Disenfranchised DNA: biochemical analysis of mutant øX174 DNA-binding proteins may further elucidate the evolutionary significance of the unessential packaging protein A.

Most icosahedral DNA viruses package and condense their genomes into pre-formed, volumetrically constrained capsids. However, concurrent genome biosynthesis and packaging are specific to single-stranded (ss) DNA micro- and parvoviruses. Before packaging, ~120 copies of the øX174 DNA-binding protein J interact with double-stranded DNA. 60 J proteins enter the procapsid with the ssDNA genome, guiding it between 60 icosahedrally ordered DNA-binding pockets formed by the capsid proteins. Although J proteins are small, 28-37 residues in length, they have two domains. The basic, positively charged N-terminus guides the genome between binding pockets, whereas the C-terminus acts as an anchor to the capsid's inner surface. Three C-terminal aromatic residues, W30, Y31, and F37, interact most extensively with the coat protein. Their corresponding codons were mutated, and the resulting strains were biochemically and genetically characterized. Depending on the mutation, the substitutions produced unstable packaging complexes, unstable virions, infectious progeny, or particles packaged with smaller genomes, the latter being a novel phenomenon. The smaller genomes contained internal deletions. The juncture sequences suggest that the unessential A* (A star) protein mediates deletion formation.IMPORTANCEUnessential but strongly conserved gene products are understudied, especially when mutations do not confer discernable phenotypes or the protein's contribution to fitness is too small to reliably determine in laboratory-based assays. Consequently, their functions and evolutionary impact remain obscure. The data presented herein suggest that microvirus A* proteins, discovered over 40 years ago, may hasten the termination of non-productive packaging events. Thus, performing a salvage function by liberating the reusable components of the failed packaging complexes, such as DNA templates and replication enzymes.

Plaque Size Tool: An automated plaque analysis tool for simplifying and standardising bacteriophage plaque morphology measurements.

Bacteriophage plaque size measurement is essential for phage characterisation, but manual size estimation requires a considerable amount of time and effort. In order to ease the work of phage researchers, we have developed an automated command-line application called Plaque Size Tool (PST) that can detect plaques of different morphology on the images of Petri dishes and measure plaque area and diameter. Plaque size measurements using PST showed no difference to those obtained with manual plaque size measurement in Fiji, indicating future results using PST are backwards compatible with prior measurements in the literature. PST can be applied to a range of lytic bacteriophages producing oval-shaped plaques, including bull's-eye and turbid morphology. The application can also be used for titer calculation if most of the plaques are stand-alone. As laboratory automation becomes more commonplace, standardised and flexible open-source analytical tools like PST will be important parts of biofoundry and cloud lab bacteriophage workflows.

Inactivation modeling of human enteric virus surrogates, MS2, Qß, and ΦX174, in water using UVC-LEDs, a novel disinfecting system.

In order to assure the microbial safety of drinking water, UVC-LED treatment has emerged as a possible technology to replace the use of conventional low pressure (LP) mercury vapor UV lamps. In this investigation, inactivation of Human Enteric Virus (HuEV) surrogates with UVC-LEDs was investigated in a water disinfection system, and kinetic model equations were applied to depict the surviving infectivities of the viruses. MS2, Qß, and ΦX 174 bacteriophages were inoculated into sterile distilled water (DW) and irradiated with UVC-LED printed circuit boards (PCBs) (266nm and 279nm) or conventional LP lamps. Infectivities of bacteriophages were effectively reduced by up to 7-log after 9mJ/cm2 treatment for MS2 and Qß, and 1mJ/cm2 for ΦX 174. UVC-LEDs showed a superior viral inactivation effect compared to conventional LP lamps at the same dose (1mJ/cm2). Non-log linear plot patterns were observed, so that Weibull, Biphasic, Log linear-tail, and Weibull-tail model equations were used to fit the virus survival curves. For MS2 and Qß, Weibull and Biphasic models fit well with R2 values approximately equal to 0.97-0.99, and the Weibull-tail equation accurately described survival of ΦX 174. The level of UV-susceptibility among coliphages measured by the inactivation rate constant, k, was statistically different (ΦX 174 (ssDNA)>MS2, Qß (ssRNA)), and indicated that sensitivity to UV was attributed to viral genetic material.

