Adverse effects of single-component measles vaccine in school children.

Measles is a highly contagious viral infection potentially with serious complications and the principal method of protection from the disease is vaccination. Measles vaccination resulted in a 79% drop in measles deaths between 2000 and 2015 worldwide. There has long been a debate about the necessity and benefit-loss ratio of routine MMR vaccination due to possible AE of MMR vaccine. Especially in developed countries which are thought to be free of measles there is an increasing tendency towards hesitation for vaccination though there have been continued outbreaks of measles in countries in which measles is considered to be eliminated. Considering those facts, we decided to publish our data about measles vaccination and adverse effects (AE) during national catch-up measles vaccination programme which took place December 8-26, 2003. A total of 152.648 children aged between seven and fourteen were vaccinated by a live attenuated measles vaccine of which 148.064 (97%) had received measles vaccine by age nine or twelve months. During one month follow-up the AE were recorded. Totally 30.302 AE were reported in 24.209 children, of which 52% of them were local and pain and swelling at injection side were the most common AE. Fever and headache were the most commonly observed systemic side effects. All AE were mild and transient except in four children in whom encephalitis was diagnosed during the one month observation period. Further investigation of the etiology of those cases revealed that they were not related to measles or measles vaccine. In conclusion, single-component measles vaccine was found to be safe in previously MMR vaccinated children in short term and long term effects may be need to be clarified by further studies.

Physicochemical and Preclinical Evaluation of a Novel Buccal Measles Vaccine.

The aim of this study is to develop an orally disintegrating film (ODF) containing a microparticulate measles vaccine formulation for buccal delivery. The measles vaccine microparticles were made with biocompatible and biodegradable bovine serum albumin (BSA) and processed by spray drying. These vaccine microparticles were incorporated in the ODF, consisting of Lycoat RS720®, Neosorb P60W® and Tween 80. The yield of the microparticles was approximately 85-95%, w/w. The mean size of the vaccine microparticles was 3.65 ± 1.89 µm and had a slightly negative surface charge of 32.65 ± 2.4 mV. The vaccine particles were nontoxic to normal cells at high concentrations (500 µg/2.5 × 105 cells) of vaccine particles. There was a significant induction of innate immune response by vaccine microparticles which was observed in vitro when compared to blank microparticles (P < 0.05). The vaccine microparticles also significantly increased the antigen presentation and co-stimulatory molecules expression on antigen presenting cells, which is a prerequisite for Th1 and Th2 immune responses. When the ODF vaccine formulation was dosed in juvenile pigs, significantly higher antibody titers were observed after week 2, with a significant increase at week 4 and plateauing through week 6 comparative to naïve predose titers. The results suggest that the ODF measles vaccine formulation is a viable dosage form alternative to noninvasive immunization that may increase patient compliance and commercial distribution.

Generation of a More Immunogenic Measles Vaccine by Increasing Its Hemagglutinin Expression.

UNLABELLED: Imported measles virus (MV) outbreaks are maintained by poor vaccine responders and unvaccinated people. A convenient but more immunogenic vaccination strategy would enhance vaccine performance, contributing to measles eradication efforts. We report here the generation of alternative pediatric vaccines against MV with increased expression of the H protein in the background of the current MV vaccine strain. We generated two recombinants: MVvac2-H2, with increased full-length H expression resulting in a 3-fold increase in H incorporation into virions, and MVvac2-Hsol, vectoring a truncated, soluble form of the H protein that is secreted into the supernatants of infected cells. Replication fitness was conserved despite the duplication of the H cistron for both vectors. The modification to the envelope of MVvac2-H2 conferred upon this virus a measurable level of resistance to in vitro neutralization by MV polyclonal immune sera without altering its thermostability. Most interestingly, both recombinant MVs with enhanced H expression were significantly more immunogenic than their parental strain in outbred mice, while MVvac2-H2 additionally proved more immunogenic after a single, human-range dose in genetically modified MV-susceptible mice. IMPORTANCE: Measles incidence was reduced drastically following the introduction of attenuated vaccines, but progress toward the eradication of this virus has stalled, and MV still threatens unvaccinated populations. Due to the contributions of primary vaccine failures and too-young-to-be-vaccinated infants to this problem, more immunogenic measles vaccines are highly desirable. We generated two experimental MV vaccines based on a current vaccine's genome but with enriched production of the H protein, the main MV antigen in provoking immunity. One vaccine incorporated H at higher rates in the viral envelope, and the other secreted a soluble H protein from infected cells. The increased expression of H by these vectors improved neutralizing responses induced in two small-animal models of MV immunogenicity. The enhanced immunogenicity of these vectors, mainly from the MV that incorporates additional H, suggests their value as potential alternative pediatric MV vaccines.

