A comprehensive synthetic library of poly-N-acetyl glucosamines enabled vaccine against lethal challenges of Staphylococcus aureus

Abstract Poly-β-(1–6)-N-acetylglucosamine (PNAG) is an important vaccine target, expressed on many pathogens. A critical hurdle in developing PNAG based vaccine is that the impacts of the number and the position of free amine vs N-acetylation on its antigenicity are not well understood. In this work, a divergent strategy is developed to synthesize a comprehensive library of 32 PNAG pentasaccharides. This library enables the identification of PNAG sequences with specific patterns of free amines as epitopes for vaccines against Staphylococcus aureus (S. aureus), an important human pathogen. Active vaccination with the conjugate of discovered PNAG epitope with mutant bacteriophage Qβ as a vaccine carrier as well as passive vaccination with diluted rabbit antisera provides mice with near complete protection against infections by S. aureus including methicillin-resistant S. aureus (MRSA). Thus, the comprehensive PNAG pentasaccharide library is an exciting tool to empower the design of next generation vaccines

Omisión de registros de causas maternas de muerte en Argentina: estudio observacional de alcance nacional

Objective: To determine the magnitude and characteristics of the omission of causes of maternal death in death certificates in Argentina, and to re-estimate the maternal mortality ratio (MMR) for the year 2014. Methods: Cross-sectional study. Retrospective review of medical records of women of childbearing age who died from causes suspected of concealing maternal deaths in public, social security, and private institutions in all jurisdictions of the country between 1 January and 31 December 2014. The cause of death recorded in the death certificate was reclassified. Outcome measures included: percentage of records with an omission, structure of causes of death, location, time of death with respect to the reproductive process, and gestational age. The RMM was re-estimated on the basis of the results. Results: Of a sample of 1,176 cases, 969 medical records (82.4%) were analyzed, identifying 60 cases in which the cause of maternal death was omitted (48 maternal deaths, 12 late maternal deaths). Omissions were found in 14.2% of maternal deaths and 33.3% of late maternal deaths. The new estimated MMR for 2014 varied between 43.3 and 47.2 deaths per 100,000 live births. Conclusions: The omission of causes of maternal death in death certificates in Argentina may be less frequent than international agencies have reported, with differences in omission between regions. Efforts must be made to emphasize the importance of filling out death certificates correctly.Fil: Abalos, Edgardo. Centro Rosarino de Estudios Perinatales; ArgentinaFil: Duhau, Mariana. No especifíca;Fil: Escobar, Paola. No especifíca;Fil: Fasola, María Letizia. No especifíca;Fil: Finkelstein, Juliana Zoe. No especifíca;Fil: Golubicki, José Luis. Sociedad Argentina de Terapia Intensiva ; ArgentinaFil: Krupitzki, Hugo Bernardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. CEMIC-CONICET. Centro de Educaciones Médicas e Investigaciones Clínicas "Norberto Quirno". CEMIC-CONICET; ArgentinaFil: Marconi, Élida. Centro Argentino de Clasificación de Enfermedades; ArgentinaFil: Santoro, Adrián. No especifíca;Fil: Vinacur, Jorge. No especifíca

Antibody to Poly-<i>N</i>-acetyl glucosamine provides protection against intracellular pathogens: Mechanism of action and validation in horse foals challenged with <i>Rhodococcus equi</i>

<div><p>Immune correlates of protection against intracellular bacterial pathogens are largely thought to be cell-mediated, although a reasonable amount of data supports a role for antibody-mediated protection. To define a role for antibody-mediated immunity against an intracellular pathogen, <i>Rhodococcus equi</i>, that causes granulomatous pneumonia in horse foals, we devised and tested an experimental system relying solely on antibody-mediated protection against this host-specific etiologic agent. Immunity was induced by vaccinating pregnant mares 6 and 3 weeks prior to predicted parturition with a conjugate vaccine targeting the highly conserved microbial surface polysaccharide, poly-<i>N</i>-acetyl glucosamine (PNAG). We ascertained antibody was transferred to foals via colostrum, the only means for foals to acquire maternal antibody. Horses lack transplacental antibody transfer. Next, a randomized, controlled, blinded challenge was conducted by inoculating at ~4 weeks of age ~10⁶ cfu of <i>R</i>. <i>equi</i> via intrabronchial challenge. Eleven of 12 (91%) foals born to immune mares did not develop clinical <i>R</i>. <i>equi</i> pneumonia, whereas 6 of 7 (86%) foals born to unvaccinated controls developed pneumonia (P = 0.0017). In a confirmatory passive immunization study, infusion of PNAG-hyperimmune plasma protected 100% of 5 foals against <i>R</i>. <i>equi</i> pneumonia whereas all 4 recipients of normal horse plasma developed clinical disease (P = 0.0079). Antibodies to PNAG mediated killing of extracellular and intracellular <i>R</i>. <i>equi</i> and other intracellular pathogens. Killing of intracellular organisms depended on antibody recognition of surface expression of PNAG on infected cells, along with complement deposition and PMN-assisted lysis of infected macrophages. Peripheral blood mononuclear cells from immune and protected foals released higher levels of interferon-γ in response to PNAG compared to controls, indicating vaccination also induced an antibody-dependent cellular release of this critical immune cytokine. Overall, antibody-mediated opsonic killing and interferon-γ release in response to PNAG may protect against diseases caused by intracellular bacterial pathogens.</p></div

Opsonic killing of intracellular <i>R</i>. <i>equi</i>.

