Immunoinformatics design of multivalent chimeric vaccine for modulation of the immune system in Pseudomonas aeruginosa infection.

Increasing in drug-resistant Pseudomonas aeruginosa and high mortality and morbidity rate have become a health challenge worldwide; therefore, developing the novel therapeutic strategies such as immunogenic vaccine candidate are required. Despite a substantial research effort, the future of immunization against P. aeruginosa due to failure in covering two separate stages of infection, and furthermore, inducing ineffective type of immune response, still remains controversial. In this study, immunoinformatics approach was utilized to design multivalent chimeric vaccine from both stages of infection containing Lectin, HIV TAT peptide, N-terminal fragment of exotoxin A and Epi8 of outer membrane protein F (OprF) with hydrophobic linkers which have a high density of B-cell, T Lymphocytes (HTL), T Lymphocytes (CTL), and IFN-γ epitopes. The physicochemical properties, antigenicity, and allergenicity for designed vaccine were analyzed. 3D model generation and refinement further validation of the final vaccine were followed by computational docking with molecular dynamics analyses that demonstrated high- affinity interaction between vaccine and TLR-4. Finally, designed vaccine was in silico cloned in pET22b. We have expected that the designed vaccine able to elucidate innate, humoral and cellular innate immune responses and control the interaction of P. aeruginosa with host and maybe overcome to P. aeruginosa vaccines drawback.

Mixing is required for uniform reconstitution of filter-dried protein antigens in a single-injection vaccine formulation.

Ambient temperature filter dried vaccine formulations have been proposed to simultaneously achieve thermostability and offer a ready-to-use immunisation device that combines reconstitution and injection. Vaccine concentration should be uniform at the point of injection, but the uniformity following direct reconstitution of filter-dried vaccines has not been reported. We present here a study of vaccine mixing and release following dissolution of filter-dried model protein and toxoid antigens within a single syringe, filter and needle unit. Release was better for filters made from glass than cellulose. Without additional mixing, uniformity was poor and only 41% of input protein was released from protein filter-dried onto glass fiber. In contrast, adding a simple glass bead and mixing by inversion, 100% release antigen solution was achieved, with uniform concentration at exit from the needle throughout a simulated injection. Adsorption onto alum adjuvant had no detectable effect on vaccine dissolution and mixing. The uniformity and yield of low doses of diphtheria and tetanus toxoid was also improved by mixing, albeit with a lower yield of 60-68%. We conclude that uniformity and mixing should be studied to ensure safety and efficacy of directly reconstituted filter-dried vaccine formulations.

A novel combination adjuvant platform for human and animal vaccines.

Adjuvants are crucial components of many vaccines. They are used to improve the immunogenicity of vaccines with the aim of conferring long-term protection, to enhance the efficacy of vaccines in newborns, elderly or immunocompromised persons, and to reduce the amount of antigen or the number of doses required to elicit effective immunity. Novel combination adjuvants have been tested in both candidate animals and humans vaccines and have generated encouraging results. Recently, we developed a combination adjuvant platform (TriAdj) comprising of three components, namely a TLR agonist, either polyI:C or CpG oligodeoxynucleotides (ODN), host defense peptide and polyphosphazene. This adjuvant platform is stable and highly effective in a wide range of animal and human vaccines tested in mice, cotton rats, pigs, sheep, and koalas. TriAdj with various vaccines antigens induced effective long-term humoral and cellular immunity. Moreover, the adjuvant platform is suitable for maternal immunization and highly effective in neonates even in the presence of maternal antibodies. This novel vaccine platform, offers excellent opportunity for use in present and future generations of vaccines against multiple infectious agents and targets challenging populations.

Multiplex immunoassay for in vitro characterization of acellular pertussis antigens in combination vaccines.

