The TLR3 Agonist Poly Inosinic:Cytidylic Acid Significantly Augments the Therapeutic Activity of an Anti-CD7 Immunotoxin for Human T-cell Leukaemia.

We have previously shown that antibody-dependent cellular cytotoxicity (ADCC) cooperates with immunotoxin (IT)-mediated killing of human leukaemia cells in an severe combined immunodeficient (SCID) mouse model of human T-cell acute lymphoblastic leukaemia (SCID-HSB-2 mice), but not in an equivalent non-obese diabetic (NOD)/SCID mouse model. In these earlier studies, we reasoned that diminished ADCC due to the functional deficit in natural killer (NK) cell activity in NOD/SCID mice resulted in a failure of effective perforin/granzyme-mediated cytotoxicity necessary for the delivery of the augmentative effect. Poly-inosinic-cytidylic acid [poly (I:C)] is a synthetic dsRNA toll-like receptor 3 (TLR3) agonist that possesses a number of biological properties that includes the in vivo activation of NK cells. We show here that intravenous (i.v.) injection of SCID mice with [poly (I:C)] results in characteristic time-related changes in serum interleukin 2 (IL-2), IL-12, and interferon γ (INFγ) cytokine levels that are consistent with TLR3 driven activation of SCID mouse NK cells. Concomitantly, there are changes in the expression levels of CD2, CD16/32 (FcγRII/RIII), CD161 (NK1.1), and F4/80 in the bulk splenocyte population. These observed changes correlate with an increase in the in vitro lytic capabilities of putative NK cells from within the splenocyte population of [poly (I:C)] treated SCID mice. We demonstrate that the in vivo activation of NK cells with [poly (I:C)] in SCID mice bearing disseminated human T-cell leukaemia xenografts resulted in a significant improvement in the therapeutic activity exerted by an intact murine monoclonal antibody against human CD7. This was also seen for a saporin-based immunotoxin constructed with the same intact antibody (HB2-SAPORIN), but not with an F(ab')2 derivative of the same antibody or of an IT constructed with the same F(ab')2 HB2 antibody derivative. This study further demonstrates the previously reported reinforcing role of ADCC for the therapeutic activity of IT in an SCID mouse model of human T-ALL and the potential to significantly boost this further with [poly (I:C)]. Our study provides the rationale to justify the exploration of the clinical utility of IT based therapeutics in combination with TLR3 agonists, such as [poly (I:C)], for the treatment of haematological, and possibly other, malignancies.

Lack of Involvement of Fenton Chemistry in Death of Methicillin-Resistant and Methicillin-Sensitive Strains of Staphylococcus aureus and Destruction of Their Genomes on Wet or Dry Copper Alloy Surfaces.

The pandemic of hospital-acquired infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has declined, but the evolution of strains with enhanced virulence and toxins and the increase of community-associated infections are still a threat. In previous studies, 10(7) MRSA bacteria applied as simulated droplet contamination were killed on copper and brass surfaces within 90 min. However, contamination of surfaces is often via finger tips and dries rapidly, and it may be overlooked by cleaning regimes (unlike visible droplets). In this new study, a 5-log reduction of a hardy epidemic strain of MRSA (epidemic methicillin-resistant S. aureus 16 [EMRSA-16]) was observed following 10 min of contact with copper, and a 4-log reduction was observed on copper nickel and cartridge brass alloys in 15 min. A methicillin-sensitive S. aureus (MSSA) strain from an osteomyelitis patient was killed on copper surfaces in 15 min, and 4-log and 3-log reductions occurred within 20 min of contact with copper nickel and cartridge brass, respectively. Bacterial respiration was compromised on copper surfaces, and superoxide was generated as part of the killing mechanism. In addition, destruction of genomic DNA occurs on copper and brass surfaces, allaying concerns about horizontal gene transfer and copper resistance. Incorporation of copper alloy biocidal surfaces may help to reduce the spread of this dangerous pathogen.

Human Coronavirus 229E Remains Infectious on Common Touch Surface Materials.

