Characterization of AMA1-RON2L complex with native gel electrophoresis and capillary isoelectric focusing.

Rhoptry neck protein 2 (RON2) binds to the hydrophobic groove of apical membrane antigen 1 (AMA1), an interaction essential for invasion of red blood cells (RBCs) by Plasmodium falciparum (Pf) parasites. Vaccination with AMA1 alone has been shown to be immunogenic, but unprotective even against homologous challenge in human trials. However, the AMA1-RON2L (L is referred to as the loop region of RON2 peptide) complex is a promising candidate, as preclinical studies with Freund's adjuvant have indicated complete protection against lethal challenge in mice and superior protection against virulent infection in Aotus monkeys. To prepare for clinical trials of the AMA1-RON2L complex, identity and integrity of the candidate vaccine must be assessed, and characterization methods must be carefully designed to not dissociate the delicate complex during evaluation. In this study, we developed a native Tris-glycine gel method to separate and identify the AMA1-RON2L complex, which was further identified and confirmed by Western blotting using anti-AMA1 monoclonal antibodies (mAbs 4G2 and 2C2) and anti-RON2L polyclonal Ab coupled with mass spectrometry. The formation of complex was also confirmed by Capillary Isoelectric Focusing (cIEF). A short-term (48 h and 72 h at 4°C) stability study of AMA1-RON2L complex was also performed. The results indicate that the complex was stable for 72 h at 4°C. Our research demonstrates that the native Tris-glycine gel separation/Western blotting coupled with mass spectrometry and cIEF can fully characterize the identity and integrity of the AMA1-RON2L complex and provide useful quality control data for the subsequent clinical trials.

Evolution of lactation: nutrition v. protection with special reference to five mammalian species.

The evolutionary origin of the mammary gland has been difficult to establish because little knowledge can be gained on the origin of soft tissue organs from fossil evidence. One approach to resolve the origin of lactation has compared the anatomy of existing primitive mammals to skin glands, whilst another has examined the metabolic and molecular synergy between mammary gland development and the innate immune system. We have reviewed the physiology of lactation in five mammalian species with special reference to these theories. In all species, milk fulfils dual functions of providing protection and nutrition to the young and, furthermore, within species the quality and quantity of milk are highly conserved despite maternal malnutrition or illness. There are vast differences in birth weight, milk production, feeding frequency, macronutrient concentration, growth rate and length of lactation between rabbits, quokkas (Setonix brachyurus), pigs, cattle and humans. The components that protect the neonate against infection do so without causing inflammation. Many protective components are not unique to the mammary gland and are shared with the innate immune system. In contrast, many of the macronutrients in milk are unique to the mammary gland, have evolved from components of the innate immune system, and have either retained or developed multiple functions including the provision of nourishment and protection of the hatchling/neonate. Thus, there is a strong argument to suggest that the mammary gland evolved from the inflammatory response; however, the extensive protection that has developed in milk to actively avoid triggering inflammation seems to be a contradiction.

Accelerated and long term stability study of Pfs25-EPA conjugates adjuvanted with Alhydrogel®.

Pfs25, a Plasmodium falciparum surface protein expressed during zygote and ookinete stages in infected mosquitoes, is a lead transmission-blocking vaccine candidate against falciparum malaria. To enhance immunogenicity, recombinant Pfs25 was chemically conjugated to recombinant nontoxic Pseudomonas aeruginosa ExoProtein A (rEPA) in conformance with current good manufacturing practices (cGMP), and formulated with the alum adjuvant Alhydrogel. In order to meet the regulatory requirements for a phase 1 human clinical trial, the vaccine product was extensively evaluated for stability at an initial time point and through the clinical trial period annually. Because basic quality control methods to characterize alum-based vaccines remain unavailable, a thermal forced degradation study was performed prior to the initial evaluation to identify the methods suitable to detect the quality of vaccine formulations. Our results show that the vaccine product Pfs25-EPA formulated on Alhydrogel is in conformance with regulatory guidelines and suitable for human trials.

Persistent Nipple Pain in Breastfeeding Mothers Associated with Abnormal Infant Tongue Movement.