Vaccines characterization is required to ensure physical, chemical, and biological integrity of antigens and adjuvants. Current analytical methods mostly require complete antigen desorption from aluminum-based adjuvants and are not always suitable to distinguish individual antigens in multivalent formulations. Here, Luminex technology is proposed to improve the analytics of vaccine characterization. As proof of concept, TdaP (tetanus, diphtheria and acellular pertussis) combination, adjuvanted with aluminum hydroxide, was chosen as model formulation to quantify and determine the level of adsorption of acellular pertussis (aP) antigens onto adjuvant surface at the same time. The assay used specific antibodies bound to magnetic microspheres presenting unique digital signatures for each pertussis antigen, allowing the simultaneous recognition of respective antigens in the whole vaccine, avoiding laborious procedures for adjuvant separation. Accurate and reproducible quantification of aP antigens in TdaP vaccine has been achieved in the range 0.78-50 ng/mL, providing simultaneously information on antigen identity, quantity, and degree of adsorption to aluminum hydroxide. The current study could further be considered as a model to set up in vitro potency assays thus supporting the replacement of animal tests accordingly to the 3Rs concept.

How common are long-lasting, intensely itching vaccination granulomas and contact allergy to aluminium induced by currently used pediatric vaccines? A prospective cohort study.

UNLABELLED: The frequency of long-lasting, intensely itching subcutaneous nodules at the injection site for aluminium (Al)-adsorbed vaccines (vaccination granulomas) was investigated in a prospective cohort study comprising 4,758 children who received either a diphtheria-tetanus-pertussis-polio-Haemophilus influenzae type b vaccine (Infanrix®, Pentavac®) alone or concomitant with a pneumococcal conjugate (Prevenar). Both vaccines were adsorbed to an Al adjuvant. Altogether 38 children (0.83 %) with itching granulomas were identified, epicutaneously tested for Al sensitisation and followed yearly. Contact allergy to Al was verified in 85 %. The median duration of symptoms was 22 months in those hitherto recovered. The frequency of granulomas induced by Infanrix® was >0.66 % and by Prevenar >0.35 %. The risk for granulomas increased from 0.63 to 1.18 % when a second Al-adsorbed vaccine was added to the schedule. CONCLUSION: Long-lasting itching vaccination granulomas are poorly understood but more frequent than previously known after infant vaccination with commonly used diphtheria-tetanus-pertussis-polio-Haemophilus influenzae type b and pneumococcal conjugate vaccines. The risk increases with the number of vaccines given. Most children with itching granulomas become contact allergic to aluminium. Itching vaccination granulomas are benign but may be troublesome and should be recognised early in primary health care to avoid unnecessary investigations, anxiety and mistrust.

A novel enzyme-linked immuno-sorbent assay (ELISA) for the quantification of total and free polysaccharide in Haemophilus influenzae b-Tetanus toxoid conjugate vaccines in monovalent and combined vaccine formulations.

Current Haemophilus influenzae b conjugate vaccines (Hib), which are made of purified capsular polysaccharide (poly-ribosyl-ribitol-phosphate; PRP) conjugated to a carrier protein, are almost completely evaluated by physico-chemical methods to ensure the integrity and stability of the vaccine and consistency of manufacture of batches. The absence of a potency assay makes the quantification of total PRP content (in SI units) and of % free polysaccharide in final fills or bulk components of Hib vaccines critical release tests for both manufacturers and national control authorities. Here we describe a simple and sensitive Enzyme-Linked Immuno-sorbent Assay (ELISA) which has been developed to quantify total and free PRP content in Hib-TT vaccine alone or when in combination with other vaccines. The assay is robust, specific and highly sensitive.

Review of a new fully liquid, hexavalent vaccine: Hexaxim.

INTRODUCTION: The introduction of injectable vaccines targeting new diseases into childhood immunization programs has resulted in the need for combination vaccines to reduce the number of injections given during early childhood and maintain acceptability of targeting multiple pathogens by vaccination. Currently, there is only one licensed hexavalent combination vaccine which targets diphtheria, polio, tetanus, Haemophilus influenzae type b, hepatitis B and pertussis. A new, fully liquid formulation hexavalent vaccine ( Hexaxim ) has been developed and is currently undergoing licensure for use in childhood immunization programs. AREAS COVERED: Safety and immunogenicity studies of Hexaxim have been undertaken in a diversity of settings, been evaluated with different dosing schedules and in comparison to the other licensed hexavalent vaccine (Infanrix hexa). This review of published journal articles and conference proceeding is focused on the studies in which Hexaxim has been evaluated and which are contributing to its pending licensure. Non-inferiority was demonstrated at the level of proportion of children developing seroprotective titers or showing seroconversion following the primary series of vaccine compared to the same target-antigens included in licensed combination vaccines. Also, Hexaxim was associated with a favorable safety and tolerability profile when administered during the first 6 months of life. Adequate and robust memory responses were elicited following a booster dose in the second year of life. EXPERT OPINION: The development of new hexavalent combination vaccines targeting established pathogens is likely to assist in improving compliance and timeliness of vaccination in infants. These formulations will, however, need to be monitored for medium- and long-term effectiveness amidst growing concern of waning immunity against diseases such as pertussis when using acellular-pertussis vaccine and possibly hepatitis B when using combination vaccines. Nevertheless, the development of such combination vaccines remains necessary to help with the introduction of other new vaccines into an already crowded childhood immunization schedules.

