A clinically parameterized mathematical model of Shigella immunity to inform vaccine design.

We refine and clinically parameterize a mathematical model of the humoral immune response against Shigella, a diarrheal bacteria that infects 80-165 million people and kills an estimated 600,000 people worldwide each year. Using Latin hypercube sampling and Monte Carlo simulations for parameter estimation, we fit our model to human immune data from two Shigella EcSf2a-2 vaccine trials and a rechallenge study in which antibody and B-cell responses against Shigella's lipopolysaccharide (LPS) and O-membrane proteins (OMP) were recorded. The clinically grounded model is used to mathematically investigate which key immune mechanisms and bacterial targets confer immunity against Shigella and to predict which humoral immune components should be elicited to create a protective vaccine against Shigella. The model offers insight into why the EcSf2a-2 vaccine had low efficacy and demonstrates that at a group level a humoral immune response induced by EcSf2a-2 vaccine or wild-type challenge against Shigella's LPS or OMP does not appear sufficient for protection. That is, the model predicts an uncontrolled infection of gut epithelial cells that is present across all best-fit model parameterizations when fit to EcSf2a-2 vaccine or wild-type challenge data. Using sensitivity analysis, we explore which model parameter values must be altered to prevent the destructive epithelial invasion by Shigella bacteria and identify four key parameter groups as potential vaccine targets or immune correlates: 1) the rate that Shigella migrates into the lamina propria or epithelium, 2) the rate that memory B cells (BM) differentiate into antibody-secreting cells (ASC), 3) the rate at which antibodies are produced by activated ASC, and 4) the Shigella-specific BM carrying capacity. This paper underscores the need for a multifaceted approach in ongoing efforts to design an effective Shigella vaccine.

Evaluation of an intragastric challenge model for Shigella dysenteriae 1 in rhesus monkeys (Macaca mulatta) for the pre-clinical assessment of Shigella vaccine formulations.

Shigellosis is a worldwide disease, characterized by abdominal pain, fever, vomiting, and the passage of blood- and mucus-streaked stools. Rhesus monkeys and other primates are the only animals that are naturally susceptible to shigellosis. A suitable animal model is required for the pre-clinical evaluation of vaccines candidates. In this study, the minimal dose of Shigella dysenteriae1 1617 strain required to produce dysentery in four of five (80% attack rate) monkeys using an escalating dose range for three groups [2 × 10(8) , 2 × 10(9) and 2 × 10(10) colony forming unit (CFU)] was determined. In addition, the monkeys were re-infected. The identified optimal challenge dose was 2 × 10(9) CFU; this dose elicited 60% protection in monkeys when they were re-challenged with a one log higher dose (2 × 10(10) CFU). The challenge dose, 2 × 10(10) CFU, produced severe dysentery in all monkeys, with one monkey dying within 24 h, elicited 100% protection when re-challenged with the same dose. All monkeys exhibited immune responses. This study concludes that the rhesus monkey model closely mimics the disease and immune response seen in humans and is a suitable animal model for the pre-clinical evaluation of Shigella vaccine candidates. Prior infection with the 1617 strain can protect monkeys against subsequent re-challenges with homologous strains.

Implementation of microfluidic sandwich ELISA for superior detection of plant pathogens.

Rapid and economical screening of plant pathogens is a high-priority need in the seed industry. Crop quality control and disease surveillance demand early and accurate detection in addition to robustness, scalability, and cost efficiency typically required for selective breeding and certification programs. Compared to conventional bench-top detection techniques routinely employed, a microfluidic-based approach offers unique benefits to address these needs simultaneously. To our knowledge, this work reports the first attempt to perform microfluidic sandwich ELISA for Acidovorax citrulli (Ac), watermelon silver mottle virus (WSMoV), and melon yellow spot virus (MYSV) screening. The immunoassay occurs on the surface of a reaction chamber represented by a microfluidic channel. The capillary force within the microchannel draws a reagent into the reaction chamber as well as facilitates assay incubation. Because the underlying pad automatically absorbs excess fluid, the only operation required is sequential loading of buffers/reagents. Buffer selection, antibody concentrations, and sample loading scheme were optimized for each pathogen. Assay optimization reveals that the 20-folds lower sample volume demanded by the microchannel structure outweighs the 2- to 4-folds higher antibody concentrations required, resulting in overall 5-10 folds of reagent savings. In addition to cutting the assay time by more than 50%, the new platform offers 65% cost savings from less reagent consumption and labor cost. Our study also shows 12.5-, 2-, and 4-fold improvement in assay sensitivity for Ac, WSMoV, and MYSV, respectively. Practical feasibility is demonstrated using 19 real plant samples. Given a standard 96-well plate format, the developed assay is compatible with commercial fluorescent plate readers and readily amendable to robotic liquid handling systems for completely hand-free assay automation.