Enteric Viral Surrogate Reduction by Chitosan.

Enteric viruses are a major problem in the food industry, especially as human noroviruses are the leading cause of nonbacterial gastroenteritis. Chitosan is known to be effective against some enteric viral surrogates, but more detailed studies are needed to determine the precise application variables. The main objective of this work was to determine the effect of increasing chitosan concentration (0.7-1.5% w/v) on the cultivable enteric viral surrogates, feline calicivirus (FCV-F9), murine norovirus (MNV-1), and bacteriophages (MS2 and phiX174) at 37 °C. Two chitosans (53 and 222 kDa) were dissolved in water (53 kDa) or 1% acetic acid (222 KDa) at 0.7-1.5%, and were then mixed with each virus to obtain a titer of ~5 log plaque-forming units (PFU)/mL. These mixtures were incubated for 3 h at 37 °C. Controls included untreated viruses in phosphate-buffered saline and viruses were enumerated by plaque assays. The 53 kDa chitosan at the concentrations tested reduced FCV-F9, MNV-1, MS2, and phi X174 by 2.6-2.9, 0.1-0.4, 2.6-2.8, and 0.7-0.9 log PFU/mL, respectively, while reduction by 222 kDa chitosan was 2.2-2.4, 0.8-1.0, 2.6-5.2, and 0.5-0.8 log PFU/mL, respectively. The 222 kDa chitosan at 1 and 0.7% w/v in acetic acid (pH 4.5) caused the greatest reductions of MS2 by 5.2 logs and 2.6 logs, respectively. Overall, chitosan treatments showed the greatest reduction of MS2, followed by FCV-F9, phi X174, and MNV-1. These two chitosans may contribute to the reduction of enteric viruses at the concentrations tested but would require use of other hurdles to eliminate food borne viruses.

Adaptive regulatory substitutions affect multiple stages in the life cycle of the bacteriophage φX174.

BACKGROUND: Previously, we showed that adaptive substitutions in one of the three promoters of the bacteriophage φX174 improved fitness at high-temperature by decreasing transcript levels three- to four-fold. To understand how such an extreme change in gene expression might lead to an almost two-fold increase in fitness at the adaptive temperature, we focused on stages in the life cycle of the phage that occur before and after the initiation of transcription. For both the ancestral strain and two single-substitution strains with down-regulated transcription, we measured seven phenotypic components of fitness (attachment, ejection, eclipse, virion assembly, latent period, lysis rate and burst size) during a single cycle of infection at each of two temperatures. The lower temperature, 37°C, is the optimal temperature at which phages are cultivated in the lab; the higher temperature, 42°C, exerts strong selection and is the condition under which these substitutions arose in evolution experiments. We augmented this study by developing an individual-based stochastic model of this same life cycle to explore potential explanations for our empirical results. RESULTS: Of the seven fitness parameters, three showed significant differences between strains that carried an adaptive substitution and the ancestor, indicating the presence of pleiotropy in regulatory evolution. 1) Eclipse was longer in the adaptive strains at both the optimal and high-temperature environments. 2) Lysis rate was greater in the adaptive strains at the high temperature. 3) Burst size for the mutants was double that of the ancestor at the high temperature, but half that at the lower temperature. Simulation results suggest that eclipse length and latent period variance can explain differences in burst sizes and fitness between the mutant and ancestral strains. CONCLUSIONS: Down-regulating transcription affects several steps in the phage life cycle, and all of these occur after the initiation of transcription. We attribute the apparent tradeoff between delayed progeny production and faster progeny release to improved host resource utilization at high temperature.