Physico-chemical properties of Salmonella typhi Vi polysaccharide-diphtheria toxoid conjugate vaccines affect immunogenicity.

In this study it was demonstrated that the immunogenicity of Vi polysaccharide-diphtheria toxoid conjugates was related to the physical and chemical structure of the conjugate. Conjugates were prepared in two steps, firstly binding adipic acid dihydrazide (ADH) spacer molecules to diphtheria toxoid (DT) carrier protein then secondly binding varying amounts of this derivatized DT to a fixed amount of Vi capsular polysaccharide purified from Salmonella enterica Serovar Typhi. As the amount of DT bound to the Vi increased the size of the conjugate increased but also the degree of cross-linking increased. The immunogenicity of the conjugates was tested in mice and measured by ELISA for anti Vi and anti DT IgG responses, and the results revealed a trend that as the amount of DT bound to the Vi increased the anti Vi responses increased. This study establishes a correlation between physico-chemical characteristics of the conjugate and the magnitude of the anti Vi and anti DT responses.

Immunogenicity and thermal stability of a combined vaccine against Haemophilus influenzae type b and Neisseria meningitidis serogroup C diseases.

The immunogenicity, structure and stability of a combined conjugate vaccine against Haemophilus influenzae type b and meningococcal serogroup C (Hib/MenC) were investigated. A rat model for immunogenicity showed that antibody responses to Hib and MenC in the combined vaccine were similar to or higher than those of individual conjugates given alone, or concomitantly at separate sites. At elevated temperatures, the combination vaccine was slightly more stable than a monovalent Hib-TT vaccine, with respect to molecular size, which could be attributed to differences in the formulations. Following 5 weeks incubation at 56 degrees C, there was some dissociation of high molecular weight conjugate without significant loss of saccharide integrity; however, this did not significantly affect the vaccine immunogenicity, demonstrating the stability of this lyophilized vaccine.

Physico-chemical characterisation and immunogenicity of a multi-valent candidate vaccine against non-typeable Haemophilus influenzae and Moraxella catarrhalis.

The physico-chemical characteristics and immunogenicity of a candidate vaccine against otitis media, prepared from recombinant lipidated outer membrane proteins (rLP4 and rLP6) from non-typeable Haemophilus influenzae (NTHi) and of the ubiquitous cell surface protein UspA2 from Moraxella catarrhalis, were evaluated. Optical spectroscopy, size exclusion chromatography and gel electrophoresis were used to characterise the purified protein components and assess their purity and molecular sizes. The results showed that the three proteins were highly purified. Possible dimers in rLP4, dimers and multimers in rLP6 and UspA2 were detected. Small amounts of rLP4 and rLP6 dimers and most of UspA2 complexes remained tightly bound even after SDS treatment under reducing conditions. Immunogenicity studies showed that all proteins induced substantial antibody responses in mice immunised with AlPO4-adsorbed rLP4, rLP6 or UspA2 or a combination of these proteins. However, combination of these proteins resulted in a reduced response to rLP4 and rLP6, but not to UspA2, suggesting interference between these proteins which should be taken into consideration during the development and evaluation of this vaccine.

An adolescent-adult formulation tetanus and diptheria toxoids adsorbed combined with acellular pertussis vaccine has comparable immunogenicity but less reactogenicity in children 4-6 years of age than a pediatric formulation acellular pertussis vaccine and diphtheria and tetanus toxoids adsorbed combined with inactivated poliomyelitis vaccine.

