Activity of vaccinia virus-neutralizing antibody in the sera of smallpox vaccinees

Individuals vaccinated against smallpox maintain substantial antiviral antibody responses for many years after vaccination. In this study, we examined the ability of antiviral antibodies from 104 unique serum samples to neutralize the two infectious forms of vaccinia virus, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). While we found direct correlations between antiviral antibody titers and the ability to neutralize IMV and EEV, correlation with EEV neutralization was weaker. To determine factors that may influence more varied EEV neutralization within a vaccinated population, we asked the following questions. (1) Does vaccinia virus-neutralizing ability remain constant over time? (2) Do multiple vaccinations boost IMV and EEV neutralization activity? We found that serum from vaccinated individuals retained ability to neutralize EEV for a relatively long time, but there was a significant drop in EEV neutralization ability in the third decade after vaccination. While all vaccinees maintained some ability to neutralize IMV, a number of individuals lost the capacity to neutralize EEV. Interestingly, the ability to neutralize either virus form was not altered by the number of vaccinations received. Since it is likely that neutralizing antibodies against both IMV and EEV are required for maximal protective immunity, a loss of anti-EEV-neutralizing ability may warrant the revaccination of individuals who have been vaccinated >20 years ago, should widespread pre-event smallpox vaccination be instituted.     

A mouse-based assay for the pre-clinical neurovirulence assessment of vaccinia virus-based smallpox vaccines

Post-vaccinal encephalitis, although relatively uncommon, is a known adverse event associated with many live, attenuated smallpox vaccines. Although smallpox vaccination ceased globally in 1980, vaccine manufacture has resumed in response to concerns over the possible use of smallpox virus as an agent of bioterrorism. To better support the production of safer smallpox vaccines, we previously reported the development of a mouse model in which a relatively attenuated vaccine strain (Dryvax^®) could be discerned from a more virulent laboratory strain (WR). Here we have further tested the performance of this assay by evaluating the neurovirulence of several vaccinia virus-based smallpox vaccines spanning a known range in neurovirulence for humans. Our data indicate that testing of 10–100 pfu of virus in mice following intracranial inoculation reliably assesses the virus's neurovirulence potential for humans.     

Rabbitpox virus and vaccinia virus infection of rabbits as a model for human smallpox

The threat of smallpox release and use as a bioweapon has encouraged the search for new vaccines and antiviral drugs, as well as development of new small-animal models in which their efficacy can be determined. Here, we reinvestigate a rabbit model in which the intradermal infection of rabbits with very low doses of either rabbitpox virus (RPV) or vaccinia virus Western Reserve (VV-WR) recapitulates many of the clinical features of human smallpox. Following intradermal inoculation with RPV, rabbits develop systemic disease characterized by extensive viremia, numerous secondary lesions on the skin and mucocutaneous tissues, severe respiratory disease, death by 9 days postinfection, and, importantly, natural aerosol transmission between animals. Contrary to previous reports, intradermal infection with VV-WR also resulted in a very similar lethal systemic disease in rabbits, again with natural aerosol transmission between animals. When sentinel and index animals were cohoused, transmission rates approached 100% with either virus, with sentinel animals exhibiting a similar, severe disease. Lower rates of transmission were observed when index and sentinel animals were housed in separate cages. Sentinel animals infected with RPV with one exception succumbed to the disease. However, the majority of VV-WR-infected sentinel animals, while becoming seriously ill, survived. Finally, we tested the efficacy of the drug 1-O-hexadecyloxypropyl-cidofovir in the RPV/rabbit model and found that an oral dose of 5 mg/kg twice a day for 5 days beginning 1 day before infection was able to completely protect rabbits from lethal disease.     

Heat shock response to vaccinia virus infection.

We have investigated the induction of heat shock proteins (HSPs) in mice infected with vaccinia virus. Vaccinia virus replicates to high levels in the ovaries of infected mice and causes a significant inhibition of host cell DNA, RNA, and protein synthesis. Many HSPs are constitutively expressed in murine ovarian tissue at low levels, consistent with their obligatory role in normal physiological events. In contrast with these events, HSP expression was augmented in virus-infected mouse ovaries 6 days postinfection. In particular, there was a dramatic increase in the expression of a protein identified as the inducible 72-kDa HSP. Analysis of cellular mRNA confirmed this protein to be the major mouse inducible HSP70 and demonstrated its presence within virus-infected cells. Hence, we have demonstrated the expression of stress proteins during poxvirus infection in vivo.     

Proteome-wide analysis of neural stem cell differentiation to facilitate transition to cell replacement therapies

Neurodegenerative diseases are devastating disorders and the demands on their treatment are set to rise in connection with higher disease incidence. Knowledge of the spatiotemporal profile of cellular protein expression during neural differentiation and definition of a set of markers highly specific for targeted neural populations is a key challenge. Intracellular proteins may be utilized as a readout for follow-up transplantation and cell surface proteins may facilitate isolation of the cell subpopulations, while secreted proteins could help unravel intercellular communication and immunomodulation. This review summarizes the potential of proteomics in revealing molecular mechanisms underlying neural differentiation of stem cells and presents novel candidate proteins of neural subpopulations, where understanding of their functionality may accelerate transition to cell replacement therapies.     