Effects of an early conformational switch defect during ϕX174 morphogenesis are belatedly manifested late in the assembly pathway.

C-terminal, aromatic amino acids in the ϕX174 internal scaffolding protein B mediate conformational switches in the viral coat protein. These switches direct the coat protein through early assembly. In addition to the aromatic amino acids, two acidic residues, D111 and E113, form salt bridges with basic, coat protein side chains. Although salt bridge formation did not appear to be critical for assembly, the substitution of an aromatic amino acid for D111 produced a lethal phenotype. This side chain is uniquely oriented toward the center of the coat-scaffolding binding pocket, which is heavily dominated by aromatic ring-ring interactions. Thus, the D111Y substitution may restructure pocket contacts. Previously characterized B(-) mutants blocked assembly before procapsid formation. However, the D111Y mutant produced an assembled particle, which contained the structural and external scaffolding proteins but lacked protein B and DNA. A suppressor within the external scaffolding protein, which mediates the later stages of particle morphogenesis, restored viability. The unique formation of a postprocapsid particle and the novel suppressor may be indicative of a novel B protein function. However, genetic data suggest that the particle represents the delayed manifestation of an early assembly error. This seemingly late-acting defect was rescued by previously characterized suppressors of early, preprocapsid, B(-) assembly mutations, which act on the level of coat protein flexibility. Likewise, the newly isolated suppressor in the external scaffolding protein also exhibited a global suppressing phenotype. Thus, the off-pathway product isolated from infected cells may not accurately reflect the temporal nature of the initial defect.

The distribution of mutational fitness effects of phage φX174 on different hosts.

Adaptation depends greatly on the distribution of mutation fitness effects (DMFE), but the phenotypic expression of mutations is often environment dependent. The environments faced by multihost pathogens are mostly governed by their hosts and therefore measuring the DMFE on multiple hosts can inform on the likelihood of short-term establishment and longer term adaptation of emerging pathogens. We explored this by measuring the growth rate of 36 mutants of the lytic bacteriophage φX174 on two host backgrounds, Escherichia coli (EcC) and Salmonella typhimurium (StGal). The DMFE showed higher mean and variance on EcC than on StGal. Most mutations were either deleterious or neutral on both hosts, but a greater proportion of mutations were deleterious on StGal. We identified two mutations with beneficial fitness effects on EcC that were neutral on StGal. Host-specific differences in fitness were associated with particular functional classes of genes involved in the initial stages of infection in accordance with previous studies of host specificity. Overall, there was a positive correlation between the effects of mutations on each host, suggesting that most new mutations will have general, rather than host-specific fitness effects. We consider these results in light of simple fitness landscape models of adaptation and discuss the relevance of context-dependent DMFE for multihost pathogens.

Nucleoside analogue mutagenesis of a single-stranded DNA virus: evolution and resistance.

It has been well established that chemical mutagenesis has adverse fitness effects in RNA viruses, often leading to population extinction. This is mainly a consequence of the high RNA virus spontaneous mutation rates, which situate them close to the extinction threshold. Single-stranded DNA viruses are the fastest-mutating DNA-based systems, with per-nucleotide mutation rates close to those of some RNA viruses, but chemical mutagenesis has been much less studied in this type of viruses. Here, we serially passaged bacteriophage X174 in the presence of the nucleoside analogue 5-fluorouracil (5-FU). We found that 5-FU was unable to trigger population extinction for the range of concentrations tested, but it negatively affected viral adaptability. The phage evolved partial drug resistance, and parallel nucleotide substitutions appearing in independently evolved lines were identified as candidate resistance mutations. Using site-directed mutagenesis, two single-nucleotide substitutions in the lysis protein E (T572C and A781G) were shown to be selectively advantageous in the presence of 5-FU. In RNA viruses, base analogue resistance is often mediated by changes in the viral polymerase, but this mechanism is not possible for X174 and other single-stranded DNA viruses because they do not encode their own polymerase. In addition to increasing mutation rates, 5-FU produces a wide variety of cytotoxic effects at the levels of replication, transcription, and translation. We found that substitutions T572C and A781G lost their ability to confer 5-FU resistance after cells were supplemented with deoxythymidine, suggesting that their mechanism of action is at the DNA level. We hypothesize that regulation of lysis time may allow the virus to optimize progeny size in cells showing defects in DNA synthesis.