Compared effectiveness of the 7-valent pneumococcal conjugate vaccine in children with the 13-valent vaccine in adults.

13-valent-pneumococcal conjugated vaccine was recently approved in the USA and Europe for adults 50 years of age or more. But this approval was followed by recommendations limiting its use to immunocompromised and asplenic patients. The extension of indications to adults was based on the well-demonstrated clinical effectiveness in infants less than 2 years of age, and on a better immune response either quantitatively or qualitatively with conjugated vaccines compared to the immunogenicity of plain polysaccharide vaccines. Nevertheless, the issue was to know whether results observed with the 7-valent pneumococcal conjugate vaccine in children are reproducible in adults with the 13-valent. The answer was given by comparing the epidemiological and physiopathological data, and the immunological response of the two populations. Very few clinical effectiveness studies in adults are available. We had for aim to assess these various issues in infants and adults. A lot of questions remain, such as the unknown impact of serotype replacement with the 13-valent pneumococcal conjugated vaccine on the clinical epidemiology and emergent Streptococcus pneumoniae pathogenicity, while waiting for the CAPITA study results expected in 2014.

Assessment of the immune responses to Treponema pallidum Gpd DNA vaccine adjuvanted with IL-2 and chitosan nanoparticles before and after Treponema pallidum challenge in rabbits.

Syphilis is a multistage, sexually transmitted disease caused by the spirochete, Treponema pallidum (Tp). A significantly high incidence of syphilis has been reported in several countries, including China, and there is an urgent need for the development of efficacious vaccines against syphilis. DNA vaccines are a major breakthrough in the field of vaccination with several advantages over traditional vaccines. Animal model studies of Tp DNA vaccines have not been reported elsewhere but our previous reports describe the development of a single-gene Tp DNA vaccine and preclinical immunization study. In this study, chitosan (CS) nanoparticles were used as a vector and an interleukin-2 expression plasmid (pIL-2) as an adjuvant to enhance a TpGpd DNA vaccine candidate (pTpGpd) in a rabbit Tp skin challenge model. At week 8 after the first immunization, three rabbits from each group were used to determine cytokine measurements and spleen lymphocyte proliferation assay. pTpGpd in combination with pIL-2 wrapped with CS led to the greatest enhancement of anti-TpGpd antibodies and T-cell proliferation. During infection, levels of anti-TpGpd antibodies and T-cell proliferation were measured. Both the serum special IgG and IL-2, interferon-γ were significantly increased by the co-injection of the IL-2 plasmid compared with the injection of TpGpd DNA alone (P<0.05). Furthermore, IL-2 plasmid coinjection efficiently enhanced the antigen-specific lymphocyte proliferation response. Additionally, the ratios of positive skin lesions and ulcer lesions in groups immunized with pTpGpd were significantly lower than those of the pIL-2, CS or pIL-2 mixed with CS control groups (P<0.001). CS vectored and pIL-2 adjuvanted pTpGpd immunized animals exhibited the lowest rates of positive skin tests (8.33%) and ulcer lesions (4.17%) and the fastest recovery (42 d). These experiments indicate that co-injection of a pIL-2 plasmid with pTpGpd DNA vaccine wrapped with CS can significantly strengthen the long-term stability of immune response during infection, efficiently improve the protective effect against T. pallidum spirochetes infection and attenuate syphilitic lesion development.

Engineering, conjugation, and immunogenicity assessment of Escherichia coli O121 O antigen for its potential use as a typhoid vaccine component.

