Search

Increasing awareness of Salmonella Paratyphi A's contribution to enteric fever episodes throughout Asia has led to the development of new S. Paratyphi A vaccines. Assays are needed to measure functional antibodies elicited by the new vaccine candidates to assess their immunogenicity and potential protective capacities. Serum bactericidal assay (SBA) is the method of choice to measure functional antibody titers against various bacterial pathogens, but it is rarely been used for large dataset and clinical samples because it is time consuming and labor-intensive. Recently we developed a high-throughput luminescence-based SBA method, against different pathogens, including Salmonella Typhimurium and Enteritidis, Shigella flexneri serovars 2a and 3a, Shigella sonnei and Neisseria meningitidis . Here we further demonstrated the applicability of such method with invasive isolates of S. Paratyphi A to assess the complement-mediated antibody-dependent killing of both preclinical and clinical standard sera. As already found for other organisms, titers obtained by the luminescence-based SBA strongly correlated with those obtained by the conventional agar plate-based assay. The SBA assay described here is a useful tool for measuring functional antibodies elicited by Salmonella vaccines, with the potential of being applied to immunogenicity assessment in clinical trials. [ABSTRACT FROM AUTHOR]

Outer membrane blebs are naturally shed by Gram-negative bacteria and are candidates of interest for vaccines development. Genetic modification of bacteria to induce hyperblebbing greatly increases the yield of blebs, called Generalized Modules for Membrane Antigens (GMMA). The composition of the GMMA from hyperblebbing mutants of Shigella flexneri 2a and Shigella sonnei were quantitatively analyzed using high-sensitivity mass spectrometry with the label-free iBAQ procedure and compared to the composition of the solubilized cells of the GMMA-producing strains. There were 2306 proteins identified, 659 in GMMA and 2239 in bacteria, of which 290 (GMMA) and 1696 (bacteria) were common to both S. flexneri 2a and S. sonnei . Predicted outer membrane and periplasmic proteins constituted 95.7% and 98.7% of the protein mass of S. flexneri 2a and S. sonnei GMMA, respectively. Among the remaining proteins, small quantities of ribosomal proteins collectively accounted for more than half of the predicted cytoplasmic protein impurities in the GMMA. In GMMA, the outer membrane and periplasmic proteins were enriched 13.3-fold ( S. flexneri 2a) and 8.3-fold ( S. sonnei ) compared to their abundance in the parent bacteria. Both periplasmic and outer membrane proteins were enriched similarly, suggesting that GMMA have a similar surface to volume ratio as the surface to periplasmic volume ratio in these mutant bacteria. Results in S. flexneri 2a and S. sonnei showed high reproducibility indicating a robust GMMA-producing process and the low contamination by cytoplasmic proteins support the use of GMMA for vaccines. Data are available via ProteomeXchange with identifier PXD002517. [ABSTRACT FROM AUTHOR]

Abstract: Aluminum based adjuvants (alum), including aluminum hydroxide (Alhydrogel®) and aluminum phosphate are the most commonly used adjuvant in the US. In order to ensure quality of vaccines, regulatory authorities require evaluation of antigen content in final vaccine products. Currently, there are no generic methods available for the determination of protein content in alum-based vaccines. Aluminum hydroxide gels exist as particles in solution, which interfere with direct quantitation of protein content in formulations using assays such as Lowry, BCA or Bradford protein assay. The present study adapts a simple fluorescent assay to directly (without the need for antigen extraction) determine antigen content on Alhydrogel® with accuracy and sensitivity using the o-phthalaldehyde (OPA) reagent. Malaria vaccine candidates AMA1-C1/Alhydrogel, AMA1-C2/Alhydrogel, MSP142-3D7/Alhydrogel, MSP142-C1/Alhydrogel or BSAM-2/Alhydrogel were used as model formulations. The results of the present study show that the OPA assay is highly accurate (87–100%), reproducible, and simple with a linear detection range of 25–400μg/mL for Alhydrogel® vaccines (except for MSP142-C1, which has a linear detection range of 31.25–500μg/mL). This assay has proven to be highly useful in our laboratory and been used in routine vaccine quality control processes. [Copyright &y& Elsevier]