Optimizing cord blood sample cryopreservation.

BACKGROUND AIMS: Cord blood (CB) banking is becoming more and more commonplace throughout the medical community, both in the USA and elsewhere. It is now generally recognized that storage of CB samples in multiple aliquots is the preferred approach to banking because it allows the greatest number of uses of the sample. However, it is unclear which are the best methodologies for cryopreservation and storage of the sample aliquots. In the current study we analyzed variables that could affect these processes. METHODS: CB were processed into mononuclear cells (MNC) and frozen in commercially available human serum albumin (HSA) or autologous CB plasma using cryovials of various sizes and cryobags. The bacteriophage phiX174 was used as a model virus to test for cross-contamination. RESULTS: We observed that cryopreservation of CB in HSA, undiluted autologous human plasma and 50% diluted plasma was equivalent in terms of cell recovery and cell viability. We also found that cryopreservation of CB samples in either cryovials or cryobags displayed equivalent thermal characteristics. Finally, we demonstrated that overwrapping the CB storage container in an impermeable plastic sheathing was sufficient to prevent cross-sample viral contamination during prolonged storage in the liquid phase of liquid nitrogen dewar storage. CONCLUSION: CB may be cryopreserved in either vials or bags without concern for temperature stability. Sample overwrapping is sufficient to prevent microbiologic contamination of the samples while in liquid-phase liquid nitrogen storage.

Enhancement of bacteriolysis of shuffled phage PhiX174 gene E.

Bacterial ghosts that are generated using the regulated PhiX174 lysis gene E offer a new avenue for the study of inactivated vaccines. Here, we constructed a library of mutant gene E using a gene-shuffling technique. After screening and recombination with the prokaryotic non-fusion expression vector pBV220, two lysis plasmids were selected. Among which, a novel mutant E gene (named mE), consisting of a 74-bp non-encoding sequence at 5'-end and a 201-bp gene ΔE, significantly increased the lysis effect on prokaryotic Escherichia coli and Salmonella enteritidis. Moreover, lysis efficiency, as measured by the OD600 value, reached 1.0 (109 CFU), avoiding the bottleneck problem observed with other bacterial lysis procedures, which results in a low concentration of bacteria in suspension, and consequent low production of bacterial ghosts. Our results may provide a promising avenue for the development of bacterial ghost vaccines.

Mechanisms responsible for a PhiX174 mutant's ability to infect Escherichia coli by phosphorylation.

The ability for a virus to expand its host range is dependent upon a successful mode of viral entry. As such, the host range of the well-studied PhiX174 bacteriophage is dictated by the presence of a particular lipopolysaccharide (LPS) on the bacterial surface. The mutant PhiX174 strain JACS-K, unlike its ancestor, is capable of infecting both its native host Escherichia coli C and E. coli K-12, which does not have the necessary LPS. The conversion of an alanine to a very reactive threonine on its virion surface was found to be responsible for the strain's expanded host range.

Detection of somatic coliphages through a bioluminescence assay measuring phage mediated release of adenylate kinase and adenosine 5'-triphosphate.

The feasibility of detecting somatic coliphages by phage infection of Escherichia coli WG5 and measurement of phage propagation by the lysis mediated release of the bacterial host adenylate kinase (AK) and adenosine 5'-triphosphate (ATP) detected by a bioluminescent signal was evaluated. After 2h of incubation, all cultures infected with reference bacteriophage phiX174 showed a significant increase in the bioluminescent signal, even with number of phages as low as less of 10 plaque forming units (PFU). Naturally occurring somatic coliphages ensured a significant bioluminescent signal after 3h of infection when >10 PFU were inoculated. These results indicate that an easy and reliable method to detect low numbers of coliphages in less than 3h is feasible.

