The standard primer sets VP7F/R and Beg9/End9 were used to amplify VP7, VP4F/R and Con2/Con3

to amplify the VP8* subunit of the VP4 gene and 10.1/10.2 to amplify NSP4 [20], [21] and [22]. PCR amplicons were purified using check details the QIAquick Gel extraction Kit (Qiagen, Inc., Hilden, Germany) according to the manufacturer’s protocol. Purified cDNA was sequenced using BigDye Sequencing Kit version 3.1 (Applied Biosystems; Foster City, CA, USA) in both directions using the oligonucleotide primer sets used in the gene amplification PCR protocol. The thermal cycling reaction consisted of 30 cycles of 96 °C for 15 s, 50 °C for 10 s and 60 °C for 4 min and the products purified by ethanol precipitation. The nucleotide sequence was determined by Applied Genetic Diagnostics (University of Melbourne, Victoria, Australia), using an ABI 3130xl Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Sequence data were analysed utilising the Sequencher® Software program version 4.1 (Gene Codes Corp Inc., Ann Arbor, MI, USA). Sequence identity was determined using the BLAST (Basic Local Alignment Search Tool)

server on the GenBank database at the National Centre for Biotechnology Information, USA (www.ncbi.nlm.nih.gov). Sequences from this study and those obtained from the GenBank Doxorubicin nmr database were aligned using ClustalW [23]. Genetic distances were calculated using the Kimura-2 correction parameters at the nucleotide level and phylogenetic trees were constructed using the Neighbor-joining method with 500 bootstrap replicates utilising the program MEGA version 4 [24]. The nucleic acid sequences for genes described in this study have been deposited in GenBank (Accession numbers JN377704-JN377721). For simplicity, samples will be referred to by abbreviate common names, AS07-obV2, AS07-obV12, AS07-obV18, AS07-obV37, AS07-obV42, AS07-obV50,

AS07-obV57, AS07-obV75, AS07-obV93, and AS07-obV121. A total of 107 stool samples were collected during a large gastroenteritis outbreak in Alice Springs (Northern Territory) were sent to the Australian Rotavirus Reference Centre for genotype analysis. Seventy-eight samples were found to be rotavirus positive. PAK6 Sixty-five samples were analysed by PAGE and silver stained to allow the visualisation and comparison of the electrophoretic pattern. An RNA electropherotype was visible in 57 samples; all samples displayed an identical long electropherotype (data not shown). Fourteen G9P[8] samples from the outbreak were selected for sequence analysis, including two samples from vaccinated infants. The coding region of the VP7 gene was determined for each of the 14 samples, and revealed a highly conserved gene, which displayed 99.9–100% nucleotide homology and 99.6–100% amino acid identity to each other. No conserved amino acid changes were observed between samples obtained from vaccinated and non-vaccinated patients.

The strategy of assessing one factor at a time while keeping the others constant may not be efficient, as it fails to take account of the interaction between the process variables and more experiments have to be done to obtain the information required. The best approach is to use experimental design, which can be used to assess the effect and interaction of the

variables involved, yielding the maximum amount of information from a minimum of experiments, while also allowing experimental errors to be assessed in order to enhance process effectiveness [13]. In recombinant bioprocesses, antibiotics like kanamycin are widely used on a bench scale to put selective BIBW2992 molecular weight pressure on the culture medium, preventing plasmid segregation, since most of the plasmids used have an antibiotic resistance STI571 concentration marker gene. Plasmid segregation may have an impact on the recombinant protein

yield, especially on an industrial scale. However, the use of these antibiotics is unfeasible on an industrial scale because they are costly and also contaminate the product and have to be completely removed in the food or drug purification process [14]. This is why studying the antibiotic concentration used in recombinant processes is so important, even though the variation of the antibiotic in the culture may affect plasmid stability. Another important variable in the process, especially on a large scale, is the inducer used in the expression system, since some inducers, like IPTG, are expensive and may be toxic to the host cell [15] and [16]. In view of these considerations, the aim of this study was to clone and express ClpP using Escherichia coli as a host, optimize protein production using experimental design and study the plasmid stability of the system. As such, central composite design was used for two variables: concentration of the inducer of the recombinant next system (IPTG) and the concentration of the antibiotic (kanamycin) in the culture medium. E. coli TOP 10 (Invitrogen) was used as the host for the cloning procedures. E. coli BL21 Star (DE3)™ (Invitrogen)