<p><b>A:</b> Maximal killing of intracellular <i>R</i>. <i>equi</i> mediated by MAb to PNAG requires both complement (C’) and PMN (C’+PMN). Background killing <5% is achieved with heat-inactivated C’ (HI C’) or PMN + HI C’. <b>B:</b> Pre-immune, normal foal sera (NFS) or representative immune foal sera with low, medium (Med) or high titers to PNAG obtained on the day of challenge with <i>R</i>. <i>equi</i> mediate killing of intracellular <i>R</i>. <i>equi</i> along with C’ and PMN. <b>C:</b> Measurement of percent cytotoxicity by LDH release shows MAb to PNAG or PNAG-immune sera plus C’ and PMN mediate lysis of infected cells. <b>D:</b> Opsonic killing of intracellular <i>R</i>. <i>equi</i> requires recognition of cell surface PNAG. Treatment of infected macrophage cultures with dispersin B to digest surface PNAG eliminates killing whereas treatment with the control enzyme, chitinase, has no effect on opsonic killing. Bars represent means of 4–6 technical replicates. Depicted data are representative of 2–3 independent experiments. Bars showing <0% kill represent data wherein the cfu counts were greater than the control of PNAG MAb + PMN + HI C’.</p

Total IgG and IgG subisotype antibody titers to PNAG in sera of horse foals.

<p>Endpoint serum titers (N = 7 controls, 12 vaccinated) of IgG or IgG subisotypes are plotted by vaccine group as a function of age in days. <b>A</b>: IgG antibody end-point titers to PNAG were significantly higher in an age-dependent matter between foals from mares that were vaccinated (filled symbols n = 12) compared with titers in sera of foals from unvaccinated, control mares (open symbols n = 7) through Days 39–42 of life. <b>B-D</b>: Concentrations of IgG_1, IgG_4/7, and IgG_3/5 to PNAG were significantly higher in foals in the vaccinated group than the unvaccinated, control group through the day indicated on the figure. Statistical comparisons made using linear mixed-effects modeling with individual foal as a random effect; NS = not significant.</p

Duration of clinical signs in foals from vaccinated or unvaccinated mares.

<p>Duration of clinical signs in foals from vaccinated or unvaccinated mares.</p

Cell-mediated immune responses of foal PBMCs.

<p><b>A:</b> Foals (N = 7 controls, 12 vaccinated) from vaccinated mares (V) had significantly (P <0.05; linear-mixed effects modeling) higher concentrations of IFN-γ produced at 28 days of age (prior to challenge) than control (C) foals in response to stimulation by a lysate of <i>R</i>. <i>equi</i>. IFN-γ production at 2 days of age was significantly (P <0.05; linear mixed-effects modeling) lower than those at all other days for both the control and vaccine groups (P values not shown on graph). <b>B:</b> IFN-γ production from PBMC from 5 vaccinated foals at 56 and 84 days of age following intrabronchial infection with virulent <i>R</i>. <i>equi</i>. Stimuli included media only (negative control), Concanavilin A (ConA; positive control), lysate of virulent <i>R</i>. <i>equi</i> strain used to infect the foals (<i>R</i>. <i>equi</i> lysate); and the same lysate treated with dispersin B to digest PNAG. All 3 stimulated groups were significantly different from the medium control at both day 56 and 84 (Overall ANOVA for repeated measures (P < 0.0001); P ≤ 0.0070 for all pairwise comparisons to media only and for pairwise comparison for <i>R</i>. <i>equi</i> lysate vs. lysate plus dispersin B (indicated on top of graph), Holm-Sidak's multiple comparisons test. <b>C:</b> Foals (N = 7 controls, 12 vaccinated) that developed pneumonia (P) had significantly (P < 0.05; linear-mixed effects modeling) lower concentrations of IFN-γ expression at each day relative to foals that remained healthy (H).</p

Duration of clinical signs in foals infused with control or PNAG-hyperimmune plasma.

<p>Duration of clinical signs in foals infused with control or PNAG-hyperimmune plasma.</p

Comparison of induction and regression of ultrasonographic lesions in foals from vaccinated or unvaccinated mares following <i>R</i>. <i>equi</i> challenge.

<p><b>A:</b> Kaplan-Meier survival plot comparing duration of detectable ultrasonographic lesions as evidence of pulmonary abscessation. Duration of pulmonary lesions identified by ultrasound was significantly (P = 0.008; Log-rank test) shorter for foals of vaccinated mares (solid line) versus those of foals from control mares (hatched line). <b>B:</b> Cumulative sum of maximum diameters of thoracic ultrasonography lesions (N = 7 Controls, 12 Vaccinated). The sums of the cumulative maximum diameters were significantly (P = 0.007; Wilcoxon rank-sum test) lower for foals from vaccinated mares (n = 12) than for unvaccinated control mares (n = 7). Open circles indicate foals diagnosed with pneumonia, filled circles indicate foals that did not develop pneumonia. Symbols with outer gray rings indicate the unvaccinated foal that did not get pneumonia and the vaccinated foal that did develop pneumonia.</p

Case definition for diagnosis of <i>R</i>. <i>equi</i> clinical pneumonia.

<p>Case definition for diagnosis of <i>R</i>. <i>equi</i> clinical pneumonia.</p