UNLABELLED: The evolution of new and reemerging historic virulent strains of respiratory viruses from animal reservoirs is a significant threat to human health. Inefficient human-to-human transmission of zoonotic strains may initially limit the spread of transmission, but an infection may be contracted by touching contaminated surfaces. Enveloped viruses are often susceptible to environmental stresses, but the human coronaviruses responsible for severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) have recently caused increasing concern of contact transmission during outbreaks. We report here that pathogenic human coronavirus 229E remained infectious in a human lung cell culture model following at least 5 days of persistence on a range of common nonbiocidal surface materials, including polytetrafluoroethylene (Teflon; PTFE), polyvinyl chloride (PVC), ceramic tiles, glass, silicone rubber, and stainless steel. We have shown previously that noroviruses are destroyed on copper alloy surfaces. In this new study, human coronavirus 229E was rapidly inactivated on a range of copper alloys (within a few minutes for simulated fingertip contamination) and Cu/Zn brasses were very effective at lower copper concentration. Exposure to copper destroyed the viral genomes and irreversibly affected virus morphology, including disintegration of envelope and dispersal of surface spikes. Cu(I) and Cu(II) moieties were responsible for the inactivation, which was enhanced by reactive oxygen species generation on alloy surfaces, resulting in even faster inactivation than was seen with nonenveloped viruses on copper. Consequently, copper alloy surfaces could be employed in communal areas and at any mass gatherings to help reduce transmission of respiratory viruses from contaminated surfaces and protect the public health. IMPORTANCE: Respiratory viruses are responsible for more deaths globally than any other infectious agent. Animal coronaviruses that "host jump" to humans result in severe infections with high mortality, such as severe acute respiratory syndrome (SARS) and, more recently, Middle East respiratory syndrome (MERS). We show here that a closely related human coronavirus, 229E, which causes upper respiratory tract infection in healthy individuals and serious disease in patients with comorbidities, remained infectious on surface materials common to public and domestic areas for several days. The low infectious dose means that this is a significant infection risk to anyone touching a contaminated surface. However, rapid inactivation, irreversible destruction of viral RNA, and massive structural damage were observed in coronavirus exposed to copper and copper alloy surfaces. Incorporation of copper alloy surfaces in conjunction with effective cleaning regimens and good clinical practice could help to control transmission of respiratory coronaviruses, including MERS and SARS.

Inactivation of murine norovirus on a range of copper alloy surfaces is accompanied by loss of capsid integrity.

Norovirus is one of the most common causes of acute viral gastroenteritis. The virus is spread via the fecal-oral route, most commonly from infected food and water, but several outbreaks have originated from contamination of surfaces with infectious virus. In this study, a close surrogate of human norovirus causing gastrointestinal disease in mice, murine norovirus type 1 (MNV-1), retained infectivity for more than 2 weeks following contact with a range of surface materials, including Teflon (polytetrafluoroethylene [PTFE]), polyvinyl chloride (PVC), ceramic tiles, glass, silicone rubber, and stainless steel. Persistence was slightly prolonged on ceramic surfaces. A previous study in our laboratory observed that dry copper and copper alloy surfaces rapidly inactivated MNV-1 and destroyed the viral genome. In this new study, we have observed that a relatively small change in the percentage of copper, between 70 and 80% in copper nickels and 60 and 70% in brasses, had a significant influence on the ability of the alloy to inactivate norovirus. Nickel alone did not affect virus, but zinc did have some antiviral effect, which was synergistic with copper and resulted in an increased efficacy of brasses with lower percentages of copper. Electron microscopy of purified MNV-1 that had been exposed to copper and stainless steel surfaces suggested that a massive breakdown of the viral capsid had occurred on copper. In addition, MNV-1 that had been exposed to copper and treated with RNase demonstrated a reduction in viral gene copy number. This suggests that capsid integrity is compromised upon contact with copper, allowing copper ion access to the viral genome.

Inactivation of norovirus on dry copper alloy surfaces.

Noroviruses (family Caliciviridae) are the primary cause of viral gastroenteritis worldwide. The virus is highly infectious and touching contaminated surfaces can contribute to infection spread. Although the virus was identified over 40 years ago the lack of methods to assess infectivity has hampered the study of the human pathogen. Recently the murine virus, MNV-1, has successfully been used as a close surrogate. Copper alloys have previously been shown to be effective antimicrobial surfaces against a range of bacteria and fungi. We now report rapid inactivation of murine norovirus on alloys, containing over 60% copper, at room temperature but no reduction of infectivity on stainless steel dry surfaces in simulated wet fomite and dry touch contamination. The rate of inactivation was initially very rapid and proportional to copper content of alloy tested. Viral inactivation was not as rapid on brass as previously observed for bacteria but copper-nickel alloy was very effective. The use of chelators and quenchers of reactive oxygen species (ROS) determined that Cu(II) and especially Cu(I) ions are still the primary effectors of toxicity but quenching superoxide and hydroxyl radicals did not confer protection. This suggests Fenton generation of ROS is not important for the inactivation mechanism. One of the targets of copper toxicity was the viral genome and a reduced copy number of the gene for a viral encoded protein, VPg (viral-protein-genome-linked), which is essential for infectivity, was observed following contact with copper and brass dry surfaces. The use of antimicrobial surfaces containing copper in high risk closed environments such as cruise ships and care facilities could help to reduce the spread of this highly infectious and costly pathogen.