was used as the bacteria for expressing the recombinant protein ClpP. Bacto™ yeast extract and tryptone were purchased from BD (Becton, Dickinson and Company), the glucose and NaCl were from Merck, the glycerol was from Invitrogen, the kanamycin was from Sigma and the IPTG (isopropyl β-d-1-thiogalactopyranoside) was purchased from Promega. The gene that codifies protein ClpP was amplified by PCR using genomic DNA from S. pneumoniae serotype 14 (strain 113/95 deposited at Instituto Adolfo Lutz) as a template. The primers used were: 5′-CCCATGGTTCCTGTAGTTATTGAACAAAC-3′ and 5′-CACTCGAGGTTCAATGAATTGTTGGC-3′. The NcoI and XhoI restriction sites are underlined in the forward and reverse primers, respectively.

This hypo-methylation was functionally linked to an increase in POMC mRNA expression possibly as a result of decreased binding of protein methyl-CpG-binding protein 2 (Mecp2) and DNA-methyltransferase

1 (DNMT1), which are involved in transcriptional repression. These epigenetic changes in the POMC gene, as a result of ELS, were still present in aged mice tested at 1 year (Patchev et al., 2014). McGowan et al. (2009) translated the animal studies described above regarding the GR gene into the human situation of child-abuse related suicide and found similar epigenetic NVP-BGJ398 changes as those identified within the hippocampal GR promoter of low-care giving rats to those present in the human hippocampal GR gene promoter (McGowan et al., 2009). Male suicide victims

abused as children had increased methylation of the hippocampal GR promoter region and an associated reduction in GR gene transcription compared with hippocampal samples from non-abused suicide victims or age-matched non-suicide non-abused controls. Later studies examining changes in the blood of children and adolescents with or without a history of childhood click here abuse have revealed that: 1. Changes in DNA methylation patterns occur shortly after the adverse experience (van der Knaap et al., 2014 and Romens et al., 2014); 2. Increases in DNA methylation within the GR promoter region as a result of childhood adversity is not exclusive to the hippocampus and can be detected in DNA extracted from whole blood (van der Knaap et al., 2014 and Romens et al., 2014); and 3. DNA methylation levels in the promoter region of the GR gene are positively correlated with the number of stressful life events (such as parental divorce, hospitalization, parental illness etc.) a child or young adult experiences in a cumulative manner (van der Knaap et al., 2014). Additional genome-wide screening studies have been performed on both human blood (Bick et al., 2012 and Suderman et al., 2014) and brain tissue (Labonte et al., 2012) to identify the sheer number

of genes differentially methylated when categorized based on experience many of childhood abuse. The relevance of long lasting epigenetic changes as a result of early life experiences could be explained by the emerging match/mismatch hypothesis of psychiatric disease (Nederhof, 2012). Studies on human development (reviewed in Belsky and Pluess (2009)) discussed the possibility that apparent ‘negative’ behavioral and or molecular changes occurring as a result of adverse environmental experience during development may, in fact, increase resilience when dealing with a matched environment of high stress in later life. These ideas forming the basis of match/mismatch hypothesis of psychiatric disease suggest that individuals are better suited when adapting to an environment which matches their early life experience (Nederhof and Schmidt, 2012).

4. The Fig. 4 (A) shows the large crystals of pure buy Vorinostat IBS. Fig. 4 (B), (C), (D), (E) and (F) of SSDs are shown to be irregular matrices due to the porous nature of the carrier with the fine particles of the drug embedded in it. Therefore it is possible that the reduced particle size, increased surface area and the close contact between

the hydrophilic carrier and the drug may be the reason for the enhanced drug solubility of the SDs. Mean dissolution time (MDT) value is used to characterize drug release rate from a dosage form, which indicates the drug release retarding efficiency of polymer. These values are shown in Table 1. SSD of IBS prepared with CP (1:10) showed lower MDT value (2.316 ± 0.5 min) in comparison to SSD prepared with SSG, MC, CC and PS which show 4.146 ± 0.7, 4.791 ± 0.1, 4.887 ± 0.2 and4.987 ± 0.05 min, respectively. This finding can be attributed to the immediate release by SSD of IBS with CP. The observed order of MDT releasing profile is as follows: crospovidone > sodium starch glycolate > microcrystalline cellulose > croscarmellose > potato starch. SSD of IBS showed good dissolution efficiency (DE = 76.36%) with

