and expression of fimbriae was investigated in wildtype and recombinant strains by transmission electron microscopy in conjunction with immunolabelling and negative staining. Negative staining was effective in revealing CFA/I and CS3, but not CS6. Although morphology of fimbriae differed between wildtype and recombinant strains, corresponding surface antigens were recognized by specific antibodies. The present study provides evidence that ETEC-specific fimbriae can adequately be expressed in an attenuated V. cholerae vaccine strain and that immunoelectron microscopy is a critical tool to validate the surface expression of antigens in view of their possible suitability for recombinant vaccines.

Functional and antigenic properties of GlpO from Mycoplasma mycoides subsp. mycoides SC: characterization of a flavin adenine dinucleotide-binding site deletion mutant

L-alpha-glycerophosphate oxidase (GlpO) plays a central role in virulence of Mycoplasma mycoides subsp. mycoides SC, a severe bacterial pathogen causing contagious bovine pleuropneumonia (CBPP). It is involved in production and translocation of toxic H(2)O(2) into the host cell, causing inflammation and cell death. The binding site on GlpO for the cofactor flavin adenine dinucleotide (FAD) has been identified as Gly(12)-Gly(13)-Gly(14)-Ile(15)-Ile(16)-Gly(17). Recombinant GlpO lacking these six amino acids (GlpODeltaFAD) was unable to bind FAD and was also devoid of glycerophosphate oxidase activity, in contrast to non-modified recombinant GlpO that binds FAD and is enzymatically active. Polyclonal monospecific antibodies directed against GlpODeltaFAD, similarly to anti-GlpO antibodies, neutralised H(2)O(2) production of M. mycoides subsp. mycoides SC grown in the presence of glycerol, as well as cytotoxicity towards embryonic calf nasal epithelial (ECaNEp) cells. The FAD-binding site of GlpO is therefore suggested as a valuable target site for the future construction of deletion mutants to yield attenuated live vaccines of M. mycoides subsp. mycoides SC necessary to efficiently combat CBPP.

Immune response of horses to vaccination with the recombinant Hc domain of botulinum neurotoxin types C and D

Botulinum neurotoxins, predominantly serotypes C and D, cause equine botulism through forage poisoning. The C-terminal part of the heavy chain of botulinum neurotoxin types C and D (HcBoNT/C and D) was expressed in Escherichia coli and evaluated as a recombinant mono- and bivalent vaccine in twelve horses in comparison to a commercially available toxoid vaccine. A three-dose subcutaneous immunization of adult horses elicited robust serum antibody response in an ELISA using the immunogen as a capture antigen. Immune sera showed dose-dependent high potency in neutralizing specifically the active BoNT/C and D in the mouse protection assay. The aluminium hydroxide based mono- and bivalent recombinant HcBoNT/C and D vaccines were characterized by good compatibility and the ability to elicit protective antibody titers similar or superior to the commercially available toxoid vaccine.

Frequency of PRRS live vaccine virus (European and North American genotype) in vaccinated and non-vaccinated pigs submitted for respiratory tract diagnostics in North-Western Germany

The frequency of PRRSV corresponding to live vaccines and wild-type was determined in 902 pigs from North-Western Germany submitted for post-mortem examination. Overall, 18.5% of the samples were positive for the EU wild-type virus. EU genotype vaccine virus was detected in 1.3% and the NA genotype vaccine virus in 8.9% of all samples. The detection of the EU vaccine was significantly higher in pigs vaccinated with the corresponding vaccine (OR=9.4). Pigs vaccinated with NA genotype had significantly higher detection chances for the corresponding vaccine virus when compared to non-vaccinated animals (OR=3.34) animals, however, NA vaccine was also frequently detected in non-vaccinated pigs. Concluding, the dynamics of NA genotype vaccine and EU wild-type virus corresponds with studies on PRRSV spread in endemically infected herds. The potential of spontaneous spread of the NA genotype vaccine should be considered in the planning of eradication programs.