Isolation and identification of the smallpox virus which caused iatrogenic vaccinia in children in the city of Vladivostok

The comparative study of the specimens of the morphological elements of exanthema obtained from 8 children with the clinical diagnosis of secondary exogenic vaccinia, dried smallpox vaccine and the cultures of other orthopoxviruses (rabbit pox, monkey pox and buffalo pox viruses) was made. The isolation and identification of the causative agents from the specimens was carried out with the use of modern virological, electron microscopic and molecular methods. The study proved the fact that 8 children had orthopoxvirus infection with its causative agent identified as vaccinia virus.     

New insights about host response to smallpox using microarray data

Background  

Smallpox is a lethal disease that was endemic in many parts of the world until eradicated by massive immunization. Due to its lethality, there are serious concerns about its use as a bioweapon. Here we analyze publicly available microarray data to further understand survival of smallpox infected macaques, using systems biology approaches. Our goal is to improve the knowledge about the progression of this disease.     

How vaccinia virus has evolved to subvert the host immune response

Viruses are obligate intracellular parasites and are some of the most rapidly evolving and diverse pathogens encountered by the host immune system. Large complicated viruses, such as poxviruses, have evolved a plethora of proteins to disrupt host immune signalling in their battle against immune surveillance. Recent X-ray crystallographic analysis of these viral immunomodulators has helped form an emerging picture of the molecular details of virus-host interactions. In this review we consider some of these immune evasion strategies as they apply to poxviruses, from a structural perspective, with specific examples from the European SPINE2-Complexes initiative. Structures of poxvirus immunomodulators reveal the capacity of viruses to mimic and compete against the host immune system, using a diverse range of structural folds that are unique or acquired from their hosts with both enhanced and unexpectedly divergent functions.     

Analyzing bioterror response logistics: the case of smallpox

To evaluate existing and alternative proposals for emergency response to a deliberate smallpox attack, we embed the key operational features of such interventions into a smallpox disease transmission model. We use probabilistic reasoning within an otherwise deterministic epidemic framework to model the 'race to trace', i.e., attempting to trace (via the infector) and vaccinate an infected person while (s)he is still vaccine-sensitive. Our model explicitly incorporates a tracing/vaccination queue, and hence can be used as a capacity planning tool. An approximate analysis of this large (16 ODE) system yields closed-form estimates for the total number of deaths and the maximum queue length. The former estimate delineates the efficacy (i.e., accuracy) and efficiency (i.e., speed) of contact tracing, while the latter estimate reveals how congestion makes the race to trace more difficult to win, thereby causing more deaths. A probabilistic analysis is also used to find an approximate closed-form expression for the total number of deaths under mass vaccination, in terms of both the basic reproductive ratio and the vaccination capacity. We also derive approximate thresholds for initially controlling the epidemic for more general interventions that include imperfect vaccination and quarantine.     

Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli

The E. coli chaperonin GroEL and its cofactor GroES promote protein folding by sequestering nonnative polypeptides in a cage-like structure. Here we define the contribution of this system to protein folding across the entire E. coli proteome. Approximately 250 different proteins interact with GroEL, but most of these can utilize either GroEL or the upstream chaperones trigger factor (TF) and DnaK for folding. Obligate GroEL-dependence is limited to only approximately 85 substrates, including 13 essential proteins, and occupying more than 75% of GroEL capacity. These proteins appear to populate kinetically trapped intermediates during folding; they are stabilized by TF/DnaK against aggregation but reach native state only upon transfer to GroEL/GroES. Interestingly, substantially enriched among the GroEL substrates are proteins with (betaalpha)8 TIM-barrel domains. We suggest that the chaperonin system may have facilitated the evolution of this fold into a versatile platform for the implementation of numerous enzymatic functions.     

Proteome-wide analysis of protein function composition reveals the clustering and phylogenetic properties of organisms

A 17-dimensional vector named the proteome vector is defined to represent an organism. The components of the vector reflect the relative contents of protein-encoding genes of the 17 cluster of orthologous groups of proteins (COGs) classes in the whole genome of the relevant organism. Based on the definition of this proteome vector, the fuzzy clustering of 36 completely sequenced organisms (8 archaea, 24 bacteria, and 4 eukarya) was performed and a proteome tree was constructed. Our results show that (1) the 36 organisms can be 100% correctly classified into three clusters corresponding to the three primary kingdoms, (2) our proteome tree is remarkably similar to that derived from 16S rRNA, and (3) the chromosomes and/or plasmids belonging to the same organism have very similar gene composition. Based on these results, we argue that the 17-dimensional proteome vector could be a good criterion for clustering approaches and to a large extent reveals the phylogenetic properties of organisms; the Three Primary Kingdoms Hypothesis is trustworthy although the existence of lateral gene transfer (LGT) brings controversy to the construction of the "universal tree of life."     

Investigation into the presence of and serological response to XMRV in CFS patients

The novel human gammaretrovirus xenotropic murine leukemia virus-related virus (XMRV), originally described in prostate cancer, has also been implicated in chronic fatigue syndrome (CFS). When later reports failed to confirm the link to CFS, they were often criticised for not using the conditions described in the original study. Here, we revisit our patient cohort to investigate the XMRV status in those patients by means of the original PCR protocol which linked the virus to CFS. In addition, sera from our CFS patients were assayed for the presence of xenotropic virus envelope protein, as well as a serological response to it. The results further strengthen our contention that there is no evidence for an association of XMRV with CFS, at least in the UK.