CP. The SSD of IBS with SSG, MC, CC and PS shows dissolution efficiency of 71.92%, 71.10%, 70.31% and 69.89% respectively. The dissolution efficiencies of commercial formulations and the pure forms are 69.45% and 58.31% respectively, which are shown in Table 1. The order of % DE releasing profile

is as follows. crospovidone > sodium starch glycolate > microcrystalline cellulose > croscarmellose > potato GS-7340 nmr starch > marketed formulation > plain drug. The dissolution profiles of the SSD and physical mixtures of CP, CC, MC, PS, SSG, marketed product and plain drug were plotted as shown in Fig. 5. The dissolution rate of IBS in physical mixtures as well as in SSD was higher for all SDs as compared with plain IBS. Plain IBS showed a poor dissolution rate whereas physical mixtures showed slight enhancement due to the presence of SD in the respective mixtures. Dissolution profiles of all also the SSD for all SD showed a trend of increase in dissolution rate with increase in SD. The Drug: SD was taken in the proportions of 1:1, 1:5, and 1:10. SSD with 1:10 proportion showed maximum drug release. The SSD drug release for various formulations is found to be CP – 98.18% (10 min), SSG – 94.29% (13 min), MC – 93.13% (12 min), CC – 93.68% (14 min), PS-93.07% (14 min), whereas for marketed formulation – 95.53% (25 min) and pure IBS – 25.21% (30 min). This shows that SSD with CP showed better dissolution profile than SSG, MC, CC and PS. The improved dissolution could be attributed to a reduction in particle size of the drug, its deposition on the surface of the SD and improved wettability. CP has very fine particle sizes and hence has large surface areas.

The initial studies in adults, children and infants with this tetravalent G1–G4 BRV formulation (BRV-TV) in the US, demonstrated that each of the components was able to infect the volunteers as determined by vaccine shedding

in the stools and the induction of immune responses [14]. The vaccine was safe; and had no impact on the immune EPZ-6438 price responses of routine childhood immunizations. Vesikari et al. in Finland compared the same tetravalent vaccine to the rhesus tetravalent vaccine (RRV-TV, later licensed as RotaShield) [15]. Both vaccines were equally efficacious, however, the BRV-TV was less reactogenic than the RRV-TV, which was associated with febrile reactions and diminished appetite. In the study, the vaccine induced immune responses in 97% of the infants tested and showed 90% efficacy against severe rotavirus gastroenteritis (SRVGE) during two rotavirus seasons (CI: 35–99%), though OSI-906 solubility dmso the size of this study was limited. These findings clearly support the immunogenicity, safety and potential for efficacy of BRV-PV. With the emergence of the G9 and G8 serotypes, the NIAID added human-bovine (UK) reassortants with G8 and G9 specificities to the tetravalent vaccine, thereby formulating a hexavalent vaccine for use in developing countries [17]. The UK bovine rotavirus G6[P5] strain was used to construct single gene substitution reassortants in which the G gene derives from

human rotavirus serotypes G1, G2, G3, G4, G8 and G9, and all the other genes derived from the UK bovine strain. Non-exclusive licenses for the production of the human-bovine

(UK) vaccine were granted to the Chengdu Institute of Biological Products (CDIBP) (China), Instituto Butantan (Brazil), Shantha Biotech (India) and Serum Institute of India Ltd. (SIIL) (India). SIIL signed the agreement with NIAID in 2005. SIIL is one of the largest vaccine manufacturers in the world. Headquartered at Pune, India, it has been manufacturing vaccines since 1971. Since 1992, SIIL has supplied vaccines to UNICEF and Pan-American Health Organization (PAHO) after receiving the mandatory WHO prequalification for the quality of its vaccines. Currently, 19 of its vaccines are WHO prequalified and Terminal deoxynucleotidyl transferase supplied to immunization programs of many developing countries. It is estimated that SIIL is the largest supplier of DTwP-HB-Hib vaccine and measles vaccine in the world. After transfer of these reassortants from NIAID, SIIL started working on them in 2007. The vaccine was formulated as a lyophilized product which was resuspended in antacid buffer just before use to address the issue of potential inactivation of rotaviruses in the stomach. The antacid buffer diluent was formulated using 9.6 g/l of citric acid and 25.6 g/l of sodium bicarbonate together with water for injection. The viruses are grown in Vero cells which are kidney epithelial cells of African green monkey (Cercopithecus aethiops) and were procured from American Type Culture Collection (ATCC), USA.