State-of-the-art production technologies for conjugate vaccines are complex, multi-step processes. An alternative approach to produce glycoconjugates is based on the bacterial N-linked protein glycosylation system first described in Campylobacter jejuni. The C. jejuni N-glycosylation system has been successfully transferred into Escherichia coli, enabling in vivo production of customized recombinant glycoproteins. However, some antigenic bacterial cell surface polysaccharides, like the Vi antigen of Salmonella enterica serovar Typhi, have not been reported to be accessible to the bacterial oligosaccharyltransferase PglB, hence hamper development of novel conjugate vaccines against typhoid fever. In this report, Vi-like polysaccharide structures that can be transferred by PglB were evaluated as typhoid vaccine components. A polysaccharide fulfilling these requirements was found in Escherichia coli serovar O121. Inactivation of the E. coli O121 O antigen cluster encoded gene wbqG resulted in expression of O polysaccharides reactive with antibodies raised against the Vi antigen. The structure of the recombinantly expressed mutant O polysaccharide was elucidated using a novel HPLC and mass spectrometry based method for purified undecaprenyl pyrophosphate (Und-PP) linked glycans, and the presence of epitopes also found in the Vi antigen was confirmed. The mutant O antigen structure was transferred to acceptor proteins using the bacterial N-glycosylation system, and immunogenicity of the resulting conjugates was evaluated in mice. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with E. coli O121 LPS. One animal developed a significant rise in serum immunoglobulin anti-Vi titer upon immunization.

Assessment of functional antibacterial opsonophagocytic antibodies elicited by 13-valent pneumococcal conjugate vaccine administered concomitantly with trivalent influenza vaccine in a randomized clinical trial in adults aged ≥65 years.

A randomized, double-blind clinical trial compared immune responses elicited by concomitant administration of 13-valent pneumococcal conjugate vaccine (PCV13) and trivalent inactivated influenza vaccine (PCV13+TIV), with PCV13 given 1 month after TIV, in healthy, pneumococcal vaccine-naïve adults aged ≥65 years. Serotype-specific pneumococcal anti-polysaccharide IgG antibody concentrations 1-month post-vaccination were uniformly lower after PCV13+TIV, than after PCV13 alone (Vaccine 2011;29:5195-202). Therefore, post hoc analyses to evaluate the functional antibody titers at 1-month post-vaccination, as measured by opsonophagocytic activity (OPA) assays, were performed. The anti-pneumococcal functional responses, while also generally lower after vaccine co-administration, nonetheless showed a greater degree of similarity between the two vaccine groups than was apparent from the anti-polysaccharide antibody responses. In particular, the proportion of OPA responders after PCV13 and TIV co-administration was similar to that observed after PCV13 alone for all serotypes evaluated. Concomitant use of PCV13 and TIV should therefore be guided by clinical circumstances.

An immunologic model for rapid vaccine assessment -- a clinical trial in a test tube.

While the duration and size of human clinical trials may be difficult to reduce, there are several parameters in pre-clinical vaccine development that may be possible to further optimise. By increasing the accuracy of the models used for pre-clinical vaccine testing, it should be possible to increase the probability that any particular vaccine candidate will be successful in human trials. In addition, an improved model will allow the collection of increasingly more-informative data in pre-clinical tests, thus aiding the rational design and formulation of candidates entered into clinical evaluation. An acceleration and increase in sophistication of pre-clinical vaccine development will thus require the advent of more physiologically-accurate models of the human immune system, coupled with substantial advances in the mechanistic understanding of vaccine efficacy, achieved by using this model. We believe the best viable option available is to use human cells and/or tissues in a functional in vitro model of human physiology. Not only will this more accurately model human diseases, it will also eliminate any ethical, moral and scientific issues involved with use of live humans and animals. An in vitro model, termed "MIMIC" (Modular IMmune In vitro Construct), was designed and developed to reflect the human immune system in a well-based format. The MIMIC System is a laboratory-based methodology that replicates the human immune system response. It is highly automated, and can be used to simulate a clinical trial for a diverse population, without putting human subjects at risk. The MIMIC System uses the circulating immune cells of individual donors to recapitulate each individual human immune response by maintaining the autonomy of the donor. Thus, an in vitro test system has been created that is functionally equivalent to the donor's own immune system and is designed to respond in a similar manner to the in vivo response.