Genomic evolution in a virus under specific selection for host recognition.

Genetic variation in viral structural proteins is often explained by evolutionary escape of strong host defenses through processes such as immune evasion, host switching, and tissue tropism. An understanding of the mechanisms driving evolutionary change in virus surface proteins is key to designing effective intervention strategies to disease emergence. This study investigated the predictability of virus genomic evolution in response to highly specific differences in host receptor structure. The bacteriophage PhiX174 was evolved on three E. coli mutant hosts, each differing only by a single sugar group in the lipopolysaccharides, used for phage attachment. Large phage populations were used in order to maximize the amount of sequence space explored by mutation, and thus the potential for parallel evolution. Repeatability was assessed by genome sequencing of multiple isolates from endpoint populations and by fitness of the endpoint population relative to its ancestor. Evolutionary lines showed similar magnitudes of fitness increase between treatments. Only one mutation, occurring in the internal DNA pilot protein H, was completely repeatable, and it appeared to be a necessary stepping stone toward further adaptive change. Substitutions in the surface accessible major capsid protein F appeared to be involved in capsid stability rather than specific interactions with host receptors, suggesting that non-specific alterations to capsid structure could be an important component of adaptation to novel hosts. 33% of mutations were synonymous and showed evidence of selection on codon usage. Lastly, results supported previous findings that evolving populations of small ssDNA viruses may maintain relatively high levels of genetic variation.

Experimental evolution and genome sequencing reveal variation in levels of clonal interference in large populations of bacteriophage phiX174.

BACKGROUND: In large asexual populations where beneficial mutations may co-occur and recombination is absent, the fate of beneficial mutations can be significantly affected by competition (i.e., clonal interference). Theoretical models predict that clonal interference (CI) can slow adaptation, alter the distribution of fixed beneficial mutations, and affect disease progression by impacting within-host evolution of pathogens. While phenotypic data support that CI is a significant determinant of adaptive outcome, genetic data are needed to verify the patterns and to inform evolutionary models. We adapted replicate populations of the bacteriophage phiX174 under two levels of CaCl2 to create benign and harsh environments. Genomic sequences of multiple individuals from evolved populations were used to detect competing beneficial mutations. RESULTS: There were several competing genotypes in most of the populations where CaCl2 was abundant, but no evidence of CI where CaCl2 was scarce, even though rates of adaptation and population sizes among the treatments were similar. The sequence data revealed that observed mutations were limited to a single portion of one gene in the harsh treatment, but spanned a different and larger region of the genome under the benign treatments, suggesting that there were more adaptive solutions to the benign treatment. CONCLUSION: Beneficial mutations with relatively large selection coefficients can be excluded by CI. CI may commonly determine the fate of beneficial mutations in large microbial populations, but its occurrence depends on selective conditions. CI was more frequent in benign selective conditions possibly due to a greater number of adaptive targets under this treatment. Additionally, the genomic sequence data showed that selection can target different types and numbers of phenotypes in environments that differ by only a single continuous variable.

[Comparative study of the resistance of bacteriophageT4, PhiX174D, MS2 and f2 to gamma radiation].

OBJECTIVE: To screen the suitable bacteriophage as virus indicator in irradiation sterilization. METHODS: Suspensions of bacteriophage T4, phiX174D, MS2, and f2, Escherichia coli 8099, and Bacillus subtilis var.niger.sp. ATCC9372 were irradiated with (60)Co-gamma ray. The mean log(10) inactivation value (LIV) and killing log value (KL) were calculated. RESULTS: (1) Under 100 Gy of gamma-radiation, the LIV levels of the bacteriophage T4, PhiX174, f2, and MS2 were 6.31, 6.92, 5.74, and 4.46 log(10) respectively, all reaching the disinfection level (LIV >/= 4.00 log(10)), (2) Under the same absorbed dose, the KL of Escherichia coli 8099 was > 7.97 log(10); (3) Under the same absorbed dose, the KL of the Bacillus subtilis var.niger.sp. ATCC9372 was 1.61 log(10). CONCLUSION: The order of resistance of the above six microorganisms to gamma-radiation from the biggest to the smallest is as follows: Bacillus subtilis var. niger. sp. > bacteriophage MS2 > bacteriophage f2 > bacteriophage T4 > bacteriophage phiX 174D > E. coli.