In Canada, the fifth dose of the routine childhood immunization schedule against diphtheria, tetanus, pertussis and polio is given at 4-6 years of age. Up to 30% of children may have significant local reactions (redness, swelling) and this may be related to pertussis and diphtheria antigen content. We sought to determine if a combination product with lower content of pertussis and diphtheria toxoids (dTap) would result in fewer local reactions and not have inferior immunogenicity to a combination vaccine with higher pertussis and diphtheria content (diphtheria-tetanus-acellular pertussis-inactivated polio virus, DTaP-IPV). Healthy children aged 4-6 years with complete primary immunization series and a fourth dose of diphtheria and tetanus toxoids component pertussis inactivated polio and Haemophilus influenzae type B conjugate vaccine were randomized to one dose of dTap, followed in 4-6 weeks by one dose of IPV or control DTaP-IPV. Immediate reactions within 30 min, solicited injection site and systemic reactions within 14 days, and unsolicited adverse events (AE) within 6 weeks post-vaccination were monitored. Serum was collected prior to immunization, and 4-6 weeks after vaccine for diphtheria, tetanus and pertussis antibodies (Ab). Sample size was designed to detect > or =10% difference in injection site erythema, pain or swelling between groups 593 children at eight Canadian sites completed the study; no participant withdrew because of an AE. All safety endpoints on days 0-14 were less frequent in children randomized to the dTap than DTaP-IPV group: erythema (34.6% versus 51.7%), swelling (24.2% versus 33.8%) and pain (39.6% versus 67.2%). Fever was also less common (8.72% versus 16.9%). All children in both study groups had seroprotective Ab levels to diphtheria and tetanus at 4-6 weeks (> or =0.10 IU/mL). The majority of children in each vaccine arm had a four-fold increase in pertussis antibodies. Fever and injection site reactions are less common in 4-6 year-old-children who receive a dTap vaccine compared to DTaP-IPV, without inferior immunogenicity.

[Complex of recombinant heat shock protein with lipopolysaccharide induces rapid protection of mice against Salmonella typhimurium and avirulent for humans avian influenza virus H5N2].

Protective effect of immunization with heat shock protein (HSP) against bacterial and viral infections in mice was studied. Recombinant HSP 70 kDa of Mycobacterium tuberculosis contaminated with lypopolysaccharide (0.185 mcg/ml) was used for experiments. One intraperitoneal injection of 100 or 400 mcg of HSP induced rapid protection against intraperitoneal challen e with 125 LD50 of Salmonella typhimurium (on 3rd-6th day) and against intranasal challenge with 10 LD50 of avirulent for humans avian influenza virus H5N2 (A/ mallard/Pennsylvania/10218/84) (on 5th-8th day). Three daily injections with 10 mcg of HSP induced rapid, significant and long-term protection against S. typhimurium. Immunization with HSP protected 100% of mice during 3 days after the challenge, 50% of immunized animals survived during 21 days (duration of the study). All nonimmunized mice died on 6th day.

A practical approach to optimization and validation of a HPLC assay for analysis of polyribosyl-ribitol phosphate in complex combination vaccines.

The use of multi-factor statistical experimental design methodology minimized the vaccine material and laboratory resources required for optimization and validation of an HPLC assay for quantitation of depolymerized and total PRP. Components of the assay selected for optimization were adjuvant dissolution, ultracentrifuge conditions including ultracentrifuge model, sample diluent, mobile phase and column oven temperature. Previous experience has shown these components of the assay to be most troublesome and therefore required optimization prior to validation. Specificity, linearity, precision, accuracy and ruggedness were confirmed through a validation of the optimized assay. The validation also established the assay to be stability indicating, by showing that changes to the integrity of the PRP-OMPC conjugate could be detected.

Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccine (part 2).

