Evaluation of live attenuated influenza a virus h6 vaccines in mice and ferrets.

Avian influenza A virus A/teal/HK/W312/97 (H6N1) possesses seven gene segments that are highly homologous to those of highly pathogenic human influenza H5N1 viruses, suggesting that a W312-like H6N1 virus might have been involved in the generation of the A/HK/97 H5N1 viruses. The continuous circulation and reassortment of influenza H6 subtype viruses in birds highlight the need to develop an H6 vaccine to prevent potential influenza pandemics caused by the H6 viruses. Based on the serum antibody cross-reactivity data obtained from 14 different H6 viruses from Eurasian and North American lineages, A/duck/HK/182/77, A/teal/HK/W312/97, and A/mallard/Alberta/89/85 were selected to produce live attenuated H6 candidate vaccines. Each of the H6 vaccine strains is a 6:2 reassortant ca virus containing HA and NA gene segments from an H6 virus and the six internal gene segments from cold-adapted A/Ann Arbor/6/60 (AA ca), the master donor virus that is used to make live attenuated influenza virus FluMist (intranasal) vaccine. All three H6 vaccine candidates exhibited phenotypic properties of temperature sensitivity (ts), ca, and attenuation (att) conferred by the internal gene segments from AA ca. Intranasal administration of a single dose of the three H6 ca vaccine viruses induced neutralizing antibodies in mice and ferrets and fully protected mice and ferrets from homologous wild-type (wt) virus challenge. Among the three H6 vaccine candidates, the A/teal/HK/W312/97 ca virus provided the broadest cross-protection against challenge with three antigenically distinct H6 wt viruses. These data support the rationale for further evaluating the A/teal/HK/W312/97 ca vaccine in humans.

Molecular studies of temperature-sensitive replication of the cold-adapted B/Ann Arbor/1/66, the master donor virus for live attenuated influenza FluMist vaccines.

Cold-adapted (ca) B/Ann Arbor/1/66 is the master donor virus for influenza B (MDV-B) vaccine component of live attenuated influenza FluMist vaccine. The six internal protein gene segments of MDV-B confer the characteristic cold-adapted (ca), temperature-sensitive (ts) and attenuated (att) phenotypes to the reassortant vaccine strains that contain the HA and NA RNA segments from the circulating wild type strains. Previously, we have mapped the loci in the NP, PA and M genes that determine the ca, ts and att phenotypes of MDV-B. In this report, the ts mechanism of MDV-B was described by comparing replication of MDV-B with its wild type counterpart at permissive and restricted temperatures. We showed that the PA and NP proteins of MDV-B are defective in RNA polymerase function at the restricted temperature of 37 degrees C resulting in greatly reduced viral RNA and protein synthesis. In addition, the two M1 residues, Q159 and V183 that are unique to MDV-B, contribute to reduced virus replication at temperatures greater than 33 degrees C, possibly due to the reduced M1 membrane association and its reduced virion M1 incorporation. Thus, the previously identified MDV-B loci not only reduce viral polymerase function at the restricted temperature but also affect virus assembly and release.

Avian influenza h6 viruses productively infect and cause illness in mice and ferrets.

Influenza pandemic preparedness has focused on influenza virus H5 and H7 subtypes. However, it is not possible to predict with certainty which subtype of avian influenza virus will cause the next pandemic, and it is prudent to include other avian influenza virus subtypes in pandemic preparedness efforts. An H6 influenza virus was identified as a potential progenitor of the H5N1 viruses that emerged in Hong Kong in 1997. This virus continues to circulate in the bird population in Asia, and other H6 viruses are prevalent in birds in North America and Asia. The high rate of reassortment observed in influenza viruses and the prevalence of H6 viruses in birds suggest that this subtype may pose a pandemic risk. Very little is known about the replicative capacity, immunogenicity, and correlates of protective immunity for low-pathogenicity H6 influenza viruses in mammals. We evaluated the antigenic and genetic relatedness of 14 H6 influenza viruses and their abilities to replicate and induce a cross-reactive immune response in two animal models: mice and ferrets. The different H6 viruses replicated to different levels in the respiratory tracts of mice and ferrets, causing varied degrees of morbidity and mortality in these two models. H6 virus infection induced similar patterns of neutralizing antibody responses in mice and ferrets; however, species-specific differences in the cross-reactivity of the antibody responses were observed. Overall, cross-reactivity of neutralizing antibodies in H6 virus-infected mice did not correlate well with protection against heterologous wild-type H6 viruses. However, we have identified an H6 virus that induces protective immunity against viruses in the North American and Eurasian lineages.