In situ ammonia production as a sanitation agent during anaerobic digestion at mesophilic temperature.

AIM: To measure the sanitizing effect of mesophilic (37 degrees C) anaerobic digestion in high ammonia concentrations produced in situ. METHODS AND RESULTS: Indicator organisms and salmonella were transferred to small-scale anaerobic batch cultures and D-values were calculated. Batch cultures were started with material from two biogas processes operating at high (46 mmol l(-1)) and low (1.6 mmol l(-1)) ammonia concentration. D-values were shortened from c. 3 days to <1 day for the bacteria. MS2 had the same D-value (1.3 days) independent of ammonia concentration whereas PhiX174 and 28B were faster inactivated in the control (1.1 and 7.9 days) than in the high ammonia (8.9 and 39 days) batch cultures. CONCLUSION: Running biogas processes at high levels of ammonia shortens the time to meet EU regulation concerning reduction of salmonella and enterococci (5 log). Unless a minimum retention time of 2 days, post-treatment digestion is needed to achieve sufficient sanitation in continuous biogas processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Running mesophilic biogas processes at high ammonia level produces residue with a high fertilizer value. With some stipulations concerning management parameters, such processes provide a method of bacterial sanitation without preceding pasteurization of the incoming organic waste.

[Numerical simulation of isothermal equilibrium adsorption of virus onto typical soils in China].

An isothermal batch experiment was conducted in the laboratory to compare adsorption of bacteriophages MS2 and phiX174 onto 6 different soils (red loam soil, red clay soil, wushan soil, huangni soil, sandy fluvo-aquic soil and loamy fluvo-aquic soil) in China. Soils with sterilized or non-sterilized treatment were used. Relative coefficients of each numerical simulation of the isotherms using three models were evaluated. The results show that the properties of the soil and virus, and presence/absence of the soil autochthonous microorganisms have pronounced effect on the virus adsorption. Both MS2 and phiX174 are almost completely adsorbed by the red clay soil, but minimal adsorption is observed in the two fluvo-aquic soils. Adsorption of phiX174 to all the non-sterilized soils is generally much greater than that of MS2, while sterilization leads to opposite results. Freundlich and Langmuir isotherms are found to have better coefficients to simulate the apparent steady-state virus concentrations. Freundlich isotherm is capable of demonstrating the effect of virus concentration on adsorption behavior. Langmuir isotherms can be used to compare relative adsorption among treatments, while the present study suggests that maximum adsorption can not be calculated when using the Langmuir isotherms.

Characterization and function of putative substrate specificity domain in microvirus external scaffolding proteins.

Microviruses (canonical members are bacteriophages phiX174, G4, and alpha3) are T=1 icosahedral virions with an assembly pathway mediated by two scaffolding proteins. The external scaffolding protein D plays a major role during morphogenesis, particularly in icosahedral shell formation. The results of previous studies, conducted with a cloned chimeric external scaffolding gene, suggest that the first alpha-helix acts as a substrate specificity domain, perhaps mediating the initial coat-external scaffolding protein interaction. However, the expression of a cloned gene could lead to protein concentrations higher than those found in typical infections. Moreover, its induction before infection could alter the timing of the protein's accumulation. Both of these factors could drive or facilitate reactions that may not occur under physiological conditions or before programmed cell lysis. In order to elucidate a more detailed mechanistic model, a chimeric external scaffolding gene was placed directly in the phiX174 genome under wild-type transcriptional and translational control, and the chimeric virus, which was not viable on the level of plaque formation, was characterized. The results of the genetic and biochemical analyses indicate that alpha-helix 1 most likely mediates the nucleation reaction for the formation of the first assembly intermediate containing the external scaffolding protein. Mutants that can more efficiently use the chimeric scaffolding protein were isolated. These second-site mutations appear to act on a kinetic level, shortening the lag phase before virion production, perhaps lowering the critical concentration of the chimeric protein required for a nucleation reaction.

