WHO working group on the quality, safety and efficacy of japanese encephalitis vaccines (live attenuated) for human use, Bangkok, Thailand, 21-23 February 2012.

Japanese encephalitis (JE) is one of the most important viral encephalitides in Asia. Two live-attenuated vaccines have been developed and licensed for use in countries in the region. Given the advancement of immunization of humans with increasing use of live-attenuated vaccines to prevent JE, there is increased interest to define quality standards for their manufacture, testing, nonclinical studies, and clinical studies to assess their efficacy and safety in humans. To this end, WHO convened a meeting with a group of international experts in February 2012 to develop guidelines for evaluating the quality, safety and efficacy of live-attenuated JE virus vaccines for prevention of human disease. This report summarizes collective views of the participants on scientific and technical issues that need to be considered in the guidelines.

Adaptation of yellow fever virus 17D to Vero cells is associated with mutations in structural and non-structural protein genes.

Serial passaging of yellow fever virus 17D in Vero cells was employed to derive seed material for a novel inactivated vaccine, XRX-001. Two independent passaging series identified a novel lysine to arginine mutation at amino acid 160 of the envelope protein, a surface-exposed residue in structural domain I. A third passage series resulted in an isoleucine to methionine mutation at residue 113 of the NS4B protein, a central membrane spanning region of the protein which has previously been associated with Vero cell adaptation of other mosquito-borne flaviviruses. These studies confirm that flavivirus adaptation to growth in Vero cells can be mediated by structural or non-structural protein mutations.

Immune correlates of protection against yellow fever determined by passive immunization and challenge in the hamster model.

Live, attenuated yellow fever (YF) 17D vaccine is highly efficacious but causes rare, serious adverse events resulting from active replication in the host and direct viral injury to vital organs. We recently reported development of a potentially safer ß-propiolactone-inactivated whole virion YF vaccine (XRX-001), which was highly immunogenic in mice, hamsters, monkeys, and humans [10,11]. To characterize the protective efficacy of neutralizing antibodies stimulated by the inactivated vaccine, graded doses of serum from hamsters immunized with inactivated XRX-001 or live 17D vaccine were transferred to hamsters by the intraperitoneal (IP) route 24h prior to virulent, viscerotropic YF virus challenge. Neutralizing antibody (PRNT(50)) titers were determined in the sera of treated animals 4h before challenge and 4 and 21 days after challenge. Neutralizing antibodies were shown to mediate protection. Animals having 50% plaque reduction neutralization test (PRNT(50)) titers of ≥40 4h before challenge were completely protected from disease as evidenced by viremia, liver enzyme elevation, and protection against illness (weight change) and death. Passive titers of 10-20 were partially protective. Immunization with the XRX-001 vaccine stimulated YF neutralizing antibodies that were equally effective (based on dose response) as antibodies stimulated by live 17D vaccine. The results will be useful in defining the level of seroprotection in clinical studies of new yellow fever vaccines.

An inactivated cell-culture vaccine against yellow fever.

BACKGROUND: Yellow fever is a lethal viral hemorrhagic fever occurring in Africa and South America. A highly effective live vaccine (17D) is widely used for travelers to and residents of areas in which yellow fever is endemic, but the vaccine can cause serious adverse events, including viscerotropic disease, which is associated with a high rate of death. A safer, nonreplicating vaccine is needed. METHODS: In a double-blind, placebo-controlled, dose-escalation, phase 1 study of 60 healthy subjects between 18 and 49 years of age, we investigated the safety and immunogenicity of XRX-001 purified whole-virus, ß-propiolactone-inactivated yellow fever vaccine produced in Vero cell cultures and adsorbed to aluminum hydroxide (alum) adjuvant. On two visits 21 days apart, subjects received intramuscular injections of vaccine that contained 0.48 µg or 4.8 µg of antigen. Levels of neutralizing antibodies were measured at baseline and on days 21, 31, and 42. RESULTS: The vaccine induced the development of neutralizing antibodies in 100% of subjects receiving 4.8 µg of antigen in each injection and in 88% of subjects receiving 0.48 µg of antigen in each injection. Antibody levels increased by day 10 after the second injection, at which time levels were significantly higher with the 4.8-µg formulation than with the 0.48-µg formulation (geometric mean titer, 146 vs. 39; P<0.001). Three adverse events occurred at a higher incidence in the two vaccine groups than in the placebo group: mild pain, tenderness, and (much less frequently) itching at the injection site. One case of urticaria was observed on day 3 after the second dose of 4.8 µg of vaccine. CONCLUSIONS: A two-dose regimen of the XRX-001 vaccine, containing inactivated yellow fever antigen with an alum adjuvant, induced neutralizing antibodies in a high percentage of subjects. XRX-001 has the potential to be a safer alternative to live attenuated 17D vaccine. (Funded by Xcellerex; ClinicalTrials.gov number, NCT00995865.).

