Development of novel vaccine formulations against pertussis for early life vaccination in the presence of maternal antibodies

Whooping cough caused by an infection with Bordetella pertussis or Bordetella parapertussis is a highly contagious respiratory disease. Globally, pertussis is the most prevalent vaccine-preventable disease. Even though the introduction of whole-cell (wP) and acellular pertussis (aP) vaccines has greatly reduced the burden of the disease, whooping cough still remains a problem in neonates and adolescents. B. pertussis is responsible for about 30 million cases of the disease each year, 90% of which are found in developing countries. About 300,000 of those infected, mostly infants, die from the infection. Various countries, especially developing nations, have reported an increase in infant morbidity due to pertussis. Recently, a rise in pertussis cases has also been observed in developed nations such as United States and Canada. Thus, novel vaccines against pertussis are urgently needed that would provide early and life-long protection. Neonatal vaccination is challenging due to the presence of maternal antibodies (MatAbs) and the bias towards mounting Th2-type immune responses following early life vaccination. Our objective was to generate a novel vaccine against whooping cough that would offer protection in infancy in the presence of vaccine-neutralizing MatAbs. In order to first establish the model of interference, we vaccinated neonatal mouse pups and piglets in the presence and absence of passive immunity. Our experiments revealed that MatAbs interfered with active immunization using pertussis toxoid (PTd) and the level of passively transferred antibodies directly correlated with the level of interference that was observed. Nevertheless, we showed that this phenomenon could be overcome by using a second booster immunization or by co-formulating the toxoid with innate stimuli such as CpG ODN. Moreover, we also demonstrated that vaccination in the presence of MatAbs does not prevent responses to booster doses given later in life. In order to improve the vaccine efficacy and immunogenicity we co-formulated the antigen with a novel adjuvant combination composed of CpG ODN, innate defense regulator peptide (IDRP) and polyphosphazene (PP). The model antigen ovalbumin (OVA) and adjuvants were formulated into PP microparticle and soluble formulations. These formulations were titrated and delivered to neonatal mice via parenteral and mucosal routes. Our experiments revealed that co-formulation of the antigen with the novel adjuvant platform resulted in a higher antibody production as compared to vaccinating with antigen alone. In addition, both the soluble and microparticle formulations composed of the adjuvant combination induced elevated anti-OVA IgG2a titers thus indicating a Th1-type response shift in neonatal mice. Intranasal route of vaccination was shown to be superior to parenteral vaccination as it resulted in the production of high concentrations of systemic IgG2a and IgA antibodies. Lastly, we co-formulated PTd and filamentous hemagglutinin (FHA) with the novel adjuvant formulation and tested them in the presence and absence of passive immunity in the murine and porcine models of pertussis. Vaccines composed of the new adjuvant formulations induced an earlier onset of immunity, superior anti-pertussis IgG2a and IgA titers, and a balanced Th1/Th2-type responses when compared to immunization with Quadracel, one of the commercially available pediatric vaccines for pertussis. Most importantly, despite having half of the antigens of the Quadracel, the novel vaccine formulations offered protection against challenge infection in the presence of passively transferred MatAbs. Taken together our results demonstrate this novel vaccine formulation and delivery to be an excellent candidate for neonatal vaccination

Oxidative stress-induced, peroxynitrite-dependent, modifications of myosin light chain 1 lead to its increased degradation by matrix metalloproteinase-2

Damage to cardiac contractile proteins such as myosin light chain 1 (MLC1), during oxidative stress is mediated by reactive oxygen species such as peroxynitrite (ONOO-), resulting in impairment of cardiac systolic function. The purpose of this study is to investigate the effects of the increased level of ONOO- on MLC1 degradation by the proteolytic enzyme matrix metalloproteinase-2 (MMP-2) during oxidative stress which ultimately decreases cardiac function. In the present study two distinct models were utilized to demonstrate the mechanism by which MLC1 is modified by ONOO- and how these post-translational modifications lead to its increased degradation by MMP-2. In a model of newborn hypoxia-reoxygenation in piglets we demonstrated that ONOO--induced nitration and nitrosylation of tyrosine and cysteine residues of MLC1 increase its degradation by MMP-2. Furthermore, we found nitration of a tyrosine residue located adjacent to the cleavage site for MMP-2. We verified these results by using a model of isolated rat heart myocytes to determine that the same mechanism responsible for cardiac dysfunction in newborn piglets occurs in isolated myocytes and that the MMP-2 involved in degradation of MLC1 is located within the myocytes. Moreover, we were able to determine that this mechanism occurs during ischemia itself before the onset of reperfusion. Furthermore, we have found that pharmacological intervention aimed at inhibition of MLC1 nitration/nitrosylation during ischemia by the ONOO- scavenger FeTPPS (5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III]), or inhition of MMP-2 activity with phenanthroline, provides an effective protection of cardiomyocyte contractility. The work presented here provides new evidence on the mechanisms of regulation of contractile proteins during the development of contractile dysfunction

