Polyclonal antibody cocktails generated using DNA vaccine technology protect in murine models of orthopoxvirus disease

<p>Abstract</p> <p>Background</p> <p>Previously we demonstrated that DNA vaccination of nonhuman primates (NHP) with a small subset of vaccinia virus (VACV) immunogens (L1, A27, A33, B5) protects against lethal monkeypox virus challenge. The L1 and A27 components of this vaccine target the mature virion (MV) whereas A33 and B5 target the enveloped virion (EV).</p> <p>Results</p> <p>Here, we demonstrated that the antibodies produced in vaccinated NHPs were sufficient to confer protection in a murine model of lethal <it>Orthopoxvirus </it>infection. We further explored the concept of using DNA vaccine technology to produce immunogen-specific polyclonal antibodies that could then be combined into cocktails as potential immunoprophylactic/therapeutics. Specifically, we used DNA vaccines delivered by muscle electroporation to produce polyclonal antibodies against the L1, A27, A33, and B5 in New Zealand white rabbits. The polyclonal antibodies neutralized both MV and EV in cell culture. The ability of antibody cocktails consisting of anti-MV, anti-EV, or a combination of anti-MV/EV to protect BALB/c mice was evaluated as was the efficacy of the anti-MV/EV mixture in a mouse model of progressive vaccinia. In addition to evaluating weight loss and lethality, bioimaging technology was used to characterize the spread of the VACV infections in mice. We found that the anti-EV cocktail, but not the anti-MV cocktail, limited virus spread and lethality.</p> <p>Conclusions</p> <p>A combination of anti-MV/EV antibodies was significantly more protective than anti-EV antibodies alone. These data suggest that DNA vaccine technology could be used to produce a polyclonal antibody cocktail as a possible product to replace vaccinia immune globulin.</p

Social Contacts and Mixing Patterns Relevant to the Spread of Infectious Diseases

Surveying 7,290 participants in eight European countries, Joël Mossong and colleagues determine patterns of person-to-person contact relevant to controlling pathogens spread by respiratory or close-contact routes

Phagocytosis of Staphylococcus aureus by Macrophages Exerts Cytoprotective Effects Manifested by the Upregulation of Antiapoptotic Factors

It is becoming increasingly apparent that Staphylococcus aureus are able to survive engulfment by macrophages, and that the intracellular environment of these host cells, which is essential to innate host defenses against invading microorganisms, may in fact provide a refuge for staphylococcal survival and dissemination. Based on this, we postulated that S. aureus might induce cytoprotective mechanisms by changing gene expression profiles inside macrophages similar to obligate intracellular pathogens, such as Mycobacterium tuberculosis. To validate our hypothesis we first ascertained whether S. aureus infection could affect programmed cell death in human (hMDMs) and mouse (RAW 264.7) macrophages and, specifically, protect these cells against apoptosis. Our findings indicate that S. aureus-infected macrophages are more resistant to staurosporine-induced cell death than control cells, an effect partly mediated via the inhibition of cytochrome c release from mitochondria. Furthermore, transcriptome analysis of human monocyte-derived macrophages during S. aureus infection revealed a significant increase in the expression of antiapoptotic genes. This was confirmed by quantitative RT-PCR analysis of selected genes involved in mitochondria-dependent cell death, clearly showing overexpression of BCL2 and MCL1. Cumulatively, the results of our experiments argue that S. aureus is able to induce a cytoprotective effect in macrophages derived from different mammal species, which can prevent host cell elimination, and thus allow intracellular bacterial survival. Ultimately, it is our contention that this process may contribute to the systemic dissemination of S. aureus infection

Characterization of Antibodies to Capsular Polysaccharide Antigens of Haemophilus influenzae Type b and Streptococcus pneumoniae in Human Immune Globulin Intravenous Preparations

