Measles Vaccine.

Measles remains an important cause of child morbidity and mortality worldwide despite the availability of a safe and efficacious vaccine. The current measles virus (MeV) vaccine was developed empirically by attenuation of wild-type (WT) MeV by in vitro passage in human and chicken cells and licensed in 1963. Additional passages led to further attenuation and the successful vaccine strains in widespread use today. Attenuation is associated with decreased replication in lymphoid tissue, but the molecular basis for this restriction has not been identified. The immune response is age dependent, inhibited by maternal antibody (Ab) and involves induction of both Ab and T cell responses that resemble the responses to WT MeV infection, but are lower in magnitude. Protective immunity is correlated with levels of neutralizing Ab, but the actual immunologic determinants of protection are not known. Because measles is highly transmissible, control requires high levels of population immunity. Delivery of the two doses of vaccine needed to achieve >90% immunity is accomplished by routine immunization of infants at 9-15 months of age followed by a second dose delivered before school entry or by periodic mass vaccination campaigns. Because delivery by injection creates hurdles to sustained high coverage, there are efforts to deliver MeV vaccine by inhalation. In addition, the safety record for the vaccine combined with advances in reverse genetics for negative strand viruses has expanded proposed uses for recombinant versions of measles vaccine as vectors for immunization against other infections and as oncolytic agents for a variety of tumors.

Stability, biophysical properties and effect of ultracentrifugation and diafiltration on measles virus and mumps virus.

Measles virus and mumps virus (MeV and MuV) are enveloped RNA viruses used for production of live attenuated vaccines for prophylaxis of measles and mumps disease, respectively. For biotechnological production of and basic research on these viruses, the preparation of highly purified and infectious viruses is a prerequisite, and to meet that aim, knowledge of their stability and biophysical properties is crucial. Our goal was to carry out a detailed investigation of the stability of MeV and MuV under various pH, temperature, shear stress, filtration and storage conditions, as well as to evaluate two commonly used purification techniques, ultracentrifugation and diafiltration, with regard to their efficiency and effect on virus properties. Virus titers were estimated by CCID50 assay, particle size and concentration were measured by Nanoparticle tracking analysis (NTA) measurements, and the host cell protein content was determined by ELISA. The results demonstrated the stability of MuV and MeV at pH <9 and above pH 4 and 5, respectively, and aggregation was observed at pH >9. Storage without stabilizer did not result in structural changes, but the reduction in infectivity after 24 hours was significant at +37 °C. Vortexing of the viruses resulted in significant particle degradation, leading to lower virus titers, whereas pipetting had much less impact on virus viability. Diafiltration resulted in higher recovery of both total and infectious virus particles than ultracentrifugation. These results provide important data for research on all upstream and downstream processes on these two viruses regarding biotechnological production and basic research.

Detection of measles, mumps and rubella viruses by immuno-colorimetric assay and its application in focus reduction neutralization tests.

Measles, mumps and rubella are vaccine-preventable diseases; however limited epidemiological data are available from low-income or developing countries. Thus, it is important to investigate the transmission of these viruses in different geographical regions. In this context, a cell culture-based rapid and reliable immuno-colorimetric assay (ICA) was established and its utility studied. Twenty-three measles, six mumps and six rubella virus isolates and three vaccine strains were studied. Detection by ICA was compared with plaque and RT-PCR assays. In addition, ICA was used to detect viruses in throat swabs (n = 24) collected from patients with suspected measles or mumps. Similarly, ICA was used in a focus reduction neutralization test (FRNT) and the results compared with those obtained by a commercial IgG enzyme immuno assay. Measles and mumps virus were detected 2 days post-infection in Vero or Vero-human signaling lymphocytic activation molecule cells, whereas rubella virus was detected 3 days post-infection in Vero cells. The blue stained viral foci were visible by the naked eye or through a magnifying glass. In conclusion, ICA was successfully used on 35 virus isolates, three vaccine strains and clinical specimens collected from suspected cases of measles and mumps. Furthermore, an application of ICA in a neutralization test (i.e., FRNT) was documented; this may be useful for sero-epidemiological, cross-neutralization and pre/post-vaccine studies.