Development and characterization of alginate coated low molecular weight chitosan nanoparticles as new carriers for oral vaccine delivery in mice.

In the present study, nanoparticles of low MW chitosan (CS) were formulated in which measles antigen was entrapped and subsequently coated with sodium alginate. The size and surface properties of the nanoparticle can be tuned with different MW of CS. In vitro release studies showed initial burst release followed by extended release, best fitted in the Makoid-Banakar model (R(2)>0.98). SDS-PAGE assay revealed that alginate coating could effectively protect antigen in acidic condition for at least 2h. Cell viability was assessed using MTT assay into HT 29 cell line. Formulations were orally administered to mice and immunological responses were evaluated using ELISA method. Obtained results showed that measles antigen-loaded CS nanoparticles induced strong immune response and significant correlation was observed between the immune response with CS MW. Protecting ability of antigen in gastric environment, sustained release kinetics, systemic and mucosal immune responses and low cytotoxicity observed for the alginate coated nanoparticles demonstrated that LMW CS could be promising platform for oral vaccine delivery.

Vaccine instability in the cold chain: mechanisms, analysis and formulation strategies.

Instability of vaccines often emerges as a key challenge during clinical development (lab to clinic) as well as commercial distribution (factory to patient). To yield stable, efficacious vaccine dosage forms for human use, successful formulation strategies must address a combination of interrelated topics including stabilization of antigens, selection of appropriate adjuvants, and development of stability-indicating analytical methods. This review covers key concepts in understanding the causes and mechanisms of vaccine instability including (1) the complex and delicate nature of antigen structures (e.g., viruses, proteins, carbohydrates, protein-carbohydrate conjugates, etc.), (2) use of adjuvants to further enhance immune responses, (3) development of physicochemical and biological assays to assess vaccine integrity and potency, and (4) stabilization strategies to protect vaccine antigens and adjuvants (and their interactions) during storage. Despite these challenges, vaccines can usually be sufficiently stabilized for use as medicines through a combination of formulation approaches combined with maintenance of an efficient cold chain (manufacturing, distribution, storage and administration). Several illustrative case studies are described regarding mechanisms of vaccine instability along with formulation approaches for stabilization within the vaccine cold chain. These include live, attenuated (measles, polio) and inactivated (influenza, polio) viral vaccines as well as recombinant protein (hepatitis B) vaccines.

Towards ambient temperature-stable vaccines: the identification of thermally stabilizing liquid formulations for measles virus using an innovative high-throughput infectivity assay.

As a result of thermal instability, some live attenuated viral (LAV) vaccines lose substantial potency from the time of manufacture to the point of administration. Developing regions lacking extensive, reliable refrigeration ("cold-chain") infrastructure are particularly vulnerable to vaccine failure, which in turn increases the burden of disease. Development of a robust, infectivity-based high throughput screening process for identifying thermostable vaccine formulations offers significant promise for vaccine development across a wide variety of LAV products. Here we describe a system that incorporates thermal stability screening into formulation design using heat labile measles virus as a prototype. The screening of >11,000 unique formulations resulted in the identification of liquid formulations with marked improvement over those used in commercial monovalent measles vaccines, with <1.0 log loss of activity after incubation for 8h at 40°C. The approach was shown to be transferable to a second unrelated virus, and therefore offers significant promise towards the optimization of formulation for LAV vaccine products.

Heat-stable measles vaccine produced by spray drying.