Inactivated yellow fever 17D vaccine: development and nonclinical safety, immunogenicity and protective activity.

In the last 10 years new concerns have arisen about safety of the live, attenuated yellow fever (YF) 17D vaccine, in particular viscerotropic adverse events, which have a case-fatality rate of 64%. A non-replicating cell culture-based vaccine would not cause these adverse events, and potentially could be used in persons with precautions or contraindications to use of the live vaccine, including age <9 months and >60 years, egg allergy, immune suppression, and pregnancy. We developed a whole virion vaccine from the 17D strain inactivated with beta-propiolactone, and adsorbed to aluminum hydroxide. The inactivated vaccine was highly immunogenic in mice, hamsters, and cynomolgus macaques. After a single dose in hamsters and macaques, neutralizing antibody titers were similar to those elicited by the live 17D vaccine (YF-VAX, Sanofi Pasteur). After two doses of inactivated vaccine, neutralizing antibody titers in hamsters were significantly higher than after a single dose of YF-VAX [geometric mean titer (GMT) 20,480 vs. 1940, respectively (P<0.001, ANOVA)]. Hamsters given a single dose or two doses of inactivated vaccine or a single dose of YF-VAX were fully protected against hepatitis, viremia, weight loss and death after challenge with YF virus (Jimenez strain). A clinical trial of the inactivated vaccine (XRX-001) has been initiated.

Construction of yellow fever/St. Louis encephalitis chimeric virus and the use of chimeras as a diagnostic tool.

St. Louis encephalitis (SLE) and West Nile (WN) flaviviruses are genetically closely related and cocirculate in the United States. Virus neutralization tests provide the most specific means for serodiagnosis of infections with these viruses. However, use of wild-type SLE and WN viral strains for laboratory testing is constrained by the biocontainment requirements. We constructed two highly attenuated yellow fever (YF) virus chimeras that contain the premembrane-envelope (prM-E) protein genes from the virulent MSI-7 (isolated in the United States) or the naturally attenuated CorAn9124 (Argentina) SLE strains. The YF/SLE (CorAn version) virus and the previously constructed YF/WN chimera were shown to specifically distinguish between confirmed human SLE and WN cases in a virus neutralization test using patient sera. These chimeras have the potential for use as diagnostic reagents and vaccines against SLE and WN.

ChimeriVax-West Nile virus live-attenuated vaccine: preclinical evaluation of safety, immunogenicity, and efficacy.

The availability of ChimeriVax vaccine technology for delivery of flavivirus protective antigens at the time West Nile (WN) virus was first detected in North America in 1999 contributed to the rapid development of the vaccine candidate against WN virus described here. ChimeriVax-Japanese encephalitis (JE), the first live- attenuated vaccine developed with this technology has successfully undergone phase I and II clinical trials. The ChimeriVax technology utilizes yellow fever virus (YF) 17D vaccine strain capsid and nonstructural genes to deliver the envelope gene of other flaviviruses as live-attenuated chimeric viruses. Amino acid sequence homology between the envelope protein (E) of JE and WN viruses facilitated targeting attenuating mutation sites to develop the WN vaccine. Here we discuss preclinical studies with the ChimeriVax-WN virus in mice and macaques. ChimeriVax-WN virus vaccine is less neurovirulent than the commercial YF 17D vaccine in mice and nonhuman primates. Attenuation of the virus is determined by the chimeric nature of the construct containing attenuating mutations in the YF 17D virus backbone and three point mutations introduced to alter residues 107, 316, and 440 in the WN virus E protein gene. The safety, immunogenicity, and efficacy of the ChimeriVax-WN(02) vaccine in the macaque model indicate the vaccine candidate is expected to be safe and immunogenic for humans.

Protection against Japanese encephalitis virus strains representing four genotypes by passive transfer of sera raised against ChimeriVax-JE experimental vaccine.

The ability of antisera raised against a candidate Japanese encephalitis virus (JEV) vaccine, ChimeriVax-JE, and the currently licensed vaccine, JE-VAX, to protect against strains of JEV representing the four major genotypes was assessed. Neutralization assays and passive protection studies in mice showed that greatest protection was provided against strains of genotypes II and III, although some protection was also afforded against genotypes I and IV strains. ChimeriVax-JE stimulated protection that was comparable or superior to the JE-VAX control.