Process optimisation for anion exchange monolithic chromatography of 4.2kbp plasmid vaccine (pcDNA3F).

Anion exchange monolithic chromatography is increasingly becoming a prominent tool for plasmid DNA purification but no generic protocol is available to purify all types of plasmid DNA. In this work, we established a simple framework and used it to specifically purify a plasmid DNA model from a clarified alkaline-lysed plasmid-containing cell lysate. The framework involved optimising ligand functionalisation temperature (30-80°C), mobile phase flow rate (0.1-1.8mL/min), monolith pore size (done by changing the porogen content in the polymerisation reaction by 50-80%), buffer pH (6-10), ionic strength of binding buffer (0.3-0.7M) and buffer gradient elution slope (1-10% buffer B/min). We concluded that preferential pcDNA3F adsorption and optimum resolution could be achieved within the tested conditions by loading the clarified cell lysate into 400nm pore size of monolith in 0.7M NaCl (pH 6) of binding buffer followed by increasing the NaCl concentration to 1.0M at 3%B/min.

[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.

Detection and characterization of pestivirus contaminations in human live viral vaccines.

In view of the use of potentially contaminated foetal calf serum (FCS) in cell cultures pestiviruses may be present in live viral vaccines. Thirty-six lots of human live viral vaccines produced by three manufacturers were tested for the presence of pestiviruses. Bovine viral diarrhoea virus (BVDV) RNA was detected in 33% of the vaccine lots. All positive results were caused by the mumps component of a single manufacturer. Partial sequences of the 5' untranslated region of BVD viral RNA were determined. The sequences were closely related to that of the NADL strain of BVDV. The amount of BVDV RNA in the vaccines was determined by real-time RT-PCR using the LightCycler. Between 3.3*10(2) and 6.2*10(5) RNA copies per dose were found to be present in the vaccine samples.Additionally, culture tests were done with FCS and human diploid cells used in the vaccine production of the manufacturer whose vaccines were positive by PCR. All attempts to detect virus antigen in MRC-5 human diploid cells or to infect these cells with BVDV failed. This suggests that BVDV RNA detected in human live viral vaccines represents passive carry over of BVDV from contaminated FCS rather than active virus replication in human diploid cells. Our results indicate that contamination with BVDV of FCS used in vaccine production does not appear to be of immediate concern to human health. Furthermore, our results indicate that gamma-irradiation of FCS destroys BVDV particles and is also effective in preventing the presence of BVDV RNA in the vaccines.

Detection of measles vaccine in the throat of a vaccinated child.

Measles vaccine is widely used, most often in association with mumps and rubella vaccines. We report here the case of a child presenting with fever 8 days after vaccination with a measles-mumps-rubella vaccine. Measles virus was isolated in a throat swab taken 4 days after fever onset. This virus was then further genetically characterised as a vaccine-type virus. Fever occurring subsequent to measles vaccination is related to the replication of the live attenuated vaccine virus. In the case presented here, the vaccine virus was isolated in the throat, showing that subcutaneous injection of an attenuated measles strain can result in respiratory excretion of this virus.

Plant-derived measles virus hemagglutinin protein induces neutralizing antibodies in mice.