Abstract: Residual host cell protein impurities in recombinant proteins intended for human use must be accurately quantified to help establish their safety. We describe a novel means of host cell protein quantitation, in which a slot blot system was employed together with scanning laser densitometry to allow picogram level sensitivity in detection of residual host cell proteins in unpurified fermentation products and final purified bulk samples. Two allelic forms of merozoite surface protein 1, a promising malaria vaccine candidate antigen currently undergoing evaluation in clinical trials, were expressed in E. coli as clinical grade proteins, refolded, and carried through several chromatographic purification steps. Several lots of these proteins were analyzed with this generic quantitative assay that uses rat polyclonal antibodies generated against soluble and insoluble E. coli proteins. The assay had a detection range of 6.1–1562 ng/mL, with a detection limit of 6.1 ng/mL, comparable to reported ELISA-based methods. This assay proved simple yet very sensitive and accurate, giving highly reproducible results. Thus it is suitable for evaluating host cell protein levels in clinical grade recombinant proteins expressed in E. coli. [Copyright &y& Elsevier]

Abstract: A dose escalating, placebo-controlled phase 1 trial was conducted to test the safety and immunogenicity of a vaccine containing recombinant Plasmodium falciparum apical membrane antigen 1 (AMA1) formulated in Montanide ISA720. Three groups of volunteers were vaccinated intramuscularly with 5μg, 20μg or 80μg of AMA1, respectively, in 0.5mL of formulation at 0, 3 and 6 months. Anti-AMA1 antibody levels and T cell stimulation indices were measured before and after each vaccination. No vaccine-related serious adverse events were recorded. Most subjects generated a mild to moderate, transient local reaction after the first vaccination. Three subjects developed a local reaction approximately 10 days following vaccination. Six of the 29 subjects seroconverted. Only one of these developed a high antibody titre. However, the interpretation of this trial was compromised by a loss of potency of the formulated vaccine during the course of the study. [Copyright &y& Elsevier]

Abstract: A mathematical model of Phase 1 vaccine trial design was used to investigate strategies for minimizing the number of serious adverse events (SAEs) that could be encountered in the first Phase 1 trials of new vaccine formulations. For a relatively standard dose escalation trial with three dose groups each with 10 subjects, an optimal balanced between risk of more than one serious adverse event and trial design is achieved by splitting each dose group into two subgroups of three and seven. Based on the modeling, for a two vaccination, dose-escalating Phase 1 trial, a design where all subjects receive the first vaccination before any subject receives a second vaccination generally carries a lower risk of multiple serious adverse events than other designs. [Copyright &y& Elsevier]

The rightful emphasis on collaboration between the public and private sectors in solving the world''s health problems, especially those of the poor in the developing world, relies on industry developing new technologies with the support of government and foundations. However, certain products, despite their potential to have a great impact on disease in the developing world, will not be developed by industry because they carry the risk of lawsuits owing to possible severe adverse reactions – risks that are not counterbalanced by potential profit of products that are of limited use in the developed world. Freund''s incomplete adjuvant (FIA) is one such example that is better developed in the public sector. In this article, we present the evidence that has led to our interest in FIA, evidence of its potential benefit in vaccines against blood-stage malaria, and the way forward to make safe, effective and affordable vaccines for malaria and other serious diseases in the developing world. [Copyright &y& Elsevier]

A major disparity in the burden of health will need to be addressed to achieve the “Grand Convergence” by 2035. In particular people living in low and middle income countries have a much higher burden of infectious diseases. Although vaccines have been very effective in reducing the global burden of infectious disease, there are no registered vaccines to address 60% of the current burden of infectious disease, especially in developing countries. Thus there is a pressing need for new vaccines and for prioritizing vaccine development given that resources for developing new vaccines are strictly limited. As part of the GLOBAL HEALTH 2035: Mission Grand Convergence meeting one working group assessed the SMART vaccine algorithm as a mechanism for prioritizing vaccine development for diseases of priority in the developing world. In particular, the working group considered which criteria in the standard SMART set were considered “key” criteria and whether other criteria should be considered, when prioritizing vaccines for this important set of countries. [ABSTRACT FROM AUTHOR]

Abstract: Many licensed vaccines are composed of live, attenuated or inactivated whole-cell microorganisms, or they comprise purified components from whole-cell extracts or culture supernatants. For some diseases, pathology is fairly well understood, and there may be known correlates of protection that provide obvious parameters for assessment of vaccine potency. However, this is not always the case, and some effective vaccines are routinely used even though the mechanisms or correlates of protection are unknown. Some more modern vaccine approaches employ purified recombinant proteins, based on molecules that appear on the surface of the pathogen. This is one of the strategies that has been adopted in the quest to develop a malaria vaccine. Use of these parasite antigens as vaccine candidates is supported by substantial epidemiological data, and some have demonstrated the ability to elicit protective responses in animal models of malaria infection. However, there is as yet no immunological correlate of protection and no functional assays or animal models that have demonstrated the ability to predict efficacy in humans. There is little precedence for the most appropriate and practical method for assessing potency of vaccines based on these recombinant molecules for malaria vaccines. This is likely because the majority of malaria vaccine candidates have only recently entered clinical evaluation. The PATH Malaria Vaccine Initiative (MVI) convened a panel with expertise in potency assay design from industry, governmental institutions, and regulatory bodies to discuss and review the rationale, available methods, and best approaches for assessing the potency of recombinant proteins, specifically for their use as malarial vaccines. The aim of this meeting was to produce a discussion document on the practical potency assessment of recombinant protein malaria vaccines, focusing on early phase potency assay development. [Copyright &y& Elsevier]

