Measles virus hemagglutinin protein expressed in transgenic lettuce induces neutralising antibodies in mice following mucosal vaccination.

Plant-made oral vaccines have the potential to overcome many of the limitations of traditional vaccines. Here we report on progress towards a lettuce-made measles vaccine. Lettuce is a palatable species which exhibits rapid growth in contained hydroponic systems and produces negligible quantities of toxins. Measles virus hemagglutinin (MV-H) protein was successfully expressed in transgenic lettuce and found to be immunogenic in mice. Lettuce extracts containing MV-H protein induced MV neutralising antibodies following intraperitoneal injection and intranasal inoculation of mice. Using a sequential prime-boost strategy in which mice were vaccinated with MV-H DNA followed by an orally delivered freeze-dried MV-H lettuce formulation a 10-fold increased in MV-specific IgG titers was observed relative to mice vaccinated with control lettuce formulations (p=0.05). MV-H protein was stable in freeze-dried lettuce for up to 13 months at room temperature, and survived at least a week at temperatures as high as 50 degrees C. This research represents a significant step towards the development of measles vaccine formulation that is effective, temperature-stable, easy to administer in a resource-poor setting and amenable to large scale manufacture.

Activation of the mu-opiate receptor by Vitex agnus-castus methanol extracts: implication for its use in PMS.

The dried ripe fruit of Vitex agnus-castus L. (VAC) is widely used for the treatment of premenstrual syndrome (PMS). A previous study reported that extracts of VAC showed affinity to opiate receptors; however, functional activity was not determined. We tested two different VAC extracts in receptor binding and functional assays. Our objectives were: (1) to confirm the opiate affinity; (2) to rule out interference by free fatty acids (FFA); (3) to determine the mode of action of VAC at the mu-opiate receptor. Methanol extracts of VAC were prepared either before (VAC-M1) or after (VAC-M2) extraction with petroleum ether to remove fatty acids. Both extracts showed significant affinities to the mu-opiate receptor, as indicated by the concentration-dependent displacement of [3H]DAMGO binding in Chinese hamster ovary (CHO)-human mu-opiate receptor (hMOR) cells. The IC50 values were estimated to be 159.8 microg/ml (VAC-M1) and 69.5 microg/ml (VAC-M2). Since the defatted extract not only retained, but exhibited a higher affinity (p<0.001), it argued against significant interference by fatty acids. In an assay to determine receptor activation, VAC-M1 and VAC-M2 stimulated [35S]GTPgammaS binding by 41 and 61% (p<0.001), respectively. These results suggested for the first time that VAC acted as an agonist at the mu-opiate receptor, supporting its beneficial action in PMS.

Crude saponins improve the immune response to an oral plant-made measles vaccine.

Millions of people live in areas where infectious diseases, such as measles, are endemic and resources are scarce. Heat-stable vaccines that are delivered orally will greatly enhance vaccination programs in these areas. A stumbling block in the development of oral vaccines is the availability of safe and effective mucosal adjuvants, especially for use with subunit vaccines. The experiments presented here examine the ability of CTB/CT, LT(R192G) and crude Quillaja saponin extracts to stimulate MV-specific immune responses in mice, following oral immunisation with plant-made measles virus hemagglutinin (MV-H) protein. LT(R192G) and crude saponin extracts both functioned as potent mucosal adjuvants when ad-mixed with plant-made MV-H protein, and were more effective than CTB/CT. MV-H protein supplemented with saponin extract induced the strongest MV-specific responses, in the greatest number of mice. Crude saponins are routinely used by the food and beverage industry at concentrations greater than those required for adjuvanticity, and as such, they have a better safety profile than bacterial enterotoxins. This study demonstrates their potential as adjuvants for use with oral plant-made vaccines.

An improved polyclonal antiserum for detecting Ryegrass mosaic rymovirus.

Ryegrass mosaic virus (RGMV) is considered the most serious and widespread virus infecting temperate pasture grasses. The use of visible symptoms to diagnose infection is unreliable and ELISA analysis requires antibodies with broad cross-reactivity. Here we describe the production of a polyclonal antiserum (PAb-cp3'Delta) using a bacterially expressed RGMV coat protein fragment. The PAb-cp3'Delta antiserum is specific for RGMV and recognises RGMV strains from each major phylogenetic cluster. PAb-cp3'Delta may be used in ELISAs for fast, accurate and inexpensive detection of RGMV.

The development of a plant-based vaccine for measles.

Plant-based vaccination strategies have the potential to overcome the limitations of the current measles vaccine. The measles virus hemagglutinin (MV-H) protein has been expressed in tobacco. Oral immunisation of mice with plant-derived MV-H protein resulted in MV-specific antibodies and secretory IgA, indicative of humoral and mucosal immune responses. In addition, boosting with oral plant-derived MV-H protein following a MV-H DNA prime, resulted in a greater response than could be induced with either vaccine alone. Collectively, this research represents a significant step towards an effective oral measles vaccine that would be temperature-stable, easy to administer and amenable to inexpensive manufacture.

Distribution and diversity of New Zealand isolates of ryegrass mosaic virus.