The most common infections in primary immune deficiency disease (PIDD) patients involve encapsulated bacteria, mainly Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae (pneumococcus). Thus, it is important to know the titers of Hib- and pneumococcus-specific antibodies that are present in immune globulin (Ig) intravenous (IGIV) preparations used to treat PIDD. In this study, seven IGIV preparations were tested by enzyme-linked immunosorbent assay and opsonophagocytic activity for antibody titers to the capsular polysaccharides of Hib and five pneumococcal serotypes. Differences in Hib- and pneumococcus-specific antibody titer were observed among various IGIV preparations, with some products having higher- or lower-than-average titers. Opsonic activity also varied among preparations. As expected, IgG2 was the most active subclass of both binding and opsonic activity except against pneumococcal serotype 6B where IgG3 was the most active. This study determines antibody titers against capsular polysaccharides of Hib and pneumococcus in seven IGIV products that have been shown to be effective in reducing infections in PIDD patients. As donor antibody levels and manufacturing methods continue to change, it may prove useful from a regulatory point of view to reassess IGIV products periodically, to ensure that products maintain antibody levels that are important for the health of IGIV recipients

POLYMOD social contact data

This data formed part of POLYMOD, a European Commission project funded within the Sixth Framework Programm

Inhibition of caspase-3 activation induced by STS in RAW 264.7 macrophages is dependent on <i>S. aureus</i> phagocytosis.

<p>The effect of live (Sa l) and heat-killed (Sa d) <i>S. aureus</i>, <i>B. subtilis</i> (Bs), <i>E. coli</i> (Ec) and latex beads (LTX) phagocytosis (MOI = 1∶5) on STS-induced caspase-3 activity in RAW 264.7. After phagocytosis cells were cultured for 24 h and treated with STS for 3 h. Caspase-3 activity was measured from cell lysates using DEVD-AFC as a substrate. STS-induced activity in control, resting cells was taken as 100%. Data are the mean±SD values from three independent experiments, each performed at least twice. *p<0.05; ***, p<0.001 significance as indicated in the figure.</p

<i>S. aureus</i>-infected hMDMs show a decreased susceptibility to the cytotoxic effects of STS.

<p>Control and <i>S. aureus</i>-infected cells (24 h post-phagocytosis) were treated with STS at a concentration of 1 µM for 24 h and cell viability was evaluated by MTT (A) and LDH (B) assays. (A) The mitochondrial metabolic activity is presented as the percentage of control hMDMs, which were considered to be 100%. (B) Plasma membrane permeabilization or cell lysis induced in mock- and <i>S. aureus-</i>infected hMDMs was determined as LDH activity levels. The experimental value was the LDH activity in the conditioned medium from the control and infected cells after STS stimulation for 24 h. The data shown is representative of at least three separate experiments, performed in triplicate, using hMDMs derived from different donors. **, p<0.01. (C) The cytotoxicity measured as the LDH activity levels in the conditioned medium from the mock- and <i>S. aureus-</i>infected hMDMs (24 and 96 h p.i.) after treatment with STS for 24 h. The data shown is representative of at least three separate experiments, performed in triplicate, using hMDMs derived from different donors. **, p<0.01.</p

hMDMs infection with <i>S. aureus</i> inhibits caspase-3 activation induced by STS and butyric acid.

<p>(A) The effect of <i>S. aureus</i> infection on caspase-3 activity was measured with DEVD-AFC as a substrate in hMDMs after 24 h stimulation with STS or BA. The diagram is a representative result of an experiment performed in triplicate using macrophages isolated from a single donor. Bars represent mean±SD of caspase-3 activity (RFU/min). The caspase-3 activity of mock-infected cells was regarded as 100%. (B) Inhibition of procaspase-3 processing induced by STS in <i>S. aureus</i>-infected hMDMs. Macrophages with or without <i>S. aureus</i> infection were STS treated, and 24 h post-infection cells were lysed for Western Blot analysis using antibodies against caspase-3. Caspase-3 antibody staining was developed with a secondary antibody conjugated to horseradish peroxidase followed by visualization using ECL as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005210#s2" target="_blank">Materials and Methods</a>.</p