BACKGROUND: Infants of breastfeeding mothers with persistent nipple pain have been shown to apply stronger vacuums to the breast and transfer less milk during one monitored feed. This may be associated with differences in the movement of the tongue. The aim was to analyse the intra-oral nipple shape and movement of the tongue of infants of mothers with and without nipple pain. METHODS: Breastfeeding infants of mothers with or without nipple pain were monitored using ultrasound and intra-oral vacuum during one breastfeed. From cine clips of the ultrasound scans measurements were made of the depth of the intra-oral space between the hard-soft palate junction (HSPJ) and the mid-tongue; the distance of the tip of the nipple to the HSPJ; and nipple diameters from the tip to the base. RESULTS: During nutritive sucking, tongue movements of infants of mothers with nipple pain resulted in a smaller intra-oral space (p = 0.040) and restricted nipple expansion compared to controls (p < 0.012). Stronger baseline and peak vacuums compared to controls were confirmed (p = 0.002). CONCLUSION: In these mothers, nipple pain was associated with restricted infant tongue movement. Ultrasound may complement measurement of intra-oral vacuum in monitoring treatment strategies in breastfeeding women experiencing nipple pain.

Identification of glutathione conjugates of troglitazone in human hepatocytes.

Troglitazone (TGZ) is an orally active antihyperglycemic agent used in the treatment of noninsulin-dependent diabetes mellitus. Several cases of liver failure following TGZ administration led to its withdrawal from the market. The mechanism of toxicity is still not understood. The formation of toxic metabolites is believed to play an important role. Herein, we report the biotransformation of TGZ in human hepatocytes. TGZ at 50 microM concentration was incubated with cryopreserved human hepatocytes. Four metabolites were found-glucuronide, sulfate, and two glutathione (GSH) conjugates of TGZ. The two GSH metabolites could be conjugation at the 6-hydroxychromane nucleus and the thiazolidinedione ring. Alternatively, the conjugation could be one of the two rings, with the two GSH metabolites are diastereomers. The sulfate conjugate was the major metabolite found. The cytochrome P450 (CYP) inhibitors furafylline (CYP1A1/2), omeprazole (CYP2C19), ketoconazole (CYP3A4), and sulfaphenazole (CYP2C9) had no inhibitory effect on the TGZ metabolism suggesting that several P450s may play a role in the TGZ metabolic pathway. Previous studies in our laboratory have shown a large interindividual variation between different donors in cytotoxicity after dosing with TGZ. Based on EC(50) values, donors were classified as sensitive or resistant. The sensitive human donors were found to form significantly less troglitazone GSH conjugates and glucuronides than the resistant donors.

Breastfeeding frequency, milk volume, and duration in mother-infant dyads with persistent nipple pain.

BACKGROUND: Nipple pain and insufficient milk supply are major causes of early weaning. We have found that persistent nipple pain was associated with strong infant sucking vacuums during breastfeeding. Several studies indicate that nipple pain and abnormal infant sucking have the potential to reduce milk transfer. We aimed to determine whether women with persistent nipple pain had low milk supply. SUBJECTS AND METHODS: The 24-hour milk production and feeding characteristics of mothers with persistent nipple pain (n=21) were compared with those mothers without nipple pain (n=21). Milk productions were measured by test-weighing the infant before and after every feed from each breast over a 24-26-hour period. Comparisons were made using Student's t tests and linear mixed models as appropriate. RESULTS: Lower milk productions were associated with longer meal durations for mothers with pain. There were no significant differences in the average 24-hour milk production or any feeding characteristics between the groups. However, four women with persistent nipple pain had milk production levels below 500 mL/day. CONCLUSIONS: The majority of breastfeeding women experiencing persistent nipple pain were able to achieve normal milk production levels. Feeding duration and frequency were similar to those of women not experiencing pain. However, longer meal durations in the pain group were associated with lower levels of milk production. Further investigation is necessary to identify mothers most affected by maternal nipple pain.

Oxygen Saturation and Suck-Swallow-Breathe Coordination of Term Infants during Breastfeeding and Feeding from a Teat Releasing Milk Only with Vacuum.

Background. Vacuum is an important factor in milk removal from the breast, yet compression is the predominant component of milk removal from bottle teats. Since bottle-feeding infants have lower oxygen saturation, vacuum levels, and different suck-swallow-breathe (SSwB) coordination to breastfeeding infants, we hypothesised that when infants fed from a teat that required a vacuum threshold of -29 mmHg for milk removal, that oxygen saturation, heart rate, and suck-swallow-breathe (SSwB) patterns would be similar to those of breastfeeding. Study Design. Infants (n = 16) were monitored during one breastfeed and one feed from the experimental teat. Simultaneous recordings were made of oxygen saturation, heart rate, vacuum, tongue movement, respiration, and swallowing. Results. There were no differences in oxygen saturation and heart rate between the breast and the teat. Infants displayed fewer sucks and breaths per swallow during nutritive sucking (NS) compared to non-nutritive sucking (NNS). The number of sucks per breath was similar for NS and NNS although respiratory rates were slower during NS. These patterns did not differ between the breast and the teat. Conclusion. These results suggest that vacuum may be conducive to safe and coordinated milk removal by the infant during both breast and bottle-feeding.