Sequences from the coat protein cistron of five ryegrass mosaic virus (RgMV) isolates indicated the presence of two distinct strains in New Zealand. The nucleotide differences between the strains, and their distribution, suggested that both strains were introduced recently, either as a mixed infection or as two independent introductions. The relationship between these New Zealand strains and other strains and isolates of RgMV, and their potential severity is discussed.

Australian isolates of ryegrass mosaic rymovirus and their relationships.

The sequences of the 3'-terminal 1.8 kb of the genomes of three Australian and three Welsh isolates of ryegrass mosaic rymovirus (RGMV) were determined, as too were the virion protein genes of two New Zealand isolates of RGMV. They were compared with each other and with the published sequences of a Danish and a South African isolate by distance and maximum likelihood methods, and found to be very closely related (mean nucleotide difference 5.5%). All three Australian isolates and one from North Island of New Zealand formed one consistent cluster, and the Danish and South African isolates formed another. However the relationships between these two clusters and the other isolates were not consistent; they depended on the method of comparison used, and on the protein, gene or codon position compared. Nonetheless the European (Welsh and Danish) sequences were 2-4 times more different from one another than those from the Antipodes, suggesting that the European RGMV population may be older than the Antipodean. The Danish isolate has 39 nucleotides of the 5'-terminal region of its virion protein gene frameshifted -1 relative to the 'common' sequence. Interestingly the South African isolate has a similar frameshift, but sequence comparisons indicate that this frameshift must have occurred independently; a possible example of 'convergent frameshifting'.

Lumbar and thoracic epidural analgesia via the caudal approach for postoperative pain relief in infants and children.

Thirty infants scheduled for a variety of gastrointestinal, genitourinary and thoracic surgical procedures were selected for insertion of lumbar or thoracic epidural catheters via the caudal approach using either an Intracath or a Burron continuous brachial plexus kit. The catheters were inserted with ease by residents in training and no catheter-related complications were encountered. Lidocaine 0.5 per cent with 1:200,000 epinephrine was then injected to assure proper placement of the catheter before narcotics were administered. Postoperative analgesia was adequate in all patients using preservative-free morphine 0.05 mg.kg-1. The mean dosing interval was 15 hr and no episodes of nausea, vomiting, hypotension or histamine release were noted. Urinary retention occurred in two infants and one infant became apnoeic three hours after epidural morphine administration but responded to naloxone and pulmonary ventilation with bag and mask. In conclusion, epidural catheters placed via the caudal approach are a safe and effective means of providing postoperative pain control in infants using preservative-free morphine. However, the use of epidural narcotics in infants less than two years of age is restricted to those who will receive intensive care unit monitoring postoperatively so that if apnoea occurs, rapid intervention can be taken by skilled nursing personnel.

Current developments in bovine mastitis treatment and control.

Mastitis in its complexity has managed to forestall all efforts of eradication in spite of years of research, antibiotics and practical control measures. This minisymposium will touch on seven topics current to treatment and control of this economically important disease.

Catabolism and biologic properties of two species of rat IgG2a Fc fragments.

Prolonged papain digestion of rat IgG2a has recently been shown to yield two species of Fc fragments, termed Fc(I) AND Fc(II). The results of structural studies indicated that Fc(II) fragment was 15 to 20% smaller than Fc(I), probably secondary to loss of a carboxy-terminal peptide. The effects of these structural laterations on the catabolism and biologic properties of the Fc fragments were determined. The results of catabolic experiments indicated that after injection into normal rats Fc(I) fragments were retained in the circulation and slowly catabolized whereas Fc(II) fragments rapidly underwent filtration in the kidneys. In nephrectomized rats, however, both Fc(I) and Fc(II) fragments possessed identical slow rates of catabolism, as determined by serum disappearance and whole body catabolic experiments. Fragment pFc, corresponding to Cgamma3 domain, was different from either of the Fc fragments in exhibiting rapid rates of catabolism in both normal and nephrectomized rats. Fc(I) was more active in complement fixation and in adherence to the Fc receptor on monocytes in comparison with Fc(II). These results support the conclusion that catabolism of Fc and maintenance in circulation are separate processes influenced by different structures in the Fc fragment. The catabolism of Fc is controlled by structures in the Cgamma2 domain. This process probably is not related either to complement fixation or to adherence to the Fc receptor on monocytes. Some correlations between the structure and biologic properties of these Fc fragments are discussed.

Polygalactolipids in spinach chloroplasts.

Two polygalactolipids, designated as components A and B, were isolated from spinach chloroplasts and were also obtained from glycolipid products synthesized with chloroplast enzymes using uridine diphosphate galactose as a galactose donor. These lipids were purified by column and thin layer chromatography. Chemical analysis of component A indicates that the lipid is trigalactosyl diglyceride, whereas component B behaves like tetragalactosyl diglyceride on a thin layer plate. The major fatty acid in trigalactosyl diglyceride was alpha-linolenic acid. Relative amount (molar ratio) of galactolipids in spinach chloroplasts was monogalactosyl diglyceride:digalactosyl diglyceride:trigalactosyl diglyceride:(tetragalactosyl diglyceride) = 60:30:5:1.