Toxicology, biodistribution and shedding profile of a recombinant measles vaccine vector expressing HIV-1 antigens, in cynomolgus macaques.

As a new human immunodeficiency virus type 1 (HIV-1) vaccine approach, the live-attenuated measles virus (MV) Schwarz vaccine strain was genetically engineered to express the F4 antigen (MV1-F4). F4 is a fusion protein comprising HIV-1 antigens p17 and p24, reverse transcriptase and Nef. This study assessed the toxicity, biodistribution and shedding profiles of MV1-F4. Cynomolgus macaques were intramuscularly immunized one or three times with the highest dose of MV1-F4 intended for clinical use, the reference (Schwarz) measles vaccine or saline, and monitored clinically for 11 or 85 days. Toxicological parameters included local and systemic clinical signs, organ weights, haematology, clinical and gross pathology and histopathology. Both vaccines were well tolerated, with no morbidity, clinical signs or gross pathological findings observed. Mean spleen weights were increased after three doses of either vaccine, which corresponded with increased numbers and/or sizes of germinal centers. This was likely a result of the immune response to the vaccines. Either vaccine virus replicated preferentially in secondary lymphoid organs and to a lesser extent in epithelium-rich tissues (e.g., intestine, urinary bladder and trachea) and the liver. At the expected peak of viremia, viral RNA was detected in some biological fluid samples from few animals immunized with either vaccine, but none of these samples contained infectious virus. In conclusion, no shedding of infectious viral particles was identified in cynomolgus monkeys after injection of MV1-F4 or Schwarz measles vaccines. Furthermore, no toxic effect in relation to the MV vaccination was found with these vaccines in this study.

Vaccine stability study design and analysis to support product licensure.

Stability evaluation supporting vaccine licensure includes studies of bulk intermediates as well as final container product. Long-term and accelerated studies are performed to support shelf life and to determine release limits for the vaccine. Vaccine shelf life is best determined utilizing a formal statistical evaluation outlined in the ICH guidelines, while minimum release is calculated to help assure adequate potency through handling and storage of the vaccine. In addition to supporting release potency determination, accelerated stability studies may be used to support a strategy to recalculate product expiry after an unintended temperature excursion such as a cold storage unit failure or mishandling during transport. Appropriate statistical evaluation of vaccine stability data promotes strategic stability study design, in order to reduce the uncertainty associated with the determination of the degradation rate, and the associated risk to the customer.

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.

Preclinical safety and biodistribution of Sindbis virus measles DNA vaccines administered as a single dose or followed by live attenuated measles vaccine in a heterologous prime-boost regimen.

Measles still causes considerable morbidity and mortality among infants and young children in developing countries. To develop a new public health tool to reduce this burden, we designed two Sindbis virus replicon vaccines encoding measles virus (MV) hemagglutinin (H) and fusion (F) proteins (pMSIN-H and pMSINHFdU). Our goal is to administer the vaccines to young infants at 6 and 10 weeks of age to prime the immune system to safely and effectively respond to subsequent immunization at age approximately 14 weeks with the licensed attenuated measles vaccine. In preparation for a phase 1 clinical trial, studies of plasmid distribution, integration, and toxicology were performed in rabbits. Biodistribution was assessed after a single DNA immunization delivered intradermally by needle-free injection. Toxicity was assessed using a heterologous prime-boost regimen consisting of a repeat-dose DNA prime followed by a live-attenuated measles vaccine boost. The only vaccine-related adverse effects observed were minimal transient erythema, edema, and inflammation confined to the injection site. Plasmids were detected in the subcutis and muscle at the site of inoculation. A small proportion of animals exhibited plasmids in the regional lymph nodes. There was no evidence of plasmid integration into the host genome. Both Sindbis-based vaccine plasmids were immunogenic in rabbits; pMSIN-H elicited higher virus-neutralizing antibody levels. Both vaccines were shown to be well tolerated and suitable for clinical trials and they are currently being tested in phase 1 studies in young adults.