Measles remains a significant problem in both the developed and developing world, and new measles vaccination strategies need to be developed. This paper examines the strategy of utilizing transgenic plants expressing a measles antigen for the development of an oral sub-unit measles vaccine. A 1.8 kb fragment encompassing the coding region of the measles virus hemagglutinin (H) protein was cloned into a plant expression cassette. Three different expression constructs were tested: pBinH (H gene alone), pBinH/KDEL (addition of a C-terminal endoplasmic reticulum-retention sequence SEKDEL) and pBinSP/H/KDEL (further addition of an authentic N-terminal plant signal peptide). The highest levels of recombinant H protein production were observed in plants transformed with pBinH/KDEL. Mice inoculated intraperitoneally with transgenic plant derived recombinant H protein produced serum anti-H protein antibodies that neutralized the measles virus (MV) in vitro. Mice gavaged with transgenic tobacco leaf extracts also developed serum H protein-specific antibodies with neutralizing activity against MV in vitro. These results indicate that the plant-derived measles H protein is immunogenic when administered orally and that, with further development, oral vaccination utilizing transgenic plants may become a viable approach to measles vaccine development.

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.

A simple method for genetic differentiation of the AIK-C vaccine strain from wild strains of measles virus.

Restriction fragment length polymorphism (RFLP) analysis was used to differentiate the AIK-C vaccine strain from the wild circulating measles viruses. Virus genomes were amplified by using reverse transcriptase-polymerase chain reaction (RT-PCR) in the part of the H-gene. The PCR product of the AIK-C strain was cut into 2 fragments after digestions with Hpall, BstPl and Avall, respectively. Otherwise, those of 17 wild-type strains isolated during 1950s-1991 were cleaved into 3 fragments only when digested with Avall. Comparison with wild strain sequences suggest that the above Hpall site and BstPl site are characteristic of the AIK-C strain. This molecular approach is also applied for the discrimination whether some symptoms after vaccination were related to the vaccine strain or not.

The pathogenetic aspects of measles virus infection: memorandum from a WHO meeting.

Over the last three years considerable progress has been made in various areas related to measles virus and infection. A meeting to discuss the currently available data on the molecular biology of measles virus, measles immunology, immunopathology, as well as animal models for measles infection, and to identify studies that need to be carried out towards developing new vaccines was organized jointly by WHO and the U.S. National Institutes of Health and held in Montreux, Switzerland, on 20-21 April 1993. This Memorandum summarizes the discussions and recommendations made by the participants.

PIP: Significant progress has been made over the past three years with regard to the measles virus and infection. A meeting was therefore jointly organized by the World Health Organization and the US National Institutes of Health for Montreux, Switzerland, on April 20-21, 1993, to discuss currently available data on the molecular biology of the measles virus, measles immunology, immunopathology, and animal models for measles infection; and to identify studies which need to be carried out in the interest of developing new vaccines. This memo summarizes the discussions and recommendations made by participants. The discussion focused upon major issues concerning immune responses to measles virus infection, the role of subclinical infection in measles transmission, adverse events associated with measles and measles vaccination, the diagnosis of measles in developing countries, and alternative vaccination strategies. There was general agreement that information is incomplete on the immune response and measles virus infection. In particular, cell-mediated immune response and its role in the recovery from infection, protection from reinfection, and lifelong immunity are poorly understood. Areas important for future research, complications of measles, how to improve the measles vaccine, and measles diagnosis were discussed. Participants concluded that measles is a potentially eradicable disease, but major efforts will be required in virology, immunology, and epidemiology. Moreover, in addition to the maximum use of currently available vaccines, new vaccines should be developed together with improved diagnostic tests to confirm measles. Research coordination and adequate funding will be required.

Molecular cloning and complete nucleotide sequence of genomic RNA of the AIK-C strain of attenuated measles virus.

Twelve cDNA clones covering the entire genome of the AIK-C strain of a seed for live measles vaccine were obtained, and the nucleotide sequences were determined. The full viral genomic RNA consists of 15,894 nucleotides. Comparisons of the nucleotide sequence and the deduced amino acid sequence between the AIK-C and other Edmonston strains revealed the following changes: 56 nucleotide differences and one C residue insertion, 31 amino acid changes, and 19 silent mutations.

Control of measles and rubella through use of attenuated vaccines.

A summary report of the current status of morbidity and mortality associated with measles and rubella vis á vis immunization practices in the United States since these vaccines were licensed in 1963 (measles) and 1969 (rubella) is presented.