Immunogenicity, safety and tolerability of meningococcal C CRM197 conjugate vaccine administered 3, 5 and 11 months post-natally to pre- and full-term infants.

A total of 79 pre-term infants with a gestational age > or =32 weeks and 74 full-term infants were studied in order to evaluate the immunogenicity, safety and tolerability of meningococcal C (MenC)-CRM(197) conjugate vaccine administered 3, 5 and 11 months post-natally. The evoked immune response seemed to be substantially similar in the pre- and full-term infants, and there were only clinically marginal differences in safety and tolerability between the groups. The results support the use of two doses of MenC-CRM(197) vaccine at 3 and 5 months of age for primary immunisation, with a booster dose being given at about 1 year. In addition to reducing costs, this scheme seems to assure global immunogenicity and potential efficacy that is better than that offered by the accelerated scheme of administration with only three doses of vaccine in the first months of life, and similar to that observed with a fourth dose used as booster after the first year.

Optimal assessment of the ability of children with recurrent respiratory tract infections to produce anti-polysaccharide antibodies.

Specific anti-polysaccharide antibody deficiency (SPAD) is an immune disorder. Diagnostic criteria have not yet been defined clearly. One hundred and seventy-six children evaluated for recurrent respiratory tract infections were analysed retrospectively. For each subject, specific anti-pneumococcal antibodies had been measured with two enzyme-linked immunosorbent assays (ELISAs), one overall assay (OA) using the 23-valent pneumococcal polysaccharide vaccine (23-PPSV) as detecting antigen and the other purified pneumococcal polysaccharide serotypes (serotype-specific assay, SSA) (serotypes 14, 19F and 23F). Antibody levels were measured before (n = 176) and after (n = 93) immunization with the 23-PPSV. Before immunization, low titres were found for 138 of 176 patients (78%) with OA, compared to 20 of 176 patients (11%) with the SSA. We found a significant correlation between OA and SSA results. After immunization, 88% (71 of 81) of the patients considered as responders in the OA test were also responders in the SSA; 93% (71 of 76) of the patients classified as responders according to the SSA were also responders in the OA. SPAD was diagnosed in 8% (seven of 93) of patients on the basis of the absence of response in both tests. Thus, we propose to use OA as a screening test for SPAD before 23-PPSV immunization. After immunization, SSA should be used only in case of a low response in OA. Only the absence of or a very low antibody response detected by both tests should be used as a diagnostic criterion for SPAD.

Analysis of antibody response in humans to the type A OspC loop 5 domain and assessment of the potential utility of the loop 5 epitope in Lyme disease vaccine development.

The OspC protein of Borrelia burgdorferi is an immunodominant antigen. Here we demonstrate that the loop 5 domain of type A OspC is surface exposed, elicits bactericidal antibody in mice, and is antigenic in humans. The data suggest that loop 5 may be suitable for inclusion in a polyvalent, chimeric OspC vaccinogen.

Production of antibodies against microcystin-RR for the assessment of purified microcystins and cyanobacterial environmental samples.

Microcystins (MC) are cyanobacterial hepatotoxins responsible for animal-poisoning and human health incidents. Immunoassays provide a sensitive means to detect these toxins, although cross-reactivity characteristics of different antibodies are variable, and most antibodies have been produced against MC-LR. Here, we have produced the first polyclonal antibodies against the commonly occurring variant, MC-RR, and compared them with MC-LR antibodies for the analysis of purified MCs and cyanobacterial environmental samples. Both antisera cross-reacted with all MCs tested, and with the related cyanobacterial hepatotoxin nodularin-R, but not with non-toxic cyanobacterial peptides. In general, better cross-reactivity characteristics were observed with the MC-RR antisera and limits of quantification were lower for most variants, with all MCs tested and nodularin-R having limits of quantification of 0.31 nM or below. The antisera had different affinities to mixtures containing pooled MC-LR and MC-RR, with MC-LR antisera underestimating total MC concentration when MC-RR represented over 70% of the total MC pool. Both antisera correlated well with HPLC-UV data when incorporated into ELISAs to screen previously characterised environmental samples from Aland, Finland. MC-RR antisera are useful for screening samples containing multiple MCs, and particularly for samples primarily containing MC-RR variants.