Many formulated vaccines, including 1790GAHB Shigella sonnei GMMA-based vaccine, contain Alhydrogel (aluminum hydroxide), consequently the antigen content must be determined in the formulated final vaccine product, as required by regulatory authorities. The direct quantification of antigens adsorbed on aluminum salts is difficult, and antigens may need to be extracted using laborious and often ineffective desorption procedures. To directly quantify the sugar vaccine target in the LPS of 1790GAHB, we have developed a new FAcE (Formulated Alhydrogel competitive ELISA) method. FAcE is an immunoassay based on the competition between S. sonnei LPS, coated on the ELISA plate, and the LPS in formulated S. sonnei GMMA, in binding a specific monoclonal antibody. To optimize the method, which is as easy to perform as a standard ELISA, we have applied a Design of Experiments (DOE) approach. A model was found to define the significant assay variables and to predict their impact on the output responses. Results obtained using the DOE optimized FAcE assay showed that the method is sensitive (0.02 μg/mL lower detection limit), precise, reproducible and can accurately quantify independently formulated drug products, making it a useful tool in routine tests of Alhydrogel-based vaccines. We are currently using this method to determine S. sonnei vaccine potency, stability and lot-to-lot variations, and are broadening its applicability to quantify active ingredients of other Alhydrogel GMMA-vaccines and in multivalent vaccines formulations. • New immune-detection based method to directly measure Alhydrogel-formulated Shigella sonnei GMMA. • Application of Design of Experiments (DOE) approach to optimize the method. • The optimized method is sensitive, precise, reproducible and accurate. [ABSTRACT FROM AUTHOR]

Background Shigella sonnei is an emergent and major diarrheal pathogen for which there is currently no vaccine. We aimed to quantify duration of maternal antibody against S. sonnei and investigate transplacental IgG transfer in a birth cohort in southern Vietnam. Methods and results Over 500-paired maternal/infant plasma samples were evaluated for presence of anti- S. sonnei -O IgG and IgM. Longitudinal plasma samples allowed for the estimation of the median half-life of maternal anti- S. sonnei -O IgG, which was 43 days (95% confidence interval: 41–45 days). Additionally, half of infants lacked a detectable titer by 19 weeks of age. Lower cord titers were associated with greater increases in S. sonnei IgG over the first year of life, and the incidence of S. sonnei seroconversion was estimated to be 4/100 infant years. Maternal IgG titer, the ratio of antibody transfer, the season of birth and gestational age were significantly associated with cord titer. Conclusions Maternal anti- S. sonnei -O IgG is efficiently transferred across the placenta and anti- S. sonnei -O maternal IgG declines rapidly after birth and is undetectable after 5 months in the majority of children. Preterm neonates and children born to mothers with low IgG titers have lower cord titers and therefore may be at greater risk of seroconversion in infancy. [ABSTRACT FROM AUTHOR]

Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural context.Wepreviously developed a high yield production process for genetically derived particles, called generalized modules for membrane antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane, they contain immunostimulatory components, especially lipopolysaccharide (LPS). We examined ways of reducing their reactogenicity by modifying lipid A, the endotoxic part of LPS, through deletion of late acyltransferase genes, msbB or htrB, in GMMA-producing Shigella sonnei and Shigella flexneri strains.GMMAwith resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line. In human peripheral blood mononuclear cells, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (S. sonnei ΔhtrB, 800-fold; ΔmsbB mutants, 300-fold). We found that the residual activity of theseGMMAis largely due to non-lipid A-related TLR2 activation. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory lipid A palmitoleoylation resulted in GMMA with hexa-acylated lipid A with ∼10-fold higher activity to stimulate peripheral blood mononuclear cells thanGMMAwith penta-acylated lipid A, mostly due to retained TLR4 activity. Thus, for use as vaccines, GMMA will likely require lipid A penta-acylation. The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development. [ABSTRACT FROM AUTHOR]