A combination of unique stabilizers and mild spray drying process conditions was employed to produce heat-stable measles vaccine powder. Live attenuated measles vaccine from Serum Institute of India was formulated with pharmaceutically approved stabilizers, including sugars, proteins, amino acids, polymers, surfactants, and plasticizers, as well as charged ions. In addition, the effects of buffer salt and pH on the storage stability of measles virus were examined. The potency of the dried vaccine stored at several temperatures was quantified by TCID(50) assay on Vero cells. As a comparison to other process methods, lead formulations were also subjected to freeze drying and foam drying. The optimized measles vaccine formulation tested at 37 degrees C was stable for approximately 8 weeks (i.e. time for 1 log TCID(50) loss). The measles titer decreased in a bi-phasic manner, with initial rapid loss within the first week but relative stability thereafter. Key stabilizers identified during the formulation screening processes were L-arginine, human serum albumin, and a combination of divalent cations. Spray drying was identified as the optimal processing method for the preparation of dried vaccine, as it generally resulted in negligible process loss and comparable, if not better storage stability, with respect to the other processes. Processing methods and formulation components were developed that produced a measles vaccine stable for up to 8 weeks at 37 degrees C, which surpassed the WHO requirement for heat stability of 1 week at that temperature.

[The establishment of method for identifying China vaccine strains and wild strains of measles virus].

OBJECTIVE: To establish a simple and quick method for identifying China vaccine strains and wild strains of Measles Virus. METHODS: To search the enzyme site in Hemagglutinin gene of measles virus for different domestic vaccine strains and wild strains of measles virus, and design the RT-PCR primer within the range covering the enzyme site, and then to confirm the specificity and sensibility of the RT-PCR method, and then identify the RT-PCR product by RFLP. RESULTS: The one-step RT-PCR method is sensitive, the measles virus of 4.64 TCID50 can be detected at least. No positive bands can be found in the non-measles virus strains, it means that the RT-PCR method has good specificity, the PCR products of Chian measles vaccine strains of Shang-191 and Chang-47 were all cut into two fragments (287 bp and 151 bp) by Afi II, but two measles wild virus strains can't be cut by Afl II. CONCLUSION: The RT-PCR-RFLP method which we established is a rapid and simple method for identifying China vaccine strain and wild strain.

Stabilizing formulations for inhalable powders of live-attenuated measles virus vaccine.

Carbon dioxide Assisted Nebulization with a Bubble Dryer((R)) (CAN-BD) processing allows particles to be made in the 3-5 mum size range, which is desirable for lung delivery, without destroying biological activity. In response to the Grand Challenge in Global Health Initiative #3, we have been developing an inhalable needle-free live-attenuated measles virus vaccine for use in developing countries. Measles was chosen because it is the number one vaccine preventable killer of children worldwide. Powders were processed by CAN-BD, where a solution containing excipients and live-attenuated measles virus in water was mixed intimately with supercritical or near superctitical carbon dioxide to form an emulsion. The emulsion was expanded to atmospheric pressure through a flow restrictor. The resulting plume was dried by heated nitrogen and the powders collected on a filter at the bottom of the drying chamber. Powders were analyzed using varying techniques including X-ray diffraction, scanning electron microscopy, Andersen cascade impaction, differential scanning calorimetery, Karl Fischer titration, and viral plaque assay. CAN-BD has been used to produce powders of live-attenuated measles virus vaccine with characteristics desirable for lung delivery. The powders retain viral activity through forming and drying the microparticles by CAN-BD, and have passed the WHO stability test for 1 week at 37 degrees C. The powders have an amorphous character and a glass transition temperature of around 60 degrees C. Lyophilization, the present standard commercial method of processing measles vaccine makes solids with a water content of less than 1%. By substituting myo-inositol for sorbitol and using the CAN-BD drying technique the water content can be lowered to 0.5%. The most successful formulations to date have been based conceptually on the current lyophilized formulation, but with modifications to the type and amounts of sugar. Of current interest are formulations containing myo-inositol, as they retain high viral activity and have low initial water content.

Stabilization of measles virus for vaccine formulation.

