Immunization with SARS coronavirus vaccines leads to pulmonary immunopathology on challenge with the SARS virus

Background

Severe acute respiratory syndrome (SARS) emerged in China in 2002 and spread to other countries before brought under control. Because of a concern for reemergence or a deliberate release of the SARS coronavirus, vaccine development was initiated. Evaluations of an inactivated whole virus vaccine in ferrets and nonhuman primates and a virus-like-particle vaccine in mice induced protection against infection but challenged animals exhibited an immunopathologic-type lung disease.

Design

Four candidate vaccines for humans with or without alum adjuvant were evaluated in a mouse model of SARS, a VLP vaccine, the vaccine given to ferrets and NHP, another whole virus vaccine and an rDNA-produced S protein. Balb/c or C57BL/6 mice were vaccinated IM on day 0 and 28 and sacrificed for serum antibody measurements or challenged with live virus on day 56. On day 58, challenged mice were sacrificed and lungs obtained for virus and histopathology.

Results

All vaccines induced serum neutralizing antibody with increasing dosages and/or alum significantly increasing responses. Significant reductions of SARS-CoV two days after challenge was seen for all vaccines and prior live SARS-CoV. All mice exhibited histopathologic changes in lungs two days after challenge including all animals vaccinated (Balb/C and C57BL/6) or given live virus, influenza vaccine, or PBS suggesting infection occurred in all. Histopathology seen in animals given one of the SARS-CoV vaccines was uniformly a Th2-type immunopathology with prominent eosinophil infiltration, confirmed with special eosinophil stains. The pathologic changes seen in all control groups lacked the eosinophil prominence.

Conclusions

These SARS-CoV vaccines all induced antibody and protection against infection with SARS-CoV. However, challenge of mice given any of the vaccines led to occurrence of Th2-type immunopathology suggesting hypersensitivity to SARS-CoV components was induced. Caution in proceeding to application of a SARS-CoV vaccine in humans is indicated.     

Oxygen- and growth rate-dependent regulation of Escherichia coli fumarase (FumA, FumB, and FumC) activity

Escherichia coli contains three biochemically distinct fumarases which catalyze the interconversion of fumarate to L-malate in the tricarboxylic acid cycle. Batch culture studies indicated that fumarase activities varied according to carbon substrate and cell doubling time. Growth rate control of fumarase activities in the wild type and mutants was demonstrated in continuous culture; FumA and FumC activities were induced four- to fivefold when the cell growth rate (k) was lowered from 1.2/h to 0.24/h at 1 and 21% O(2), respectively. There was a twofold induction of FumA and FumC activities when acetate was utilized instead of glucose as the sole carbon source. However, these fumarase activities were still shown to be under growth rate control. Thus, the activity of the fumarases is regulated by the cell growth rate and carbon source utilization independently. Further examination of FumA and FumC activities in a cya mutant suggested that growth rate control of FumA and FumC activities is cyclic AMP dependent. Although the total fumarase activity increased under aerobic conditions, the individual fumarase activities varied under different oxygen levels. While FumB activity was maximal during anaerobic growth (k = 0.6/h), FumA was the major enzyme under anaerobic cell growth, and the maximum activity was achieved when oxygen was elevated to 1 to 2%. Further increase in the oxygen level caused inactivation of FumA and FumB activities by the high oxidized state, but FumC activity increased simultaneously when the oxygen level was higher than 4%. The same regulation of the activities of fumarases in response to different oxygen levels was also found in mutants. Therefore, synthesis of the three fumarase enzymes is controlled in a hierarchical fashion depending on the environmental oxygen that the cell encounters.     

Aerosol infectivity of a Baculovirus to Trichoplusia ni larvae: An alternative larval inoculation strategy for recombinant protein production

The baculovirus–insect expression system is a popular tool for recombinant protein production. The standard method for infecting insect larvae with recombinant baculovirus for protein production involves either feeding occlusion bodies or injecting budded virus into the cuticle. In this study, we showed that the recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) at titers >10⁸ pfu/mL efficiently infected Trichoplusia ni (T. ni) larvae through aerosol inoculation of budded virus at a pressure of 5.5 × 10⁴ Pa. The dipping T. ni larvae in virus‐containing solution efficiently infected them. These results indicate that surface contamination, either by aerosol or dipping, lead to infection via spiracles. The aerosol infection route for AcMNPV was restricted to T. ni and Plutella xylostella larvae, whereas Spodoptera litura and Helicoverpa armigera larvae were resistant to this inoculation process. The yields of the reporter proteins DsRed and EGFP from T. ni larvae following aerosol infection were nearly identical to those following oral feeding or injection. This alternative baculovirus infection strategy facilitates recombinant protein and virus production by insect larvae. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The effect of human C-reactive protein on the cell-attachment activity of fibronectin and laminin