Highlights: [•] An affordable broadly-protective vaccine against meningococcus is needed for Africa. [•] Generalised modules for membrane antigens (GMMA) are a novel vaccine approach. [•] We engineered a GMMA-based vaccine with over-expressed factor H binding protein. [•] Mutation led to capsule deletion, reduced reactogenicity and increased GMMA release. [•] Vaccination induced bactericidal antibodies against African A, W and X strains. [Copyright &y& Elsevier]

Summary: Background: Typhoid vaccination is a public health priority in developing countries where young children are greatly affected by typhoid fever. Because present vaccines are not recommended for children younger than 2 years, the Novartis Vaccines Institute for Global Health developed a conjugate vaccine (Vi-CRM197) for infant immunisation. We aimed to assess the immunogenicity and safety of Vi-CRM197 in participants of various ages in endemic countries in south and southeast Asia. Methods: We did two randomised, observer-blind, age de-escalation, phase 2 trials at two sites in Pakistan and India (study A), and at one site in the Philippines (study B), between March 2, 2011, and Aug 9, 2012. Adults aged 18–45 years, children aged 24–59 months, older infants aged 9–12 months, and infants aged 6–8 weeks were randomly assigned (1:1) with a computer-generated randomisation list (block size of four) to receive either 5 μg Vi-CRM197 or 25 μg Vi-polysaccharide vaccine (or 13-valent pneumococcal conjugate vaccine in children younger than 2 years). Both infant populations received Vi-CRM197 concomitantly with vaccines of the Expanded Programme on Immunization (EPI), according to WHO schedule. With the exception of designated study site personnel responsible for vaccine preparation, study investigators, those assessing outcomes, and data analysts were masked to treatment allocation. We specified no a-priori null hypothesis for the immunogenicity or safety objectives and all analyses were descriptive. Analyses were by modified intention-to-treat. These studies are registered with ClinicalTrials.gov, numbers NCT01229176 and NCT01437267. Findings: 320 participants were enrolled and vaccinated in the two trials: 200 in study A (all age groups) and 120 in study B (children and infants only), of whom 317 (99%) were included in the modified intention-to-treat analysis. One dose of Vi-CRM197 significantly increased concentrations of anti-Vi antibody in adults (from 113 U/mL [95% CI 67–190] to 208 U/mL [117–369]), children (201 U/mL [138–294] to 368 U/mL [234–580]), and older infants (179 U/mL [129–250] to 249 U/mL [130–477]). However, in children and older infants, a second dose of conjugate vaccine had no incremental effect on antibody titres and, at all ages, concentrations of antibodies increased substantially 6 months after vaccination (from 55 U/mL [33–94] to 63 U/mL [35–114] in adults, from 23 U/mL [15–34] to 51 U/mL [34–76] in children, and from 21 U/mL [14–31] to 22 U/mL [14–33] in older infants). Immune response in infants aged 6–8 weeks was lower than that in older participants and, 6 months after third vaccination, antibody concentrations were significantly higher than pre-vaccination concentrations in Filipino (21 U/mL [16–28] vs 2·88 U/mL [1·95–4·25]), but not Pakistani (3·76 U/mL [2·77–5·08] vs 2·77 U/mL [2·1–3·66]), infants. Vi-CRM197 was safe and well tolerated and did not induce any significant interference with EPI vaccines. No deaths or vaccine-related serious adverse events were reported throughout the studies. Interpretation: Vi-CRM197 is safe and immunogenic in endemic populations of all ages. Given at 9 months of age, concomitantly with measles vaccine, Vi-CRM197 shows a promise for potential inclusion in EPI schedules of countries endemic for typhoid. An apparent absence of booster response and a reduction in antibody titres 6 months after immunisation should be further investigated, but data show that an immunogenic typhoid vaccine can be safely delivered to infants during EPI visits recommended by WHO. Funding: Sclavo Vaccines Association and Regione Toscana. [ABSTRACT FROM AUTHOR]

Abstract: The present study reports a method to determine the total protein concentration or concentration of a protein of interest in a protein–protein conjugate using ultraviolet absorption, after determining the molar ratio of proteins in the conjugates, from which an extinction coefficient can be calculated. A Microsoft Excel solver-based template using amino acid analysis data was developed for determining the molar ratio. The percent mass of each protein in the conjugate is calculated from the amino acid composition data using the least squares method in the Microsoft Excel solver function, and the percent mass is converted to molar portion of each protein using corresponding molecular weight. A molar ratio is obtained by dividing the molar portion of protein 1 by the molar portion of protein 2. A weighted extinction coefficient is calculated using the molar ratio, and the total protein concentration is determined using ultraviolet absorption at 280nm. The accuracy of the method was verified using mixtures of known proteins. The present study provides a rapid, simple and accurate method for determining protein concentration in protein–protein conjugates. [Copyright &y& Elsevier]