Evaluation of a Pourquier ELISA kit in relation to agar gel immunodiffusion (AGID) test for assessment of the humoral immune response in sheep and goats with and without Mycobacterium paratuberculosis infection.

The present study was designed to evaluate a commercial ELISA kit (Institut Pourquier) for the diagnosis of ovine and caprine paratuberculosis under Australian conditions and to compare its accuracy with the existing AGID test. The sensitivity of the ELISA in sheep and goats was 34.9% and 56.4%, with a specificity of 98.8% and 100.0%, respectively. Sensitivity of AGID was 13.8% for sheep and 39.5% for goats, with specificity of 100.0% for both species. The sensitivity of the ELISA in sheep depended on the category of histological lesions. AGID and ELISA were conditionally independent, and appeared to detect overlapping but distinct subgroups of infected animals. The ELISA was significantly more sensitive than the AGID. The ELISA was simple to perform, robust and repeatable. Coefficients of variation of <12.0% were observed for positive and negative controls included on 193 plates over a 10-month period and there was a high level of intraassay repeatability with 12.0% of the duplicate samples having CV of >15.0%.

Assessment of the compatibility of co-administered 7-valent pneumococcal conjugate, DTaP.IPV/PRP-T Hib and hepatitis B vaccines in infants 2-7 months of age.

This study assessed compatibility of concurrently administered 7-valent pneumococcal conjugate (PCV7), hepatitis B (HB) and DTaP.IPV/Hib vaccines. Infants were given DTaP.IPV/Hib and HB at 2, 4, 6 months and randomly assigned (2:1) to receive PCV7 concurrently or sequentially (at 3, 5, 7 months). Antibody levels were compared in 246 concurrent and 122 sequential vaccinees. Responses to PCV7, DTaP.IPV/Hib and HB were generally unaltered with concurrent administration except that Hib responses were increased (p=0.008) and HB responses were reduced (p=0.006) with concurrent dosing, the latter possibly from same thigh injection with DTaP.IPV/Hib. We conclude that PCV7, DTaP.IPV/Hib and HB are compatible with concurrent, separate injections.

Identification and assessment of new vaccine candidates for group A streptococcal infections.

Group A Streptococcus (GAS) is a human-specific pathogen responsible for a wide variety of human diseases. Numerous GAS surface antigens interact with the human immune system and only some of these proteins have been studied in depth. A few of these may elicit protective response against GAS infection. In this study, we have used an in silico approach to identify antigenic peptides from GAS surface proteins. Putative GAS surface proteins from the M1 GAS genome were identified by the presence on LPxTG cell-wall anchoring motif and an export signal sequence. This technique identified 17 proteins of known or putative function, and another 11 which do not have known homologues. Peptides derived from predicted antigenic sequences near the amino terminus of six of these proteins, and another seven peptides derived from the two known surface proteins, GRAB and MtsA, were conjugated to keyhole lymphocyanin (KLH), and investigated for their capacity to induce opsonic antibody responses in outbred Quackenbush mice. All peptide-KLH antisera demonstrated opsonic capacity against both 88/30 and M1 GAS. However, KLH sera alone was also able to induce opsonic antibodies, suggesting that anti-KLH antibodies contributed to the opsonisation seen in the peptide-KLH antisera. KLH is therefore a promising carrier molecule for potential GAS peptide vaccines.

Increased efficacy of immersion vaccination in fish with hyperosmotic pretreatment.

Immersion vaccination is common practice in aquaculture, because of its convenience for mass vaccination with sufficient protection. However, the mechanisms of antigen uptake and presentation, resulting in a protective immune response and the role of the innate immune system therein are largely unknown. The impact of immersion vaccination on fish physiology and on the ensuing innate and specific immune response was characterized with fluorescently labeled particulate and soluble model antigens. Vaccination of common carp by direct immersion (DI) or hyperosmotic immersion (HI; direct immersion, preceded by a brief immersion in a hypertonic solution) greatly enhanced the uptake of soluble, but not particulate antigen through temporary disruption of the integrity of the epithelia of gills and skin. Damage induced is mild and does not impose additional stress over the handling associated with immersion vaccination. Especially HI briefly but strongly activates the innate immune system. We conclude that HI more effectively increased the uptake of vaccine and enhanced the efficacy by which vaccine components are processed and presented by the innate immune system, dually enhancing the mucosal immune response. Understanding the mechanisms involved in uptake and processing of vaccine in the early phase of the immune response will greatly benefit the design of immersion vaccination.