We have previously reported that purified human C-reactive protein (CRP) specifically binds to the cell-binding region of plasma fibronectin (Fn) in a Ca2+-dependent reaction that is saturable at a molar ratio of CRP/Fn of approximately 9. In this study, the binding of CRP to Fn was found to interfere with the cell-attachment promoting activity of Fn. The inhibition of cell attachment was dependent on the concentration of the CRP and involved the phosphorylcholine (PC) binding site of CRP since inhibition was prevented by allowing the CRP to react with either PC (or closely related monophosphate compounds) or a mAb specific for the PC-binding site of CRP. Binding of CRP to laminin was also Ca2+-dependent; however, this binding did not alter the cell-attachment promoting activity of laminin. CRP by itself does not mediate cell attachment. Since CRP is selectively deposited at sites of tissue damage along with plasma Fn and has the ability to bind to Fn and alter its cell-binding activity, CRP may modulate early events in tissue repair.     

Apoptosis is required during early stages of tail regeneration in Xenopus laevis

The Xenopus tadpole is able to regenerate its tail, including skin, muscle, notochord, spinal cord and neurons and blood vessels. This process requires rapid tissue growth and morphogenesis. Here we show that a focus of apoptotic cells appears in the regeneration bud within 12 h of amputation. Surprisingly, when caspase-3 activity is specifically inhibited, regeneration is abolished. This is true of tails both before and after the refractory period. Programmed cell death is only required during the first 24 h after amputation, as later inhibition has no effect on regeneration. Inhibition of caspase-dependent apoptosis results in a failure to induce proliferation in the growth zone, a mispatterning of axons in the regenerate, and the appearance of ectopic otoliths in the neural tube, in the context of otherwise normal continued development of the larva. Larvae amputated during the refractory stage exhibit a much broader domain of caspase-3-positive cells, suggesting a window for the amount of apoptosis that is compatible with normal regeneration. These data reveal novel roles for apoptosis in development and indicate that a degree of apoptosis is an early and obligate component of normal tail regeneration, suggesting the possibility of the existence of endogenous inhibitory cells that must be destroyed by programmed cell death for regeneration to occur.     

Mutation in the Xanthomonas campestris xanA gene required for synthesis of xanthan and lipopolysaccharide drastically reduces the efficiency of bacteriophage (phi)L7 adsorption

(Phi)L7 is a lytic phage infecting the gram-negative Xanthomonas campestis pv. campestris, a plant pathogen. To study phage-host interaction, a (phi)L7-resistant mutant was isolated from strain Xc17 by mini-Tn5 transposition and designated CH7LR. CH7LR could not plate (phi)L7 in double-layered assay and formed turbid clearing zones when the cell lawn was dropped with a high titer of (phi)L7. Sequence analysis showed that the mutated gene is xanA coding for phosphoglucomutase/phosphomannomutase, required for the synthesis of lipopolysaccharide and exopolysaccharide (xanthan). The involvement of xanA was confirmed by isolating another mutant with interrupted xanA and complementing with the cloned wild-type gene. Nonmucoid mutants are still sensitive to (phi)L7, indicating that xanthan is not involved in (phi)L7 adsorption. Since the mutants still exhibited low efficiencies of phage adsorption, we predict, by analogy with the cases in other bacteriophages of gram-negative bacteria, that other outer membrane components such as a protein are required for the formation of a complex receptor.     