Abstract: Nontyphoidal Salmonellae (NTS) are a common cause of bacteraemia in children and HIV-infected adults in Sub-Saharan Africa. We have previously shown that antibodies play a key role in both bactericidal and cellular mechanisms of immunity to NTS, but found that high concentrations of antibody to Salmonella Typhimurium O antigen (OAg) in the serum of some HIV-infected African adults is associated with impaired killing of NTS. To further investigate the function of antibodies to the OAg of NTS, we developed a method to purify these antibodies from human serum by affinity chromatography. Purified Salmonella Typhimurium OAg was activated with adipic acid dihydrazide (ADH) via two different chemistries before linking to N-hydroxysuccinamide-Sepharose resin: one ADH molecule was introduced per OAg chain on its terminal 3-deoxy-D-manno-octulosonic acid sugar (OAg–ADH), or multiple ADH molecules were attached along the OAg chain after oxidation with sodium periodate (OAgoxADH). Both resulting columns worked well when tested with commercial polyclonal anti-O:4,5 antibodies from rabbit serum. Over 90% of the applied antibodies bound to the resin and 89% of these antibodies were then eluted as detected by ELISA. OAg–ADH was preferred as the method for OAg derivatisation as it does not modify the saccharide chain and can be applied to OAg from different bacteria. Both columns were able to bind OAg-specific antibodies in human serum, but antibody recovery was initially low. Different elution buffers were tested and different amounts of OAg–ADH were linked to the resin to improve the yield. Optimal recovery (51%) was obtained by loading 1mg of activated OAg per ml of resin and eluting with 0.1M glycine, 0.1M NaCl pH2.4. The column matrix could be regenerated following elution with no detectable loss in performance for over ten uses. This method offers the potential to purify antibodies to Salmonella OAg from polyclonal serum following vaccination or natural exposure to Salmonella and so investigate the functionality and diversity of the antibody response to OAg. [Copyright &y& Elsevier]

Abstract: 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 30mM sodium dodecyl sulfate (SDS) or 20mM cetylpyridinium chloride in the extraction buffer (0.60M citrate, 0.55M 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. [Copyright &y& Elsevier]

Abstract: A Phase 1 study was conducted in 24 malaria naïve adults to assess the safety and immunogenicity of the recombinant protein vaccine apical membrane antigen 1-Combination 1 (AMA1-C1)/Alhydrogel with CPG 7909 in two different formulations (phosphate buffer and saline), and given at two different dosing schedules, 0 and 1 month or 0 and 2 months. Both formulations were well tolerated and frequency of local reactions and solicited adverse events was similar among the groups. Peak antibody levels in the groups receiving CPG 7909 in saline were not significantly different than those receiving CPG 7909 in phosphate. Peak antibody levels in the groups vaccinated at a 0,2 month interval were 2.52-fold higher than those vaccinated at a 0,1 month interval (p =0.037, 95% CI 1.03, 4.28). In vitro growth inhibition followed the antibody level: median inhibition was 51% (0,1 month interval) versus 85% (0,2 month interval) in antibody from samples taken 2 weeks post-second vaccination (p =0.056). [Copyright &y& Elsevier]

Abstract: 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 µg/ml), reproducible, and simple with a linear detection range of 0.16–10 µg/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. [Copyright &y& Elsevier]

Abstract: Immunization with a recombinant yeast-expressed Plasmodium falciparum merozoite surface protein 3 (MSP3) protected Aotus nancymai monkeys against a virulent challenge infection. Unfortunately, the production process for this yeast-expressed material was not optimal for human trials. In an effort to produce a recombinant MSP3 protein in a scaleable manner, we expressed and purified near-full-length MSP3 in Escherichia coli (EcMSP3). Purified EcMSP3 formed non-globular dimers as determined by analytical size-exclusion HPLC with in-line multi-angle light scatter and quasi-elastic light scatter detection and velocity sedimentation (R h 7.6±0.2nm and 6.9nm, respectively). Evaluation by high-resolution atomic force microscopy revealed non-linear asymmetric structures, with beaded domains and flexible loops that were recognized predominantly as dimers, although monomers and larger multimers were observed. The beaded substructure corresponds to predicted structural domains, which explains the velocity sedimentation results and improves the conceptual model of the protein. Vaccination with EcMSP3 in Freund''s adjuvant-induced antibodies that recognized native MSP3 in parasitized erythrocytes by an immunofluorescence assay and gave delayed time to treatment in a group of Aotus monkeys in a virulent challenge infection with the FVO strain of P. falciparum. Three of the seven monkeys vaccinated with EcMSP3 had low peak parasitemias. EcMSP3, which likely mimics the native MSP3 structure located on the merozoite surface, is a viable candidate for inclusion in a multi-component malaria vaccine. [Copyright &y& Elsevier]