Nipple pain during breastfeeding with or without visible trauma.

BACKGROUND: Nipple pain is a major cause of early weaning. The causes of nipple pain are diverse, and most treatments involve experience-based assessment. There is little knowledge of the intensity or variation in pain experienced by breastfeeding women. Given the high breastfeeding initiation rates, it is important to evaluate pain experienced by lactating women in detail. OBJECTIVE: To investigate and compare the pain experienced by breastfeeding women using objective measures. METHODS: The type, effect, and severity of pain were measured using the McGill Pain Questionnaire, Brief Pain Inventory, and Visual Analogue Scale, respectively, for 2 groups of breastfeeding women. One group were experiencing persistent nipple pain despite treatment, and the other had obvious signs of nipple trauma. RESULTS: Pain intensity and interference scores were highly variable for both groups. Mothers with nipple trauma reported significantly higher mean pain intensity and breastfeeding interference. Higher pain intensity scores were related to higher interference scores. After accounting for pain intensity, higher interference with general activity, mood, and sleep interference was related to longer duration of pain. There was no difference in MPQ class scores. CONCLUSIONS: The ramifications of nipple pain extend far beyond the act of breastfeeding, particularly for women whose pain lasts several months. Given the lack of evidence-based treatments, it is not surprising that pain is a major contributor to premature weaning. Further research into the causes of nipple pain is necessary to enable the implementation of effective interventions, thus reducing further complications such as infection and postnatal depression. Detailed pain analysis may assist in assessing the success of these interventions.

Tongue movement and intra-oral vacuum of term infants during breastfeeding and feeding from an experimental teat that released milk under vacuum only.

BACKGROUND: Recent literature supports the theory that vacuum is integral to the removal of milk from the breast rather than peristaltic compression of the breast. AIM: We aimed to determine if breastfed infants could remove breast milk from an experimental teat designed to release milk only when a vacuum is applied. METHODS: Submental ultrasound images and intra-oral vacuum measurements were recorded simultaneously during both a breastfeed and a feed with the experimental teat. RESULTS: Infants placed the nipple and teat a similar distance from the nipple hard-soft palate junction when the tongue was lowered (4.7 mm vs 5.3 mm). As the tongue lowered the nipple and teat expanded evenly although the nipple expanded more than the teat (3.1mm vs 1.5 mm). Both baseline (-31 mm Hg vs -12 mm Hg) and peak vacuum (-122 mm Hg vs -67 mm Hg) applied to the breast were significantly higher than for the teat. CONCLUSION: Breastfed infants are able to remove milk from a teat using only vacuum with a similar tongue movement to that of breastfeeding. This evidence supports the theory that vacuum is a critical factor in the removal of milk from the breast.

Efficient extraction of vaccines formulated in aluminum hydroxide gel by including surfactants in the extraction buffer.

Efficient antigen extraction from vaccines formulated on aluminum hydroxide gels is a critical step for the evaluation of the quality of vaccines following formulation. It has been shown in our laboratory that the efficiency of antigen extraction from vaccines formulated on Alhydrogel decreased significantly with increased storage time. To increase antigen extraction efficiency, the present study determined the effect of surfactants on antigen recovery from vaccine formulations. The Plasmodium falciparum apical membrane antigen 1 (AMA1) formulated on Alhydrogel and stored at 2-8°C for 3 years was used as a model in this study. The AMA1 on Alhydrogel was extracted in the presence or absence of 30 mM sodium dodecyl sulfate (SDS) or 20mM cetylpyridinium chloride in the extraction buffer (0.60 M citrate, 0.55 M phosphate, pH 8.5) using our standard antigen extraction protocols. Extracted AMA1 antigen was analyzed by 4-20% Tris-glycine SDS-PAGE followed by silver staining or western blotting. The results showed that inclusion of SDS or cetylpyridinium chloride in extraction buffer increased the antigen recovery dramatically and can be used for efficient characterization of Alhydrogel vaccines.

Long term stability of a recombinant Plasmodium falciparum AMA1 malaria vaccine adjuvanted with Montanide(®) ISA 720 and stabilized with glycine.