For the comparison of inter-genotype neutralization data a heatmap representation of log10

titers (range 1.0–6.0 log10) was employed with titers below the assay threshold of 20 being censored with a value of 10 (1.0 log10). The phylogenetic relationship between L1 amino acid sequences (neighbor-joining [NJ] tree) and inter-genotype distance matrices (n = 500 bootstrap replicates; heatmap range 0.0–1.0) were created using Mega v4.1 [37]. As both HPV vaccines consistently generate HPV31 cross-neutralizing antibodies following immunization, we used this as a benchmark for selecting an appropriate animal model for our pre-clinical immunization studies. selleck chemicals llc BALB/c mice were immunized intra-muscularly with Cervarix® over a 7 week schedule resulting in a median HPV16 neutralizing antibody titer of 10,416 (IQR 7943–16,862; n = 10) ( Fig. 1). Cross-neutralization of HPV31, however, was only apparent in one mouse (HPV31 titer of 733) with a very high HPV16 neutralizing titer of 543,122. Cervarix® immunization of BALB/c mice sub-cutaneously or intra-muscularly over a 12 week schedule did not elicit neutralizing antibodies against HPV31 (data not shown). Conversely, immunization of NZW rabbits with Cervarix® over the same 12 week schedule generated a median HPV16 neutralizing antibody titer of 40,792 (IQR 28,214–57,869;

n = 8) accompanied by a median HPV31 titer of 152 (IQR 35–346; n = 8). Although differences in dosing levels between mice and rabbits GSK1349572 chemical structure may impact on the antibody responses elicited here, HPV31 neutralizing antibody titers generated in rabbits were similar to the titers found in human vaccinees ( Fig. 1) [20], suggesting that NZW rabbits were an appropriate model for examining the generation of cross-neutralizing antibodies Calpain following immunization with L1 VLP. NZW rabbits were immunized with L1 VLP representing individual HPV genotypes

from the Alpha-7 and Alpha-9 species groups and the control BPV. As expected, immunization with L1 VLP induced predominantly high titer neutralizing antibodies against the immunizing genotype resulting in a median type-specific titer of 100,287 (IQR 64,478–246,691) (Fig. 2). However, there were several cases wherein L1 VLP elicited antibodies capable of neutralizing pseudoviruses representing other genotypes. Some of these responses were weak and sporadic, while some were of a reasonable titer and consistent between animals in the same group. For example, HPV33 and HPV58 appeared to share common neutralization epitopes resulting in a median reciprocal neutralization titer of 553 (IQR 520–3594). Similarly, although of a lesser magnitude, VLP representing HPV39 and HPV59 also appeared to share common neutralization epitopes. A phylogenetic representation of the amino acid sequences used for the Alpha-7 and Alpha-9 VLP and pseudovirus L1 proteins demonstrates the close relationship between certain genotypes within each of these two species groups (Fig. 3A).

Education and advice to return to activity and exercise will still remain the cornerstones of early treatment for WAD, but they require further

investigation to determine the most effective form of exercise, dose, and ways to deliver these approaches. Activity and exercise will likely be sufficient for patients at low risk of developing chronic pain, although this is yet to be formally tested. Those patients at medium or high risk of poor recovery will likely need additional treatments PI3K inhibitor drugs to the basic advice/activity/exercise approach. This may include medication to target pain and nociceptive processes as well as methods to address early psychological responses to injury. As was seen in the aforementioned interdisciplinary trial for acute WAD, this is not so easy to achieve.71 The participants of this trial not only found the

side effects of medication unacceptable, but also were less compliant with attendance to a clinical psychologist (46% of participants attended fewer than 4 of 10 sessions) compared to attendance with the physiotherapist (12% attended fewer than four sessions over 10 weeks). It is possible that people with acute whiplash injury see themselves as having a ‘physical’ injury and thus, are more accepting of physiotherapy. selleckchem The burden of requiring visits with several practitioners may also lead to poor compliance. Physiotherapists may be the health care providers best placed to deliver psychological interventions for acute WAD. This approach has been investigated in mainly chronic conditions such as arthritis,73 and recently, in