Substitutions near the hemagglutinin receptor-binding site determine the antigenic evolution of influenza A H3N2 viruses in U.S. swine

Swine influenza A virus is an endemic and economically important pathogen in pigs, with the potential to infect other host species. The hemagglutinin (HA) protein is the primary target of protective immune responses and the major component in swine influenza A vaccines. However, as a result of antigenic drift, vaccine strains must be regularly updated to reflect currently circulating strains. Characterizing the cross-reactivity between strains in pigs and seasonal influenza virus strains in humans is also important in assessing the relative risk of interspecies transmission of viruses from one host population to the other. Hemagglutination inhibition (HI) assay data for swine and human H3N2 viruses were used with antigenic cartography to quantify the antigenic differences among H3N2 viruses isolated from pigs in the United States from 1998 to 2013 and the relative cross-reactivity between these viruses and current human seasonal influenza A virus strains. Two primary antigenic clusters were found circulating in the pig population, but with enough diversity within and between the clusters to suggest updates in vaccine strains are needed. We identified single amino acid substitutions that are likely responsible for antigenic differences between the two primary antigenic clusters and between each antigenic cluster and outliers. The antigenic distance between current seasonal influenza virus H3 strains in humans and those endemic in swine suggests that population immunity may not prevent the introduction of human viruses into pigs, and possibly vice versa, reinforcing the need to monitor and prepare for potential incursions

Neonatal asphyxia induces the nitration of cardiac myosin light chain 2 that is associated with cardiac systolic dysfunction

Hypoxia followed by reoxygenation (H-R) observed during perinatal asphyxia is a serious complication with high mortality and morbidity rates that may cause adverse cardiovascular effects in neonates. Our aim was to determine if oxidative stress related to H-R induces peroxynitrite-dependent modifications of the cardiac contractile protein, myosin regulatory light chain 2 (MLC2), and whether this is associated with development of cardiac systolic dysfunction. Twelve newborn piglets were acutely instrumented for hemodynamic monitoring and randomized to a control group ventilated with only atmospheric air or to the H-R study group exposed to alveolar normocapnic hypoxia followed by reoxygenation. Afterward, animals were euthanized, and the hearts were harvested for biochemical analyses. Systolic function as well as cardiac MLC2 levels decreased in H-R animals, whereas nitrates and nitrotyrosine levels increased. Negative correlations between nitrates, nitrotyrosine, and MLC2 levels were observed. Moreover, H-R induced nitration of two tyrosine residues within the MLC2 protein. Similarly, in vitro exposure of MLC2 to peroxynitrite resulted in the nitration of tyrosine, which increased the susceptibility of MLC2 to subsequent degradation by matrix metalloproteinase 2. Substitution of this tyrosine with phenylalanine prevented the matrix metalloproteinase 2-dependent degradation of MLC2. In addition, a large decrease in MLC2 phosphorylation caused by H-R was observed. Oxidative stress related to asphyxia induces nitration of cardiac MLC2 protein and thus increases its degradation. This and a large decrease in MLC2 phosphorylation contribute to the development of systolic dysfunction. Inhibition of MLC2 nitration and/or direct inhibition of its degradation by MMP-2 could be potential therapeutic targets aiming at reduction of myocardial damage during resuscitation of asphyxiated newborns

Effects of MMP-9 inhibition by doxycycline on proteome of lungs in high tidal volume mechanical ventilation-induced acute lung injury