Summary: Both the blood-stage protein apical membrane antigen 1 (AMA1) and the 25-kDa sexual-stage protein (Pfs25) of Plasmodium falciparum are two leading candidates in malarial vaccine development. We have previously demonstrated that conjugation of these malarial antigens to recombinant Pseudomonas aeruginosa ExoProtein A (rEPA) significantly increased the mean-specific functional antibody responses in mice; however, some mice responded poorly and were unable to demonstrate a functional response. We hypothesized that the immunogenicities of these two malarial antigens could be further enhanced by the inclusion of a CpG oligodeoxynucleotide in the formulation. Mice were immunized with either rEPA-conjugated or unconjugated AMA1 and Pfs25 formulated on Alhydrogel with or without the addition of CPG 7909. Mice received the formulations on days 0 and 28, and mouse sera were collected on day 42. ELISA analyses on these sera showed that the addition of CPG 7909 to AMA1–rEPA and Pfs25–rEPA formulated on Alhydrogel induced significantly higher mean antibody titers than the formulations without CPG 7909, and led to a mixed Th1/Th2 response as demonstrated by the production of mouse IgG1 and IgG2a subclasses. The presence of CPG 7909 in the formulations of both conjugated antigens greatly increased the proportion of responders with antibody titers sufficient to inhibit blood-stage parasite growth in vitro or block transmission of sexual-stage parasites to mosquitoes. The results obtained in this study indicate the potential use of a combination strategy to increase the number of responders to malarial antigens in humans. [Copyright &y& Elsevier]

Summary: Enzyme linked immunosorbent assay (ELISA) has been widely used to measure antibody titers for evaluating the immunogenicity of a vaccine. However, there is as yet no generally accepted way of expressing the ELISA results in the case of experimental vaccines, since there is usually no uniform standard. Both end point and single dilution methods have significant disadvantages. In this paper, we obtained reproducible data with fewer dilutions of samples by addition of serially diluted standard serum to each ELISA plate. Since this ELISA method gives reliable antibody titer with less labor than other methods, it can strongly support vaccine development. [Copyright &y& Elsevier]

Abstract: CpG oligodeoxynucleotides are potent immunostimulants. In this study, CPG 7909 was formulated with the recombinant Plasmodium falciparum protein AMA1-C1 adsorbed to Alhydrogel (aluminum hydroxide) and used to immunize mice. Mice receiving free CPG 7909 in a separate same site injection to the AMA1-C1/Alhydrogel had the same antibody responses as mice receiving AMA1-C1/Alhydrogel alone. For mice immunized with CPG 7909 bound to the AMA1-C1/Alhydrogel formulation, there was a bell shaped CPG 7909 dose–response curve with the highest antibody response co-incident with the concentration of CPG 7909 that saturated binding to the Alhydrogel. At a higher CPG 7909 dose where 74% was unbound, there was no enhancement of response over AMA1-C1/Alhydrogel alone. Our results suggest that the adjuvant effects of CpGs are optimal when adsorbed to Alhydrogel and highlight the need for careful characterization of the vaccine formulation. [Copyright &y& Elsevier]

Abstract: CpG oligodeoxynucleotides are potent immunostimulants. For parenterally delivered alum-based vaccines, the immunostimulatory effect of CpG depends on the association of the CpG and antigen to the alum. We describe effects of buffer components on the binding of CPG 7909 to aluminum hydroxide (Alhydrogel), assays for measuring binding of CPG 7909 to alum and CPG 7909 induced dissociation of antigen from the alum. Free CPG 7909 is a potent inducer of IP-10 in mice. However the lack of IP-10 production from formulations containing bound CPG 7909 suggested that CPG 7909 does not rapidly dissociate from the alum after injection. It also suggests that IP-10 assays are not a good basis for potency assays for alum-based vaccines containing CPG 7909. [Copyright &y& Elsevier]