Horizontal transfer of antibiotic resistance genes on abiotic touch surfaces: implications for public health.

UNLABELLED: Horizontal gene transfer (HGT) is largely responsible for increasing the incidence of antibiotic-resistant infections worldwide. While studies have focused on HGT in vivo, this work investigates whether the ability of pathogens to persist in the environment, particularly on touch surfaces, may also play an important role. Escherichia coli, virulent clone ST131, and Klebsiella pneumoniae harboring extended-spectrum-ß-lactamase (ESBL) bla(CTX-M-15) and metallo-ß-lactamase bla(NDM-1), respectively, exhibited prolonged survival on stainless steel, with approximately 10(4) viable cells remaining from an inoculum of 10(7) CFU per cm(2) after 1 month at 21°C. HGT of bla to an antibiotic-sensitive but azide-resistant recipient E. coli strain occurred on stainless steel dry touch surfaces and in suspension but not on dry copper. The conjugation frequency was approximately 10 to 50 times greater and occurred immediately, and resulting transconjugants were more stable with ESBL E. coli as the donor cell than with K. pneumoniae, but bla(NDM-1) transfer increased with time. Transconjugants also exhibited the same resistance profile as the donor, suggesting multiple gene transfer. Rapid death, inhibition of respiration, and destruction of genomic and plasmid DNA of both pathogens occurred on copper alloys accompanied by a reduction in bla copy number. Naked E. coli DNA degraded on copper at 21°C and 37°C but slowly at 4°C, suggesting a direct role for the metal. Persistence of viable pathogenic bacteria on touch surfaces may not only increase the risk of infection transmission but may also contribute to the spread of antibiotic resistance by HGT. The use of copper alloys as antimicrobial touch surfaces may help reduce infection and HGT. IMPORTANCE: Horizontal gene transfer (HGT) conferring resistance to many classes of antimicrobials has resulted in a worldwide epidemic of nosocomial and community infections caused by multidrug-resistant microorganisms, leading to suggestions that we are in effect returning to the preantibiotic era. While studies have focused on HGT in vivo, this work investigates whether the ability of pathogens to persist in the environment, particularly on touch surfaces, may also play an important role. Here we show prolonged (several-week) survival of multidrug-resistant Escherichia coli and Klebsiella pneumoniae on stainless steel surfaces. Plasmid-mediated HGT of ß-lactamase genes to an azide-resistant recipient E. coli strain occurred when the donor and recipient cells were mixed together on stainless steel and in suspension but not on copper surfaces. In addition, rapid death of both antibiotic-resistant strains and destruction of plasmid and genomic DNA were observed on copper and copper alloy surfaces, which could be useful in the prevention of infection spread and gene transfer.

The anti-CD20 antibody rituximab augments the immunospecific therapeutic effectiveness of an anti-CD19 immunotoxin directed against human B-cell lymphoma.

The chimaeric anti-CD20 antibody rituximab (Rituxan) sensitises lymphoma cells to small molecule cytotoxic drugs and to protein toxins. We have explored the augmentive effect of rituximab on the anti-CD19 immunotoxin BU12-SAPORIN in a model of human lymphoma. Intact rituximab and its F(ab)2 derivative both augmented the immunospecific protein synthesis inhibitory effects of BU12-SAPORIN in a complement-independent manner. A combination of rituximab + BU12-SAPORIN completely abolished the proliferation of Ramos cells in vitro and also induced a significantly greater degree of apoptosis in these cells. Treatment with rituximab, BU12-SAPORIN or a combination of both induced poly(ADPribose) polymerase and caspase 3 cleavage, although this was always consistently greater in combination-treated cells. zVAD almost completely inhibited apoptosis in rituximab- or BU12-SAPORIN-treated cells but only partially in combination-treated cells. In severe combined immunodeficient (SCID)-Ramos mice the combination of rituximab + BU12-SAPORIN was significantly better therapeutically than either single agent. The immunological fidelity of the therapeutic effect because of combination treatment was demonstrated through the failure of rituximab to augment an irrelevant anti-CD7 immunotoxin. The therapeutic efficacy of rituximab and combination treatment was reduced in SCID-Ramos mice depleted of serum complement while natural killer cell depletion failed to show any convincing role for antibody-dependent cellular cytotoxicity. This study shows a clear therapeutic advantage from using rituximab to immunospecifically augment immunotoxin cytotoxicity warranting further investigation.