An attenuated live measles virus (MV) was characterized by several biophysical methods as a function of temperature and pH. Following a method developed previously, the resultant light scattering and spectroscopic data were synthesized into an empirical phase diagram that visually and simultaneously represents the entire data set. Using this empirically-based phase diagram, screening assays were developed to identify potential vaccine stabilizers. Various compounds are shown by these assays to inhibit the temperature-induced aggregation of viral particles, and also to protect the integrity of the viral envelope. Accelerated stability assays show that, upon thermal challenge, MV formulated with these excipients retains its infectivity to a significant extent. Thus, the enhanced physical stability produced by this method is shown to protect the biological activity of this important but labile vaccine.

Dose-dependent protection against or exacerbation of disease by a polylactide glycolide microparticle-adsorbed, alphavirus-based measles virus DNA vaccine in rhesus macaques.

Measles remains an important cause of vaccine-preventable child mortality. Development of a low-cost, heat-stable vaccine for infants under the age of 6 months could improve measles control by facilitating delivery at the time of other vaccines and by closing a window of susceptibility prior to immunization at 9 months of age. DNA vaccines hold promise for development, but achieving protective levels of antibody has been difficult and there is an incomplete understanding of protective immunity. In the current study, we evaluated the use of a layered alphavirus DNA/RNA vector encoding measles virus H (SINCP-H) adsorbed onto polylactide glycolide (PLG) microparticles. In mice, antibody and T-cell responses to PLG-formulated DNA were substantially improved compared to those to naked DNA. Rhesus macaques received two doses of PLG/SINCP-H delivered either intramuscularly (0.5 mg) or intradermally (0.5 or 0.1 mg). Antibody and T-cell responses were induced but not sustained. On challenge, the intramuscularly vaccinated monkeys did not develop rashes and had lower viremias than vector-treated control monkeys. Monkeys vaccinated with the same dose intradermally developed rashes and viremia. Monkeys vaccinated intradermally with the low dose developed more severe rashes, with histopathologic evidence of syncytia and intense dermal and epidermal inflammation, eosinophilia, and higher viremia compared to vector-treated control monkeys. Protection after challenge correlated with gamma interferon-producing T cells and with early production of high-avidity antibody that bound wild-type H protein. We conclude that PLG/SINCP-H is most efficacious when delivered intramuscularly but does not provide an advantage over standard DNA vaccines for protection against measles.

Development of a dendritic cell vaccine against measles for patients following hematopoietic cell transplantation.

Most patients who have undergone hematopoietic cell transplantation (HCT) lose specific immunity to measles. However, due to its immunosuppressive potential, it has been recommended that a live attenuated measles vaccination be administered two years following HCT. Measles virus (MV) glycoproteins including hemagglutinin (HA) are expressed on MV-infected dendritic cells (DCs), and they impair efficient antigen presentation between the DC and T cell. We produced a DC-based vaccine against MV by loading DCs with MV-infected autologous DCs. MV in the infected DCs was inactivated using ultraviolet-B. The DC-based vaccine neither expressed HA nor inhibited allogeneic T cell proliferation, while it induced the production of interferon-gamma (IFN-gamma) by autologous CD4 and CD8 naive T cells ex vivo. Importantly, the vaccine derived from patients who had undergone HCT also efficiently induced IFN-gamma producing cells. These findings indicate that our DC-based MV vaccine induces MV-specific immunity even in post-HCT patients without causing immunosuppression.

Identification of class II HLA-DRB1*03-bound measles virus peptides by 2D-liquid chromatography tandem mass spectrometry.

Two-dimensional liquid chromatography (2D-LC), combined with gas phase fractionation tandem mass spectrometry, was used to identify 13 naturally processed peptides originating from measles virus that were presented by HLA-DRB1*03 class II molecules. The peptides are from three of the six measles structural proteins: phosphoprotein, nucleocapsid, and hemagglutinin. These peptides provide an important first step toward understanding the mechanism of immune response to measles virus and development of a new generation of peptide-based vaccines.

[The morphology of the micro-capsulated polyacrylic acid-based formulation of measles vaccine].