Stabilizing the glycosylation pattern of influenza B hemagglutinin following adaptation to growth in eggs.

The currently circulating influenza B viruses from both antigenic lineages contain an N-linked glycosylation site in the hemagglutinin (HA) protein at positions of 196 or 197. However, egg adaptation caused the loss of the glycosylation site that could impact virus antigenicity and vaccine efficacy. The effect of the 196/197 glycosylation site on influenza B virus growth and antigenicity was systemically evaluated in this study by the molecular approach. Paired recombinant 6:2 reassortant influenza B vaccine strains, with or without the 196/197 glycosylation site, were generated by reverse genetics and the glycosylation site was retained in MDCK cells. In contrast, all the viruses that contained the introduced glycosylation site were unable to grow in eggs and rapidly lost the glycosylation site once adapted to grow in eggs. We showed that glycosylation affected virus binding to the alpha-2,3-linked sialic acid receptor and affected virus antigenicity as tested by postinfected ferret sera. We have further identified that the Arginine residue at amino acid position 141 (141R) can stabilize the 196/197 glycosylation site without affecting virus antigenicity. Thus, the 141R could be introduced into vaccine strains to retain the 196/197 glycosylation site for influenza B vaccines.

Genetic mapping of the cold-adapted phenotype of B/Ann Arbor/1/66, the master donor virus for live attenuated influenza vaccines (FluMist).

Cold adapted (ca) B/Ann Arbor/1/66 is the master donor virus for the influenza B (MDV-B) vaccine component of the live attenuated influenza vaccine (FluMist). The six internal genes contributed by MDV-B confer the characteristic cold-adapted (ca), temperature-sensitive (ts) and attenuated (att) phenotypes to the vaccine strains. Previously, it has been determined that the PA and NP segments of MDV-B control the ts phenotype while the att phenotype requires the M segment in addition to PA and NP. Here, we show that the PA, NP and PB2 segments are responsible for the ca phenotype of MDV-B when examined in chicken cell lines. Five loci in three RNA segments, R630 in PB2, M431 in PA and A114, H410 and T509 in NP, are sufficient to allow efficient virus growth at 25 degrees C. Substitution of these five amino acids with wt (wild type) residues completely reverted the MDV-B ca phenotype. Conversely, introduction of these five ca amino acids into B/Yamanashi/166/98 imparted the ca phenotype to this heterologous wt virus. In addition, we also found that the MDV-B M1 gene affected virus replication in chicken cells at 33 and 37 degrees C. Recombinant viruses containing the two MDV-B M1 residues (Q159, V183) replicated less efficiently than those containing wt M1 residues (H159, M183) at 33 and 37 degrees C, implicating the role of the MDV-B M segment to the att phenotype. The complexity of the multigenic signatures controlling the ca, ts and att phenotypes of MDV-B provides the molecular basis for the observed genetic stability of the FluMist vaccines.

Tumor-specific intravenous gene delivery using oncolytic adenoviruses.

In this report, we describe a vector system that specifically delivers transgene products to tumors following intravenous (i.v.) administration. The Escherichia coli cytosine deaminase (CD) gene was placed in the E3B region of the tumor-selective, replication-competent adenovirus ONYX-411, under the control of endogenous viral late gene regulatory elements. Thus, CD expression was directly coupled to the tumor-selective replication of the viral vector. In vitro, CD was expressed efficiently in various human cancer cell lines tested but not in cultured normal human cells, including human hepatocytes. Following i.v. administration into nude mice carrying human tumor xenografts, robust CD activity was detected only in tumors but not in liver or other normal tissues. Levels of CD activity in the tumors increased progressively following i.v. virus administration, correlating closely with virus replication in vivo. Subsequent administration of 5-fluorocytosine (5-FC) demonstrated a trend to improve the antitumor efficacy of these viruses in a mouse xenograft model, presumably due to the intratumoral conversion of 5-FC to the chemotherapeutic drug 5-fluorouracil. We show that the combination of a highly selective oncolytic virus, ONYX-411, with the strategic use of the viral E3B region for transgene insertion provides a powerful platform that allows for tumor-specific, persistent and robust transgene expression after i.v. administration. This technology provides an opportunity to enhance greatly both safety and efficacy of cancer gene therapy.