High fidelity of yellow fever virus RNA polymerase.

Three consecutive plaque purifications of four chimeric yellow fever virus-dengue virus (ChimeriVax-DEN) vaccine candidates against dengue virus types 1 to 4 were performed. The genome of each candidate was sequenced by the consensus approach after plaque purification and additional passages in cell culture. Our data suggest that the nucleotide sequence error rate for SP6 RNA polymerase used in the in vitro transcription step to initiate virus replication was as high as 1.34 x 10(-4) per copied nucleotide and that the error rate of the yellow fever virus RNA polymerase employed by the chimeras for genome replication in infected cells was as low as 1.9 x 10(-7) to 2.3 x 10(-7). Clustering of beneficial mutations that accumulated after multiple virus passages suggests that the N-terminal part of the prM protein, a specific site in the middle of the E protein, and the NS4B protein may be essential for nucleocapsid-envelope interaction during flavivirus assembly.

Traditional and novel approaches to flavivirus vaccines.

Yellow fever, dengue, Japanese encephalitis and tick-borne encephalitis viruses are the medically most important members of the Flavivirus genus composed primarily of arboviruses. In this paper, we review the commercially available traditional flavivirus vaccines against yellow fever, Japanese encephalitis, and tick-borne encephalitis, as well as modern approaches to flavivirus vaccines. Formalin inactivation technology has been employed to produce killed vaccines. Flaviviruses have been attenuated by multiple passages in animal tissues and cell cultures to produce empirical live attenuated vaccines. The use of traditional methods is being pursued to develop vaccines against other flavivirus diseases, such as dengue, and to improve existing vaccines, such as for Japanese encephalitis. With the recent development of infectious clones, rational approaches to attenuated flavivirus vaccines have employed the introduction of specific mutations into wild type viruses and chimerisation between different viruses. Novel methods for delivery of live vaccines, such as inoculation of infectious DNA or RNA, have been described. Other approaches, such as the construction of protein subunit, expression vector-based and naked DNA vaccines, have been proposed to create alternate vaccine candidates.

Molecular evidence for the origin of the widespread Venezuelan equine encephalitis epizootic of 1969 to 1972

Venezuelan equine encephalitis (VEE) is a mosquito-borne pathogen that has caused encephalitis in equine species and humans during sporadic outbreaks in the western hemisphere. The last, and most widespread, VEE outbreak occurred in South America, Central America, Mexico and the U.S.A. (Texas) during 1969 to 1972. We have cloned and sequenced the genome of a virulent VEE subtype I-AB virus, strain 71-180, isolated in Texas in 1971. Thirty four nucleotide differences were detected between the genome of 71-80 virus and that of subtype I-AB Trinidad donkey (TRD) virus isolated during the 1943 VEE epizootic in Trinidad. Fifteen nucleotide changes occurred in the non-structural genes, 16 in the structural genes and three in the 3' non-coding region. Only six of the nucleotide diferences resulted in amino acid substitutions: one change in each of non-structural proteins nsP1 and nsP3, two in the E2 envelope glycoprotein, one in the 6K popypeptide and one in the E1 envelope glycoprotein. The close genetic relationship between 71-180 virus and TRD virus, commonly used for production of formalin-inactivated VEE vaccines, suggests that incompletely inactivated virulent vaccine virus may have been the source of this and other VEE outbreaks. Use of formalized virulent virus was discontinued during the 1969 to 1972 panzootic. No VEE epizootics have been reported since the introduction of the live attenuated TC-83 vaccine virus (AU)

Genetic and epidemiological studies of dengue type 2 viruses by hybridization using synthetic deoxyoligonucleotides as probes

A rapid nucleic acid hybridization procedure was developed for examining the genotypic variation of dengue type 2 viruses (DEN 2) having distinct RNase T1 fingerprints and isolated from different geographical areas. Synthetic DNA hybridization probes were constructed complementary in nucleotide sequence to common and unique RNase T1 oligonecleotides of topotype viruses from Puerto Rico/South Pacific, Jamaica, the Seychelles, Thailand/Burma and Africa. Hybridization probes with both type- and topotype-specific reactivities were observed, as were probes specific for two or more of the DEN 2 topotypes. These results confirm geographical movement of topotype virus strains and suggest possible origins. Detection of DEN 2 RNA by hybridization is a rapid and reproducible method that can be modified and applied as a viable alternative to the labourious T1 oligonucleotide fingerprinting.(AU)