The study is a contribution to the EDQM's efforts to meet some of the expectations of the 3 Rs: Replacement, Reduction and Refinement of animal assays as proposed by Russell and Burch in 1959 and adopted by the European Union in 1986, and specifically to validate alternative assays to replace, for batch-release purposes, the European Pharmacopoeia (Ph. Eur.) in vivo direct challenge procedures for the potency determination of diphtheria toxoid vaccines. The study results may be used in support of the replacement of the multi-dilution direct challenge procedures in different animal models by a single dilution serology test, where appropriate, and to use sera from the same animals for potency testing of several components in combined vaccines. With regard to the latter, the present study explores the possibility of testing both diphtheria and tetanus toxoid potencies using serum from the same animals.

Combined DNA vaccines formulated either in DDA or in saline protect cattle from Mycobacterium bovis infection.

We tested the effectiveness of combined DNA vaccines encoding antigens Ag85B, MPT64 and MPT83 from Mycobacterium tuberculosis on cattle. Our results showed that calves treated with combined DNA vaccines in the presence of dimethyldioctyldecyl ammonium bromide (DDA) or saline elicited a strong gamma interferon (IFN-gamma) response 1 or 2 months after the third vaccination. All three antigens induced substantial levels of IFN-gamma production 1 month after the bacterial challenge, when the BCG-driven IFN-gamma levels dropped to less than one third of their peak values. Animals receiving combined DNA vaccines produced highest amounts of IgG antibody titer 2 months after the third vaccination. Steady state low IgG levels were found 2 months after bacterial challenge. A few small lung and lymph node lesions were detected in 1/5 animals treated with combined DNA vaccines, whereas 3/5 of BCG-treated and 5/5 of vector-control calves showed larger and significantly more lesions. About 70- to 100-fold fewer bacteria were found in the lungs and lymph nodes of combined DNA vaccine-treated animals compared to those of the control group. Histopathological analyses showed that vaccinated calves possessed substantially improved post-infection lung and lymph node pathology relative to the controls. Our data indicate that combined DNA vaccines may be used in cattle to combat bovine tuberculosis.

IgE reactivity to vaccine components in dogs that developed immediate-type allergic reactions after vaccination.

Allergic reactions after vaccination are considered as an important practical problem in dogs; however, their immunological mechanism has not been well understood. The present study was designed to investigate the relationship between IgE reactivity to the vaccines and immediate-type allergic reactions after vaccination in dogs. Sera from 10 dogs that developed immediate-type allergic reactions such as circulatory collapse, cyanosis, dyspnea, facial edema, and vomiting within 1h after vaccination with non-rabies monovalent or combined vaccines and sera from 50 dogs that did not develop allergic reactions after vaccination were collected. Serum IgE reactivity to the injected vaccines was measured by fluorometric ELISA using a mouse monoclonal anti-dog IgE antibody. Then, IgE reactivity to fetal calf serum (FCS) and stabilizer proteins (gelatin, casein, and peptone) included in the vaccines was measured in sera that had high levels of IgE to the vaccines. Levels of serum specific IgE to the vaccines in dogs with immediate-type allergic reactions (59-4173 fluorescence units [FU], mean +/- S.D.: 992.5 +/- 1181.9 FU) were significantly higher than those in control dogs (38-192 FU, 92.4 +/- 43.3 FU) (P < 0.001). Of the eight dogs that developed immediate-type allergic reactions and had high levels of serum specific IgE to the vaccines, seven had specific IgE directed to FCS. The IgE reactivity to the vaccines in sera from these dogs was almost completely inhibited by FCS. The other one dog had serum IgE directed to gelatin and casein included in the vaccine as stabilizers. The results obtained in this study suggest that immediate-type allergic reactions after vaccination in dogs were induced by type I hypersensitivity mediated by IgE directed to vaccine components. In addition, FCS, gelatin, and casein included in vaccines could be the causative allergens that induced immediate-type allergic reactions after vaccination in dogs.

A new DTPw-HBV/Hib vaccine is immunogenic and safe when administered according to the EPI (Expanded Programme for Immunization) schedule and following hepatitis B vaccination at birth.