The morphology and virus localization were studied in the microcapsulated measles vaccine formulation involving polyacrylic acid (PAA) copolymers as a matrix. Transmission electron microscopy and phosphotungsic staining at a pH value of 2 to 7 showed that the morphology of microparticles was related to the value of pH and to the concentration of a polymer in the matrix. In the neutral medium, the microcapsules had the sizes of 0.5 to 10 microm, which were optimal for transport through the intestinal wall when immunization was orally used. Immunocytochemistry revealed measles virus antigen within the microparticles. The specific activity of the microcapsulated formulation of measles virus was as high as 3.36 and 4.31 lg of TCD50/0.5 ml for the samples containing 1 and 0.1% polymer, respectively. The findings suggest that the microparticles of the vaccine contain live measles virus.

Modulation of disease, T cell responses, and measles virus clearance in monkeys vaccinated with H-encoding alphavirus replicon particles.

Measles remains a major worldwide problem partly because of difficulties with vaccination of young infants. New vaccine strategies need to be safe and to provide sustained protective immunity. We have developed Sindbis virus replicon particles that express the measles virus (MV) hemagglutinin (SIN-H) or fusion (SIN-F) proteins. In mice, SIN-H induced high-titered, dose-dependent, MV-neutralizing antibody after a single vaccination. SIN-F, or SIN-H and SIN-F combined, induced somewhat lower responses. To assess protective efficacy, juvenile macaques were vaccinated with a single dose of 10(6) or 10(8) SIN-H particles and infant macaques with two doses of 10(8) particles. A dose of 10(8) particles induced sustained levels of high-titered, MV-neutralizing antibody and IFN-gamma-producing memory T cells, and most monkeys were protected from rash when challenged with wild-type MV 18 months later. After challenge, there was a biphasic appearance of H- and F-specific IFN-gamma-secreting CD4+ and CD8+ T cells in vaccinated monkeys, with peaks approximately 1 and 3-4 months after challenge. Viremia was cleared within 14 days, but MV RNA was detectable for 4-5 months. These studies suggest that complete clearance of MV after infection is a prolonged, phased, and complex process influenced by prior vaccination.

A powder formulation of measles vaccine for aerosol delivery.

Both the mortality rate for measles and the risks associated with injection continue to be high in the developing world. In response to the need for safe, cost-effective vaccine delivery technologies, a powder formulation of measles vaccine has been developed to test the feasibility of administering measles vaccine as an aerosol. The first challenge in aerosol formulation development is to produce fine particles without damaging the activity of the virus or inducing physical changes. In this study, live attenuated measles vaccine is micronized by jet milling to generate particle sizes appropriate for pulmonary delivery (1-5 microm). Milling does not induce detectable physical changes and significant viral potency is maintained. Potency retention of milled vaccine ranges from 31 to 89%, demonstrating that the standard dose of vaccine can easily be achieved. Following size reduction, particles are blended with an inert carrier to improve handling and aerosol dispersion. The measles vaccine formulation is dispersable, as shown by laser light particle size analysis of vaccine aerosols. Thus, evaluation of both the potency retention and the aerosol characteristics of the current formulation clearly demonstrates the feasibility of delivering measles vaccine as a powder aerosol for immunization.

In vivo antibody response and in vitro CTL activation induced by selected measles vaccine candidates, prepared with purified Quil A components.

Semipurified Quil A and purified Quil A were used to prepare well-characterized subunit vaccine candidates against measles. Variation in the relative amounts of the measles virus (MV) fusion (F) protein, Quil A-components and lipids did not influence induction of antibody responses in mice, but had a pronounced effect on the capacity to induce cytotoxic T cell (CTL) activity of a CD8(+) MV F-protein specific human T cell clone in vitro. A characteristic MV iscom preparation based on the combined use of HPLC-purified Quil A-components QA-3 and QA-22 (QA-3/22) efficiently induced CTL activity in vitro. Comparable results were obtained by mixing beta-propiolactone inactivated MV with iscom-matrix QA-3/22 or free QA-22. On the basis of the data presented it was concluded that these three preparations are interesting MV vaccine candidates for further evaluation in pre-clinical experiments in a primate model.