the management of acute low back pain,74 with results showing some early promise. This is not to say that patients with a diagnosed psychopathology such as depression or post-traumatic stress disorder should be managed by physiotherapists, and of course, these patients will require referral to an appropriately trained professional. Physiotherapists may also no need to take a greater role in the overall care plan of the patient with acute WAD. This would mean having expertise in the assessment of risk factors and an understanding of when additional treatments such as medication and psychological interventions are required. Whilst this has traditionally been the role of general practitioners, it is difficult to see how the busy structure of medical primary care will allow for the appropriate assessment of patients to first identify those at risk, develop a treatment plan, follow the patient’s progress, and modify treatment as necessary. In the case of chronic WAD, more effective interventions need development and testing. It is becoming clear that management approaches that focus predominantly on physical rehabilitation are achieving only small effect sizes.

Mutations Y30A and Y196A (amino acid numbering corresponds to prototoxin without the 13 amino acids N-terminal peptide sequence) were introduced into Epigenetics Compound Library solubility dmso the gene encoding epsilon prototoxin (P-Etx) using the QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Inc. Santa Clara, US) according to the manufacturer’s instructions. Recombinant P-Etx with Y30A and Y196A mutations is termed Y30A-Y196A. Recombinant Y30A-Y196A was expressed, purified and its thermostability assessed as described previously

[14]. Purified recombinant Etx prototoxin was activated with trypsin, TPCK treated from bovine pancreas (Sigma-Aldrich Company Ltd., Gillingham, UK) for 1 h at room temperature and removal of

the C-terminal peptide sequence was assessed by SDS-PAGE as described previously [14]. MDCK.2 cells check details (ATCC-LGC Standards, Teddington, UK) and ACHN cells (ECACC, Salisbury, UK) were routinely cultured in Eagle’s Minimum Essential Medium (EMEM; ATCC-LGC Standards, Teddington, UK) supplemented with 10% Foetal Bovine Serum Gold (PAA, Pasching, Austria) at 37 °C in a humidified atmosphere of 95% air/5% CO2. The culture medium was replaced every 2–3 days. Cells were routinely detached by incubation in trypsin/EDTA and split as appropriate (typically 1:6 dilutions). The cytotoxic activity of trypsin-activated toxin toward MDCK.2 and ACHN cells was determined by measuring the amount of lactate dehydrogenase (LDH) released from the cytosol of lysed cells into the cell culture medium using the CytoTox 96 nonradioactive cytotoxicity assay kit (Promega UK, Southampton, UK) as described previously [14]. The toxin dose required to kill 50% of the cell monolayer (CT50) was determined by nonlinear regression analysis using GraphPad

Prism 6 software (GraphPad Software, La Jolla, USA). All experiments were performed in triplicate with three technical replicates each. To measure binding of prototoxin to MDCK.2 and ACHN cells the On-Cell Western assay was used as described previously next [14]. Bound prototoxin was detected with mouse anti-Etx monoclonal Bio355 antibody (Bio-X Diagnostics S.P.R.L, Belgium) and IRDye 800CW goat anti-mouse IgG (H + L) antibody (LI-COR Biosciences, Lincoln, USA) at 1:500 dilution each. To quantify the amount of fluorescent signal, plates were imaged at 800 nm using the Odyssey CLx infrared imaging system (LI-COR Biosciences, Lincoln, USA). The binding activity of the mutant prototoxin was expressed as the percentage of fluorescence intensity relative to wild type prototoxin. To compare the means of the On-Cell Western assay data, Two-Way ANOVA analysis followed by Dunnett’s multiple comparisons test was carried out using the GraphPad Prism 6 software (GraphPad Software, La Jolla).

However, few clinical trials had been performed in low-income Asian countries with high childhood mortality for either vaccine. At the advice of WHO’s Strategic Advisory Group of Experts (SAGE) [14], a multi-country, placebo-controlled, double-blind Phase III efficacy trial of PRV was conducted in two Asian countries eligible for GAVI Alliance co-financing, Bangladesh Apoptosis Compound Library high throughput and Vietnam. As reported by Zaman et al. [15], PRV was well tolerated, and over an efficacy follow-up period

of nearly 2 years, the vaccine was 48.3% efficacious (95% confidence interval [CI]: 22.3–66.1) against severe rotavirus gastroenteritis. For evaluation of a rotavirus vaccine, measurements of serum anti-rotavirus immunoglobulin (Ig)A and/or serum neutralizing antibody (SNA) responses are considered as the standard for assessing immune responses following rotavirus vaccination [16], [17], [18], [19] and [20].