New combination vaccines and reliable sources of vaccine components are essential to ensure the success of mass immunisation programmes in the 21st century. We evaluated a new combined diphtheria-tetanus-whole-cell-pertussis-hepatitis B vaccine, extemporaneously mixed with a Haemophilus influenzae type b conjugate vaccine (DTPw-HBV/Hib) containing 2.5 microg PRP in 913 Philippino infants, administered according to the EPI schedule at 6, 10 and 14 weeks of age after a birth dose of hepatitis B vaccine (HBV; trial DTPw-HBV/Hib-001). One month after the third dose of DTPw-HBV/Hib (N = 182), 99.4% and 94.2% of subjects had anti-PRP antibody levels > or =0.15 microg/mL and > or =1.0 microg/mL, respectively. In addition, 95.9%, 100.0% and 87.6% of subjects had seroprotective antibody concentrations against diphtheria, tetanus and hepatitis B, respectively. The seroprotection rate to hepatitis B increased significantly to 94.3% in subjects who received a dose of HBV at birth. The pertussis vaccine response rate was > or =95%. Seroprotection/vaccine response rates to all antigens after DTPw-HBV/Hib were at least as good as those observed after vaccination with GSK Biologicals' licensed Tritanrix HepB/Hiberix (containing 10 microg PRP) which was used as comparator. Although redness >20 mm in diameter and fever > or = 37.5 degrees C (axillary route) occurred more often after the new DTPw-HBV/Hib vaccine (p < 0.05), other Grade 3 adverse events occurred similarly between the groups. The new DTPw-HBV/Hib vaccine was as immunogenic and well tolerated as the licensed control vaccine when administered according to the immunologically challenging EPI schedule. A birth dose of HBV is important to maximize protection against hepatitis B in endemic regions where the EPI schedule is in place.

Comparative investigation of the safety of hexavalent vaccines for primary scheduled infant immunizations in Germany over a time period of 2 years.

BACKGROUND: Confidence in hexavalent vaccines has recently been shattered in Germany, after 5 infant deaths occurred shortly after such a vaccination. The aim of this study was to evaluate the safety of hexavalent vaccines over a time period of 2 years. MATERIAL/METHODS: With the written informed consent of the parents, we enrolled in the study all healthy infants who attended our office for a primary scheduled infant immunization during the study period from July 1, 2001, to June 30, 2003. This period was divided into two parts: (1) In a pilot study (July 1 to December 31, 2001), the two hexavalent vaccines licensed in Germany, Hexavac and Infanrix hexa, were compared to each other to evaluate if one of these vac-cines would be better tolerated. (2) During the follow-up study period (January 1, 2002, to June 30, 2003), that hexavalent vaccine was to be used, if any, which had been shown to have lower side effects during the pilot period. The trivalent measles-mumps-rubella vaccine Priorix was applied to all infants during both study periods. The parents were advised to immediately consult our office in any case of suspected or proven side effect from the vaccination. RESULTS: 3,658 polyvalent (hexavalent and trivalent) vaccinations were applied to 1997 infants. Local reactions were observed after application of Priorix in 0.0%, of Infanrix hexa in 0.46%, and of Hexavac in 3.1%, respectively. CONCLUSIONS: The safety of Priorix and Infanrix hexa in particular could be established. Hexavalent vaccines can be recommended for introduction in all European countries for primary scheduled infant immunizations.

Distribution of adsorbed antigen in mono-valent and combination vaccines.

The distribution of alpha-casein, bovine serum albumin (BSA), myoglobin and recombinant protective antigen (rPA) in mono-valent and combination vaccines containing aluminum hydroxide adjuvant was studied by fluorescence microscopy and flow cytometry. Green and red fluorescent probes were conjugated to the antigens. Adsorption isotherms of the fluorescently labeled proteins to aluminum hydroxide adjuvant demonstrated that incorporation of the fluorescent probe did not significantly affect the adsorption. In mono-valent vaccine systems, antigen adsorption occurred within one minute and uniform surface coverage of the adjuvant aggregates was observed within 1h. Content uniformity was achieved through a cycle of de-aggregation and re-aggregation of the aluminum hydroxide adjuvant aggregates caused by mixing. For combination vaccines, two antigens were adsorbed separately to the aluminum hydroxide adjuvant prior to combination. Following combination, cycles of de-aggregation and re-aggregation occurred due to mixing, which led to uniform distribution of both antigens. The results of this study indicate that content uniformity should not be an issue during the production of mono-valent or combination vaccines as long as adequate mixing procedures are followed.