Use of the next generation pharmaceutical impactor for particle size distribution measurements of live viral aerosol vaccines.

Aerosol administration of live measles vaccine virus has proven to be extremely efficacious in field trials using an industrial compressor coupled to a disposable nebulizer (IPI). To develop a new system for administration, it is necessary to characterize the operating characteristics of the old system. There are no standardized techniques for measuring particle size of live biological agents. This study evaluated the Next Generation Pharmaceutical Impactor's (NGI) ability to particle size wet aerosols in an effort to measure the particle size distribution of live measles vaccine from the IPI nebulizer. As a control albuterol was aerosolized using a Pari LC Star, since the soluble albuterol is evenly distributed throughout the droplets and laser diffraction measurements should agree with those from the NGI, as long as the NGI is cooled to prevent heat transfer to the aerosol. Albuterol was also used as a control for the IPI using quantitative ultraviolet spectrophotometry. There was close agreement in MMD (mean +/- 95% CI) for the LC Star, measured by laser diffraction (3.24 +/- 0.06 microm) and the NGI (2.93 +/- 0.22 microm) and the IPI (4.26 +/- 0.17 and 4.26 +/- 0.24 microm, respectively). For the measles vaccine assayed for plaque forming units, there were significant differences between the NGI MMD (6.14 +/- 0.39 microm) compared to laser diffraction (4.95 +/- 0.16 microm) indicating that the vaccine is not evenly distributed among the droplets of various sizes. This is likely clumping of the virus due to gelatin in the formulation. These data indicate that the NGI is capable of particle sizing live biological agents.

MMR vaccination, measles epidemiology and sero-surveillance in the Republic of Ireland.

OBJECTIVE: Following the introduction of a national measles and subsequent MMR vaccination programme, to determine the susceptibility of 3-14-year-old children to measles, mumps and rubella and to relate the results to the epidemiology of measles and the need for vaccination policy changes. DESIGN: Cross-sectional sero-survey and trends in measles notifications and mortality. SETTING: Paediatric hospital outpatient departments in Dublin. SUBJECTS: Sera were collected from 837 children attending the clinics in 1991 and 1992. RESULTS: The prevalence of antibodies in children aged 3-6, 7-10 and 11-14 years was 84, 83 and 95% for measles; 48, 60 and 65% for mumps; and 78, 63 and 74% for rubella, respectively. The prevalence of mumps antibodies may be underestimated. Ninety-six per cent of girls aged 13-14 years had rubella antibodies. A widespread outbreak of measles occurred in 1993. Over recent years, an increasing proportion of measles notifications were in older children. CONCLUSION: Given sub-optimal uptake of MMR vaccine, outbreaks of infection in pre- and primary school children are inevitable. In such circumstances, a 2-shot MMR vaccine programme with high uptake is essential to prevent a shift of disease into older age groups.

Measles--mumps--rubella immunization: the role of the general practitioner in achieving a high uptake.

A survey of all general practitioners in Fife conducted prior to the introduction of measles-mumps-rubella immunization on a pilot basis in May 1987 showed that 85% considered mumps worth preventing and 94% believed the rubella programme worth augmenting with universal childhood immunization. Ninety seven per cent considered measles worth preventing and 98% were prepared to recommend measles-mumps-rubella immunization to parents instead of measles vaccine. One year after introducing the measles-mumps-rubella vaccine in Fife, 91% of children had been immunized with the combined vaccine or measles vaccine before their second birthday. This compares with the 83% that received measles vaccine before the combined vaccine was introduced. Eighty per cent of preschool children were also immunized with the combined vaccine at school entry in a catch-up exercise. This study demonstrates that there are few major professional barriers to achieving a high uptake of the measles-mumps-rubella vaccine in this area. The vaccine was introduced nationally on 1 October 1988 and its uptake is likely to exceed the current unsatisfactory level achieved with measles vaccine. However, this outcome will largely depend on the commitment of doctors to the programme.