Gwendolyn A. Myers

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

ABSTRACT 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-WN02 vaccine in the macaque model indicate the vaccine candidate is expected to be safe and immunogenic for humans.

A Single Amino Acid Substitution in the Envelope Protein of Chimeric Yellow Fever-Dengue 1 Vaccine Virus Reduces Neurovirulence for Suckling Mice and Viremia/Viscerotropism for Monkeys

ABSTRACT A chimeric yellow fever-dengue 1 (ChimeriVax-DEN1) virus was produced by the transfection of Vero cells with chimeric in vitro RNA transcripts. The cell culture supernatant was subjected to plaque purification for the identification of a vaccine candidate without mutations. Of 10 plaque-purified clones, 1 containing no mutation (clone J) was selected for production of the vaccine virus. During subsequent cell culture passaging of this clone for vaccine production, a single amino acid substitution (K to R) occurred in the envelope (E) protein at residue 204 (E204) (F. Guirakhoo, K. Pugachev, Z. Zhang, G. Myers, I. Levenbook, K. Draper, J. Lang, S. Ocran, F. Mitchell, M. Parsons, N. Brown, S. Brandler, C. Fournier, B. Barrere, F. Rizvi, A. Travassos, R. Nichols, D. Trent, and T. Monath, J. Virol. 78:4761-4775, 2004). The same mutation was observed in another clone (clone E). This mutation attenuated the virus in 4-day-old suckling mice inoculated by the intracerebral (i.c.) route and led to reduced viremia in monkeys inoculated by the subcutaneous or i.c. route. The histopathology scores of lesions in the brain tissue of monkeys inoculated with either the E204K or E204R virus were reduced compared to those for monkeys inoculated with the reference virus, a commercial yellow fever 17D vaccine (YF-VAX). Both viruses grew to significantly lower titers than YF-VAX in HepG2, a human hepatoma cell line. After intrathoracic inoculation into mosquitoes, both viruses grew to a similar level as YF-VAX, which was significantly lower than that of their wild-type DEN1 parent virus. A comparison of the E-protein structures of nonmutant and mutant viruses suggested the appearance of new intramolecular bonds between residues 204R, 261H, and 257E in the mutant virus. These changes may be responsible for virus attenuation through a change in the pH threshold for virus envelope fusion with the host cell membrane.

Sterilizing immunity against experimental Helicobacter pylori infection is challenge-strain dependent.

The development of a murine model of Helicobacter pylori infection through serial in vivo passage of candidate strains has enabled a quantitative assessment of vaccine efficacy. In this study we compare infection with and protection against challenge from both CagA(+) type I, and CagA(-) type II in vivo adapted isolates. In vivo passage of a type II H. pylori isolate resulted in a highly infectious strain (X47-2AL), capable of reproducibly infecting mice to high density (10(7) CFU/g of gastric tissue). Similarly adapted type I strains were found to colonize mice at a significantly lower level (10(4)-10(5) CFU/g tissue). Mucosal immunization with recombinant urease (rUre) significantly protected animals against both types. Protection against X47-2AL was characterized by a > or =100-fold (or 2 log) reduction in bacterial density. However, the presence of a residual infection highlighted the inability to achieve sterilizing immunity against this strain. The level of protection appeared independent of challenge dose, and was stable for up to 6 months, all animals exhibiting a low-level residual infection that did not recrudesce with time. Similarly immunized mice challenged with isolates representing the residual infection were also protected, confirming that they did not represent a sub-population of H. pylori that could escape immunity. Immunization and challenge studies with type I adapted-isolates, demonstrated a similar 2-3 log reduction in the bacterial burden, but that in this instance resulted in sterilizing immunity. These results suggest varied specificity for the murine host by different Helicobacter strains that can influence the outcome of both infection and immunity.

Oral immunization with recombinant Helicobacter pylori urease confers long-lasting immunity against Helicobacter felis infection.

Recombinant Helicobacter pylori urease (rUre) has been shown to confer protection against challenge with Helicobacter felis in mice. The purpose of the present study was to examine duration of the immune response and long-term protective efficacy of immunization with rUre. Swiss Webster mice were orally immunized four times at weekly intervals with 100 microg rUre plus 5 microg heat-labile enterotoxin of Escherichia coli (LT) adjuvant, or with LT only. At 4, 10, 20 or 40 weeks post immunization, 25 rUre-immunized mice and control mice were challenged with H. felis and sacrificed at 2 or 10 weeks post-challenge. H. felis infection was assessed by gastric urease assay and by histology. Anti-H. pylori urease specific antibody levels were measured in serum and saliva both pre- and post-challenge. Over the 40 week time period, the infection rates in rUre-immunized mice were significantly lower than those in controls (p < 0.05) as assessed by gastric urease activity. Protection ranged from 79 100% at 2 weeks post-challenge and 63-78% at 10 weeks post-challenge. Gastric bacterial density in rUre-immunized mice was significantly lower than that of controls (p < 0.03) as determined by histologic assessment. Anti-urease antibody levels remained elevated in the serum and mucosal compartments at 39 weeks following immunization. This study shows that immunization with rUre plus LT results in long-lasting protective immunity against challenge with H. felis.