Assessment of the antibody response in 110 healthy individuals who have been subject to Vi capsular polysaccharide vaccine.

Typhoid fever is a disease predominant in underdeveloped and developing countries. Typhoid fever is more prevalent, in fact endemic, in countries where fecal contamination of water and food sources are very common. The majority of the reported cases are in the adult age group. There are three different vaccines which can be used to prevent typhoid fever. In this study, we have used the parenteral Vi vaccine which was developed using the polysaccharide Vi antigen that covers the bacterial surface, thus, concealing the O antigen protecting the bacteria against Anti-O antibodies and regarded as virulence factor. A total of 110 individuals whose sera were negative for seroconversion prior to vaccination were included in this study in which we have assessed Anti-Vi antibodies by tube agglutination. Serum and stool samples of 110 individuals were assessed 1 month after the vaccination. A total of 105 (95.5%) of the vaccinated people were considered to have positive (1/40 and higher) response and this result was regarded as prophylactic seroconversion. None of the people in the study group had Salmonella typhi, S. paratyphi A,B,C isolated from their stool cultures.

Economic comparison between conventional and disposables-based technology for the production of biopharmaceuticals.

Time to market, cost effectiveness, and flexibility are key issues in today's biopharmaceutical market. Bioprocessing plants based on fully disposable, presterilized, and prevalidated components appear as an attractive alternative to conventional stainless steel plants, potentially allowing for shorter implementation times, smaller initial investments, and increased flexibility. To evaluate the economic case of such an alternative it was necessary to develop an appropriate costing model which allows an economic comparison between conventional and disposables-based engineering to be made. The production of an antibody fragment from an E. coli fermentation was used to provide a case study for both routes. The conventional bioprocessing option was costed through available models, which were then modified to account for the intrinsic differences observed in a disposables-based option. The outcome of the analysis indicates that the capital investment required for a disposables-based option is substantially reduced at less than 60% of that for a conventional option. The disposables-based running costs were evaluated as being 70% higher than those of the conventional equivalent. Despite this higher value, the net present value (NPV) of the disposables-based plant is positive and within 25% of that for the conventional plant. Sensitivity analysis performed on key variables indicated the robustness of the economic analysis presented. In particular a 9-month reduction in time to market arising from the adoption of a disposables-based approach, results in a NPV which is identical to that of the conventional option. Finally, the effect of any possible loss in yield resulting from the use of disposables was also examined. This had only a limited impact on the NPV: for example, a 50% lower yield in the disposable chromatography step results in a 10% reduction of the disposable NPV. The results provide the necessary framework for the economic comparison of disposables and conventional bioprocessing technologies.

Kinetics of antibody concentration and avidity for the assessment of immune response to pneumococcal vaccine among children with bone marrow transplants.

The kinetics of the immune response to the 23-valent pneumococcal polysaccharide vaccine (PPV) were studied in 38 children who received bone marrow transplants (BMTs). Anti-pneumococcal antibody concentrations increased 1 and 3 months after vaccination for all 5 serotypes tested, but, in 21 children, the vaccine was not adequately immunogenic. Children vaccinated <18 months after receiving a BMT had a 4.2-fold increased odds of poor response (P=. 06). Antibody concentrations returned close to baseline levels 9 months after vaccination. Avidity declined significantly as early as 1 month after vaccination and remained low thereafter. Antibody concentration responses to PPV were superior among 9 healthy control children (P=.001); 37 of 38 children with a BMT elicited adequate, persistent immune responses to Haemophilus influenzae conjugate vaccine. Immune responses to PPV in children with a BMT are suboptimal, short lived, and associated with declining avidity. The different kinetics of antibody concentration and avidity indicate that both markers should be used for evaluating pneumococcal vaccines in this high-risk population.