Plasmodium falciparum apical membrane antigen 1 (AMA1) is an asexual blood-stage vaccine candidate against the malaria parasite. AMA1-C1/ISA 720 refers to a mixture of recombinant AMA1 proteins representing the FVO and 3D7 alleles in 1:1 mass ratio, formulated with Montanide(®) ISA 720 as a water-in oil emulsion. In order to develop the AMA1-C1/ISA 720 vaccine for human use, it was important to determine the shelf life of this formulation. Previously it was found 267 mM glycine stabilized the proteins in Montanide(®) ISA 720 formulations for a short period of time at 2-8°C [25]. We now test the long term stability of AMA1-C1 at 10 and 40 µg/mL formulated with Montanide(®) ISA 720 with 50mM glycine as a stabilizer. Stability of AMA1-C1/ISA 720 at different time points following formulation (0, 5, 12 or 18 months) was evaluated by determining the mean particle size (diameter of the mean droplet volume), total protein content by a Modified Lowry assay, identity and integrity using western blot and SDS-PAGE. Our results showed that the mean particle size of these emulsions increased over time, whereas protein content, as determined by an ELISA method using a monoclonal antibody against penta-his, decreased over time. For the 10 µg/mL AMA1-C1/ISA 720 vaccine, the protein content was 6.5±2.2 µg/mL, and for the 40 µg/mL AMA1-C1/ISA 720 vaccine, the protein content was only 8.2±2.3 µg/mL after 18 months of storage at 2-8°C. These results suggest that the integrity of the protein was affected by long-term storage. The results of the present study indicate that the AMA1-C1/ISA 720 emulsion was unstable after 12 months of storage, after which AMA1-C1 proteins were partially degraded.

Validation of nipple diameter and tongue movement measurements with B-mode ultrasound during breastfeeding.

Infant feeding problems are extremely common during breastfeeding establishment. To objectively assess infant sucking, consistent methods to analyze ultrasound images of the infant's oral cavity are required. We developed and assessed the reliability of an extensive ultrasound measurement protocol by measuring nipple diameter and placement. Midline submental ultrasound scans of 30 term breastfed infants were analyzed by two raters. Nipple diameter, nipple hard-soft palate junction distance and tongue hard-soft palate junction distance were measured on two frames: tongue-up and tongue-down. No evidence of measurement bias was found between raters and inter-rater agreement and consistency scores were high. The changes in nipple diameter and placement were consistent with previous descriptions; however, the diameter of the nipple was not consistent in either position. This method provides objective measurements representative of tongue movement, and further investigation is required to ensure usefulness when examining sucking difficulties.

Development of a Direct Alhydrogel Formulation Immunoassay (DAFIA).

Alhydrogel (aluminum hydroxide) is a widely used adjuvant in the US. Regulatory authorities require that vaccines be tested to determine the antigen content in the final vaccine product. The level of formulated antigen is currently determined in our laboratory by the o-Phthalaldehyde (OPA) fluorescent protein assay, and antigen identity and integrity are determined by Western blot and SDS-PAGE. However, OPA assay is non-specific and only limited to detection of total protein content, and it is often not sensitive enough to detect antigens in low dose formulations. Furthermore, antigens used in identity and integrity tests must be extracted from vaccines using an extraction procedure which is time-consuming and may not completely recover antigens for analysis or may alter the structures of antigens during extraction. The present study developed a Direct Alum Formulation Immunoassay (DAFIA) which was designed to directly (without antigen extraction), accurately, and sensitively determine the antigen content, identity and integrity on alum. The AMA1-C1/Alhydrogel formulation was used as a model vaccine in assay development and validation. The results showed that the DAFIA is highly antigen-specific, accurate (87-100%), sensitive (0.16 microg/ml), reproducible, and simple with a linear detection range of 0.16-10 microg/ml. These results demonstrate that DAFIA is an excellent assay to determine antigen content, identity and integrity of antigens bound to alum and may be used in routine vaccine quality control for testing antigens in Alhydrogel-based vaccines.

Montanide ISA 720 vaccines: quality control of emulsions, stability of formulated antigens, and comparative immunogenicity of vaccine formulations.

Montanide ISA 720 is an experimental adjuvant, formulated as water-in-oil emulsions, that induces high antibody titers in several animal species. It has been used in human vaccine trials with malaria and HIV vaccines. The heightened response is likely due, in part, to the formation of a depot at the injection site. However, post-formulation modifications were seen with seven proteins tested during storage of ISA 720 formulations at 37 degrees C for 1 week and two proteins stored longer at 4 degrees C. Potency studies in mice, in which the stored vaccines were diluted into placebo emulsions for appropriate dosing, indicated that this instability could lead to loss of immunogenicity in the post-injection depot, limiting the allowable storage time of preformed vaccines. We describe point-of-injection formulation for ISA 720 vaccines that meets the requirement for in vitro stability. For preformed vaccines, addition of glycine or glycylglycine prevented antigen modification on storage at 37 degrees C, providing a potential way of stabilizing antigen/ISA 720 formulations for in vitro storage and the post-injection depot.