Abstract Background Although mechanical ventilation (MV) is a major supportive therapy for patients with acute respiratory distress syndrome, it may result in side effects including lung injury. In this study we hypothesize that MMP-9 inhibition by doxycycline might reduce MV-related lung damage. Using a proteomic approach we identified the pulmonary proteins altered in high volume ventilation-induced lung injury (VILI). Forty Wistar rats were randomized to an orally pretreated with doxycycline group (n = 20) or to a placebo group (n = 20) each of which was followed by instrumentation prior to either low or high tidal volume mechanical ventilation. Afterwards, animals were euthanized and lungs were harvested for subsequent analyses. Results Mechanical function and gas exchange parameters improved following treatment with doxycycline in the high volume ventilated group as compared to the placebo group. Nine pulmonary proteins have shown significant changes between the two biochemically analysed (high volume ventilated) groups. Treatment with doxycycline resulted in a decrease of pulmonary MMP-9 activity as well as in an increase in the levels of soluble receptor for advanced glycation endproduct, apoliporotein A-I, peroxiredoxin II, four molecular forms of albumin and two unnamed proteins. Using the pharmacoproteomic approach we have shown that treatment with doxycycline leads to an increase in levels of several proteins, which could potentially be part of a defense mechanism. Conclusion Administration of doxycycline might be a significant supportive therapeutic strategy in prevention of VILI.</p

Ischemia induced peroxynitrite dependent modifications of cardiomyocyte MLC1 increases its degradation by MMP-2 leading to contractile dysfunction

Damage to cardiac contractile proteins during ischemia followed by reperfusion is mediated by reactive oxygen species such as peroxynitrite (ONOO − ), resulting in impairment of cardiac systolic function. However, the pathophysiology of systolic dysfunction during ischemia only, before reperfusion, remains unclear. We suggest that increased ONOO − generation during ischemia leads to nitration/nitrosylation of myosin light chain 1 (MLC1) and its increased degradation by matrix metalloproteinase-2 (MMP-2), which leads to impairment of cardiomyocyte contractility. We also postulate that inhibition of ONOO − action by use of a ONOO − scavenger results in improved recovery from ischemic injury. Isolated rat cardiomyocytes were subjected to 15 and 60 min. of simulated ischemia. Intact MLC1 levels, measured by 2D gel electrophoresis and immunoblot, were shown to decrease with increasing duration of ischemia, which correlated with increasing levels of nitrotyrosine and nitrite/nitrate. In vitro degradation of human recombinant MLC1 by MMP-2 increased after ONOO − exposure of MLC1 in a concentration-dependent manner. Mass spectrometry analysis of ischemic rat cardiomyocyte MLC1 showed nitration of tyrosines 78 and 190, as well as of corresponding tyrosines 73 and 185 within recombinant human cardiac MLC1 treated with ONOO − . Recombinant human cardiac MLC1 was additionally nitrosylated at cysteine 67 and 76 corresponding to cysteine 81 of rat MLC1. Here we show that increased ONOO − production during ischemia induces MLC1 nitration/nitrosylation leading to its increased degradation by MMP-2. Inhibition of MLC1 nitration/nitrosylation during ischemia by the ONOO − scavenger FeTPPS (5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III]), or inhition of MMP-2 activity with phenanthroline, provides an effective protection of cardiomyocyte contractility

Ischemia/reperfusion-induced myosin light chain 1 phosphorylation increases its degradation by matrix metalloproteinase 2

Degradation of myosin light chain 1 (MLC1) by matrix metalloproteinase-2 (MMP-2) during myocardial ischemia/reperfusion (I/R) injury has been established. However, the exact mechanisms controlling this process remain unknown. I/R increases the phosphorylation of MLC1, but the consequences of this modification are not known. We hypothesized that phosphorylation of MLC1 plays an important role in its degradation by MMP-2. To examine this, isolated perfused rat hearts were subjected to 20 min global ischemia followed by 30 min of aerobic reperfusion. I/R increased phosphorylation of MLC1 (as measured by mass spectrometry). If hearts were subjected to I/R in the presence of ML-7 (a myosin light chain kinase (MLCK) inhibitor) or doxycycline (a MMP inhibitor) an improved recovery of contractile function was seen compared to aerobic hearts and MLC1 was protected from degradation. Enzyme kinetic studies revealed an increased affinity of MMP-2 for the phosphorylated form of MLC1 compared to non-phosphorylated MLC1. We conclude that MLC1 phosphorylation is important mechanism controlling the intracellular action of MMP-2 and promoting the degradation of MLC1. These results further support previous findings implicating posttranslational modifications of contractile proteins as a key factor in the pathology of cardiac dysfunction during and following ischemia. [Image: see text