Identification of an interacting coat-external scaffolding protein domain required for both the initiation of phiX174 procapsid morphogenesis and the completion of DNA packaging.

The phiX174 external scaffolding protein D mediates the assembly of coat protein pentamers into procapsids. There are four external scaffolding subunits per coat protein. Organized as pairs of asymmetric dimers, the arrangement is unrelated to quasi-equivalence. The external scaffolding protein contains seven alpha-helices. The protein's core, alpha-helices 2 to 6, mediates the vast majority of intra- and interdimer contacts and is strongly conserved in all Microviridae (canonical members are phiX174, G4, and alpha3) external scaffolding proteins. On the other hand, the primary sequences of the first alpha-helices have diverged. The results of previous studies with alpha3/phiX174 chimeric external scaffolding proteins suggest that alpha-helix 1 may act as a substrate specificity domain, mediating the initial coat scaffolding protein recognition in a species-specific manner. However, the low sequence conservation between the two phages impeded genetic analyses. In an effort to elucidate a more mechanistic model, chimeric external scaffolding proteins were constructed between the more closely related phages G4 and phiX174. The results of biochemical analyses indicate that the chimeric external scaffolding protein inhibits two morphogenetic steps: the initiation of procapsid formation and DNA packaging. phiX174 mutants that can efficiently utilize the chimeric protein were isolated and characterized. The substitutions appear to suppress both morphogenetic defects and are located in threefold-related coat protein sequences that most likely form the pores in the viral procapsid. These results identify coat-external scaffolding domains needed to initiate procapsid formation and provide more evidence, albeit indirect, that the pores are the site of DNA entry during the packaging reaction.

Antigen discovery and delivery of subunit vaccines by nonliving bacterial ghost vectors.

The bacterial ghost (BG) platform system is a novel vaccine delivery system endowed with intrinsic adjuvant properties. BGs are nonliving Gram-negative bacterial cell envelopes which are devoid of their cytoplasmic contents, yet maintain their cellular morphology and antigenic structures, including bioadhesive properties. The main advantages of BGs as carriers of subunit vaccines include their ability to stimulate a high immune response and to target the carrier itself to primary antigen-presenting cells. The intrinsic adjuvant properties of BGs enhance the immune response to target antigens, including T-cell activation and mucosal immunity. Since native and foreign antigens can be carried in the envelope complex of BGs, combination vaccines with multiple antigens of diverse origin can be presented to the immune system simultaneously. Beside the capacity of BGs to function as carriers of protein antigens, they also have a high loading capacity for DNA. Thus, loading BGs with recombinant DNA takes advantage of the excellent bioavailability for DNA-based vaccines and the high expression rates of the DNA-encoded antigens in target cell types such as macrophages and dendritic cells. There are many spaces within BGs including the inner and outer membranes, the periplasmic space and the internal lumen which can carry antigens, DNA or mediators of the immune response. All can be used for subunit antigen to design new vaccine candidates with particle presentation technology. In addition, the fact that BGs can also carry piggyback large-size foreign antigen particles, increases the technologic usefulness of BGs as combination vaccines against viral and bacterial pathogens. Furthermore, the BG antigen carriers can be stored as freeze-dried preparations at room temperature for extended periods without loss of efficacy. The potency, safety and relatively low production cost of BGs offer a significant technical advantage over currently utilized vaccine technologies.