In vitro thermal stress induces apoptosis and reduces development of porcine parthenotes

The precise physiological causes that result in reduced development of oocytes after heat shock (HS) are not clear. In this study, apoptosis, heat shock protein70 (hsp70), and in vitro development of porcine oocytes were evaluated after HS. Porcine cumulus-oocyte complexes (COCs) were subjected to in vitro maturation for 42 h. The matured oocytes were then heated at 41.5 degrees C for 0 h (control, C0h), 1 h (HS1h), 2 h (HS2h), or 4 h (HS4h). An additional group of oocytes was cultured for 4 h without HS (control, C4h). In Experiment 1, expression of hsp70 was detected by Western-blotting and no difference between controls and HS groups was observed. In Experiment 2, apoptosis of matured oocytes after HS was examined by Annexin V-FITC and TUNEL. No significant TUNEL-positive signals were detected in the heated oocytes compared to the controls, but the intensity of Annexin V-FITC labeling among different groups increased with length of HS and in vitro culture (P<0.05). Oocytes were parthenogenetically activated by an electric pulse plus 6-DMAP (Experiment 3). Mean (+/-S.E.M.) embryonic development in HS2h (cleavage: 42+/-29%; blastocyst: 11+/-10%) and HS4h (cleavage: 36+/-28%; blastocyst: 11+/-8%) were decreased when compared to those in C0h (cleavage: 63+/-12%; blastocyst: 24+/-14%) and C4h (cleavage: 66+/-8%; blastocyst: 21+/-11%). Numbers of blastocysts with TUNEL-positive signals were similar among groups, but the signals increased before the eight-cell stage in HS groups (P<0.05). In conclusion, developmental competence of matured pig oocytes was compromised after heat shock, but it was not closely associated with the expression of oocyte hsp70. However, there may be a link between apoptosis and developmental competence of porcine oocytes.     

Vacuolar Ca2+/H+ transport activity is required for systemic phosphate homeostasis involving shoot-to-root signaling in Arabidopsis

Calcium ions (Ca(2+)) and Ca(2+)-related proteins mediate a wide array of downstream processes involved in plant responses to abiotic stresses. In Arabidopsis (Arabidopsis thaliana), disruption of the vacuolar Ca(2+)/H(+) transporters CAX1 and CAX3 causes notable alterations in the shoot ionome, including phosphate (P(i)) content. In this study, we showed that the cax1/cax3 double mutant displays an elevated P(i) level in shoots as a result of increased P(i) uptake in a miR399/PHO2-independent signaling pathway. Microarray analysis of the cax1/cax3 mutant suggests the regulatory function of CAX1 and CAX3 in suppressing the expression of a subset of shoot P(i) starvation-responsive genes, including genes encoding the PHT1;4 P(i) transporter and two SPX domain-containing proteins, SPX1 and SPX3. Moreover, although the expression of several PHT1 genes and PHT1;1/2/3 proteins is not up-regulated in the root of cax1/cax3, results from reciprocal grafting experiments indicate that the cax1/cax3 scion is responsible for high P(i) accumulation in grafted plants and that the pht1;1 rootstock is sufficient to moderately repress such P(i) accumulation. Based on these findings, we propose that CAX1 and CAX3 mediate a shoot-derived signal that modulates the activity of the root P(i) transporter system, likely in part via posttranslational regulation of PHT1;1 P(i) transporters.     

Does leukotriene C4 mediate hypoxic vasoconstriction in isolated ferret lungs?

To evaluate leukotriene (LT) C4 as a mediator of hypoxic pulmonary vasoconstriction, we examined the effects of FPL55712, a putative LT antagonist, and indomethacin, a cyclooxygenase inhibitor, on vasopressor responses to LTC4 and hypoxia (inspired O2 tension = 25 Torr) in isolated ferret lungs perfused with a constant flow (50 ml.kg-1.min-1). Pulmonary arterial injections of LTC4 caused dose-related increases in pulmonary arterial pressure during perfusion with physiological salt solution containing Ficoll (4 g/dl). FPL55712 caused concentration-related inhibition of the pressor response to LTC4 (0.6 micrograms). Although 10 micrograms/ml FPL55712 inhibited the LTC4 pressor response by 61%, it did not alter the response to hypoxia. At 100 microgram/ml, FPL55712 inhibited the responses to LTC4 and hypoxia by 73 and 71%, respectively, but also attenuated the vasoconstrictor responses to prostaglandin F2 alpha (78% at 8 micrograms), phenylephrine (68% at 100 micrograms), and KCl (51% at 40 mM). At 0.5 microgram/ml, indomethacin significantly attenuated the pressor response to arachidonic acid but did not alter responses to LTC4 or hypoxia. These results suggest that in isolated ferret lungs 1) the vasoconstrictor response to LTC4 did not depend on release of cyclooxygenase products and 2) LTC4 did not mediate hypoxic vasoconstriction.