Abstract: Conjugation of polysaccharides to carrier proteins has been a successful approach for producing safe and effective vaccines. In an attempt to increase the immunogenicity of two malarial vaccine candidate proteins of Plasmodium falciparum, apical membrane antigen 1 (AMA1) to a blood stage vaccine candidate and surface protein 25 (Pfs25) a mosquito stage vaccine candidate, were each independently chemically conjugated to the mutant, nontoxic Pseudomonas aeruginosa ExoProtein A (rEPA). AMA1 is a large (66kD) relatively good immunogen in mice; Pfs25 is a poorly immunogenic protein when presented on alum to mice. Mice were immunized on days 0 and 28 with AMA1– or Pfs25–rEPA conjugates or unconjugated AMA1 or Pfs25, all formulated on Alhydrogel. Remarkably, sera from mice 14 days after the second immunization with Pfs25–rEPA conjugates displayed over a 1000-fold higher antibody titers as compared to unconjugated Pfs25. In contrast, AMA1 conjugated under the same conditions induced only a three-fold increase in antibody titers. When tested for functional activity, antibodies elicited by the AMA1–rEPA inhibited invasion of erythrocytes by blood-stage parasites and antibodies elicited by the Pfs25–rEPA conjugates blocked the development of the sexual stage parasites in the mosquito midgut. These results demonstrate that conjugation to rEPA induces a marked improvement in the antibody titer in mice for the poor immunogen (Pfs25) and for the larger protein (AMA1). These conjugates now need to be tested in humans to determine if mice are predictive of the response in humans. [Copyright &y& Elsevier]

Abstract: P25 is a major surface protein of Plasmodium ookinetes. Antibodies against P25 prevent the formation of oocysts in the mosquito and thereby block transmission of the parasite through an endemic population. Plasmodium vivax transmission-blocking vaccines based on Pv25 have undergone human trials and inhibit transmission significantly. The current assay to determine transmission-blocking activity (TBA) of these sera, the ‘standard membrane feeding assay’, is complex and can be performed by few groups worldwide that require both mosquito breeding facilities and access to volunteers naturally infected with P.vivax—a costly, and uncontrolled source of parasites. Here we report the development of novel assays to determine TBA using two clones (Pv25DR and Pv25DR3) of transgenic rodent parasites (Plasmodium berghei) expressing Pv25. We show that oocyst development of the transgenic parasites is inhibited by monoclonal antibody against Pv25 with the same kinetics exhibited by wild type parasites when exposed to mouse monoclonal antibodies targeted to a paralogous protein P28. Human transmission-blocking sera from a clinical vaccine trial of Pv25 inhibited oocyst development of Pv25DR and Pv25DR3, whereas non-blocking sera did not. We further show transmission-blocking activity can be determined in a simple assays of ookinete development in vitro, assays that obviate the need for mosquito colonies. These results demonstrate that transgenic rodent malarias expressing proteins from human Plasmodium species can be cheap, safe, and simple tools for testing TBA from sera. To this end the cloned lines have been deposited with, and are freely available from, MR4. [Copyright &y& Elsevier]

Abstract: Apical membrane antigen 1 (AMA1) has been shown to be a promising malaria vaccine candidate. The multiallelic AMA1-C1 vaccine currently in Phase 1 trials in the US and Mali contains an equal mixture of the ectodomain portion of recombinant AMA1 from the FVO and 3D7 clones of Plasmodium falciparum, formulated on Alhydrogel. It is hoped that inclusion of a human-optimized CpG oligodeoxynucleotide (ODN) (CPG 7909) with our existing AMA1-C1/Alhydrogel vaccine will lead to a higher concentration of functional AMA1-C1 antibodies. Preclinical studies were performed in mice, rats and guinea pigs to assess the safety, immunogenicity and functionality of the immune response to AMA1-C1 with Alhydrogel+CPG 7909 compared to antigen with Alhydrogel alone. Day 42 mean anti-AMA1 ELISA titer values derived from individual animals were compared between Alhydrogel and Alhydrogel+CPG 7909 groups at each antigen dose for each species. Sera from Alhydrogel+CPG 7909 groups displayed significantly higher antibody titers (P <0.025) than their comparable Alhydrogel alone group. Mouse IgG isotype analysis showed that AMA1-C1/Alhydrogel induced a predominately Th2 type response while AMA1-C1/Alhydrogel+CPG 7909 gave a mixed Th1/Th2 type response. When tested for functional activity by in vitro inhibition of parasite invasion, IgG isolated from serum pools of AMA1-C1/Alhydrogel+CPG 7909 animals was more effective against both FVO and 3D7 parasites than an equal concentration of IgG from animals receiving vaccines adjuvanted with Alhydrogel alone. These promising preclinical results have recently led to the start of a Phase 1 trial in the US. [Copyright &y& Elsevier]