Thus, the Phase III efficacy trial of PRV in two Asian countries also aimed to measure the anti-rotavirus IgA and SNA responses to human rotavirus serotypes contained in the vaccine (G1, G2, G3, G4, and P1A[8]) at approximately 14 days after Selleck PLX 4720 the third dose. The availability of such immunogenicity data, coupled with efficacy data from the same population, might contribute to identification of an immune correlate of protection or to design of clinical trials of additional rotavirus vaccine candidates. Here we report the detailed findings of the immune responses to a 3-dose regimen of PRV among infants in the two GAVI-eligible Asian countries, Bangladesh and Vietnam, where the pivotal Phase III efficacy trial of PRV was conducted. As previously reported [15], a placebo-controlled, randomized, double-blinded trial all to evaluate the efficacy of three doses of PRV against severe RVGE among infants in low-income populations in Asia was conducted in rural Matlab, Bangladesh, and in urban and peri-urban Nha Trang, Vietnam from March 2007

through March 2009. The study was approved by the investigators’ corresponding institutional review boards and the Western Institutional Review Board. The study was conducted in accordance with the principles of the Declaration of Helsinki and in compliance with Good Clinical Practice guidelines. After obtaining informed consent, infants were randomized in a 1:1 ratio to receive three oral doses of PRV or placebo given with other routine pediatric vaccines, including oral poliovirus vaccine (OPV) and diphtheria-tetanus-whole cell pertussis (DTwP) vaccine, according to local Expanded Program on Immunization schedules (approximately 6-, 10-, and 14-weeks of age). Participants were followed from the moment they were enrolled until the end of the study. Trial enrollment in Bangladesh began in March 2007, while in Vietnam the enrollment began in September 2007.

[1] and [43]. But infection rates are just one indicator of disease burden. Although STIs are reported to have a devastating impact in Sub-saharan Africa [44], there are few reliable data to support these observations. Mortality directly linked to STIs is low, even though these infections may be responsible for an important percentage of HIV acquisition. Morbidity is

not fully evaluated. There are various types of complications, from recurrent pain associated with genital herpes symptoms, to pregnancy complications, and sterility. Data on the incidence of each type of complication are scarce. The economic, psychological and social impact of STIs is not fully documented. As STIs are associated with shame and disgrace, victims tend to hide their disease. As a consequence, the burden of STIs expressed as disability-adjusted years (DALYs) is considered by funding agencies as not high enough to deserve support selleck inhibitor for vaccine development. The introduction of

vaccines targeting sexually transmitted infections is contentious, and STI vaccination programs for adolescents are difficult to implement and often result in low coverage. Another barrier to the perception of the STI burden and the need for a vaccine is the fact that these diseases are still thought to be easily controllable with inexpensive treatments or other interventions. Syphilis is not considered http://www.selleckchem.com/products/fg-4592.html as a candidate for vaccine development as it can be easily cured, although it is still a prevalent disease in developing countries and has re-emerged in developed countries [1] and [45]. Approaches based on screening and treatment of chlamydia, gonorrhea and trichomonas have shown their limitations or failure, while antibiotic resistance is dramatically

increasing [1]. Gonorrhea is now resistant to almost all available antibiotics. Antivirals are effective in reducing the length and severity of HSV-2 reactivations, but they do not totally suppress viral shedding and transmission [46]. A final point: STIs are a public health problem in both developed and developing countries. But most of until the STIs are more prevalent in the poor communities of the society and in developing countries; therefore, these populations are unlikely to be able to pay for the vaccine against these infections. Emerging-country manufacturers can often create a viable business model for these low-income countries with large volume and low prices. However, STI vaccines are not on their radar screen, not due to any scientific issues, but due to the fact that there is little concern for the need for these vaccines in their markets, therefore no justification whatsoever for investment. Vaccine producers are regularly re-evaluating the interest of developing specific vaccines in the light of new data and scientific breakthroughs, and identified pulling and pushing forces.