Abstract: Plasmodium vivax is responsible for the majority of malaria cases outside of Africa, and results in substantial morbidity. Transmission blocking vaccines are a potentially powerful component of a multi-faceted public health approach to controlling or eliminating malaria. We report the first phase 1 clinical trial of a P. vivax transmission blocking vaccine in humans. The Pvs25H vaccine is a recombinant protein derived from the Pvs25 surface antigen of P. vivax ookinetes. The protein was expressed in Saccharomyces cerevisiae, purified, and adsorbed onto Alhydrogel®. Ten volunteers in each of three dose groups (5, 20, or 80μg) were vaccinated by intramuscular injection in an open-label study at 0, 28 and 180 days. No vaccine-related serious adverse events were observed. The majority of adverse events causally related to vaccination were mild or moderate in severity. Injection site tenderness was the most commonly observed adverse event. Anti-Pvs25H antibody levels measured by ELISA peaked after the third vaccination. Vaccine-induced antibody is functionally active as evidenced by significant transmission blocking activity in the membrane feeding assay. Correlation between antibody concentration and degree of inhibition was observed. Pvs25H generates transmission blocking immunity in humans against P. vivax demonstrating the potential of this antigen as a component of a transmission blocking vaccine. [Copyright &y& Elsevier]

Abstract: 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°C for 1 week and two proteins stored longer at 4°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°C, providing a potential way of stabilizing antigen/ISA 720 formulations for in vitro storage and the post-injection depot. [Copyright &y& Elsevier]

The EphA3 receptor tyrosine kinase preferentially binds ephrin-A5, a member of the corresponding subfamily of membrane-associated ligands. Their interaction regulates critical cell communication functions in normal development and may play a role in neoplasia. Here we describe a random mutagenesis approach, which we employed to study the molecular determinants of the EphA3/ephrin-A5 recognition. Selection and functional characterization of EphA3 point mutants with impaired ephrin-A5 binding from a yeast expression library defined three EphA3 surface areas that are essential for the EphA3/ephrin-A5 interaction. Two of these map to regions identified previously in the crystal structure of the homologous EphB2-ephrin-B2 complex as potential ligand/receptor interfaces. In addition, we identify a third EphA3/ephrin-A5 interface that falls outside the structurally characterized interaction domains. Functional analysis of EphA3 mutants reveals that all three Eph/ephrin contact areas are essential for the assembly of signaling-competent, oligomeric receptor-ligand complexes. [ABSTRACT FROM AUTHOR]

Combination B is a malaria vaccine that comprises recombinant Plasmodium falciparum (P. falciparum) blood-stage proteins MSP1, MSP2 and RESA, formulated with the adjuvant Montanide ISA 720. A phase I-IIb double-blind randomised placebo-controlled trial was undertaken in 120 children aged 5–9 years. Subjects were randomised in four groups: (i) No sulphadoxine-pyrimethamine (SP)+vaccine, (ii) No SP+placebo, (iii) SP+vaccine, (iv) SP+placebo. 15 μg of each protein were given in the thigh, at both first and second injection (4 weeks apart). The placebo was adjuvant emulsified with saline.No serious or severe AEs occurred. Moderate AEs were seen in 3% of the vaccine and 3% of the placebo recipients after first injection and in 12 and 10% after second injection. The vaccine induced significant antibody responses to all three antigens but triggered an IFN-γ response to MSP1 only. At Week 12, the IFN-γ response to MSP1 was substantially higher in the vaccine group where No SP had been given.Combination B proved to be safe and immunogenic in children aged 5–9 years. Vaccine immunogenicity was neither impaired by circulating parasites nor increased after pre-treatment with SP and pre-treatment is not advisable in future trials of malaria vaccines, at least for those including blood-stage antigens. [Copyright &y& Elsevier]

Group B Streptococcus (GBS) causes pneumonia, meningitis and sepsis in neonates. The current distribution pattern of GBS serotypes in developing countries such as India, China and Brazil is not clear. In order to appropriately plan for vaccination programs to address the burden of this disease in these countries, prospective population based studies are urgently needed. In our discussions, we focused on India, China and Brazil because of the membership of our workgroup, but data on other countries are also presented here. Further studies in developing countries are needed so as to better formulate appropriate public health interventions. [ABSTRACT FROM AUTHOR]