Live attenuated tetravalent (G1-G4) bovine-human reassortant rotavirus vaccine (BRV-TV): Randomized, controlled phase III study in Indian infants.

BACKGROUND: Rotavirus remains the leading cause of diarrhoea among children <5years. We assessed immunogenic non-inferiority of a tetravalent bovine-human reassortant rotavirus vaccine (BRV-TV) over the licensed human-bovine pentavalent rotavirus vaccine RV5. METHODS: Phase III single-blind study (parents blinded) in healthy infants randomized (1:1) to receive three doses of BRV-TV or RV5 at 6-8, 10-12, and 14-16weeks of age. All concomitantly received a licensed diphtheria, tetanus, pertussis, hepatitis B, Haemophilus influenzae type b conjugate vaccine (DTwP-HepB-Hib) and oral polio vaccine (OPV). Immunogenic non-inferiority was evaluated in terms of the inter-group difference in anti-rotavirus serum IgA seroresponse (primary endpoint), and seroprotection/seroresponse rates to DTwP-HepB-Hib and OPV vaccines. Seroresponse was defined as a ≥4-fold increase in titers from baseline to D28 post-dose 3. Non-inferiority was declared if the difference between groups (based on the lower limit of the 95% confidence interval [CI]) was above -10%. Each subject was evaluated for solicited adverse events 7days and unsolicited & serious adverse events 28days following each dose of vaccination. RESULTS: Of 1195 infants screened, 1182 were randomized (590 to BRV-TV; 592 to RV5). Non-inferiority for rotavirus serum IgA seroresponse was not established: BRV-TV, 47.1% (95%CI: 42.8; 51.5) versus RV5, 61.2% (95%CI: 56.8; 65.5); difference between groups, -14.08% (95%CI: -20.4; -7.98). Serum IgA geometric mean concentrations at D28 post-dose 3 were 28.4 and 50.1U/ml in BRV-TV and RV5 groups, respectively. For all DTwP-HepB-Hib and OPV antigens, seroprotection/seroresponse was elicited in both groups and the -10% non-inferiority criterion between groups was met. There were 16 serious adverse events, 10 in BRV-TV group and 6 in RV5 group; none were classified as vaccine related. Both groups had similar vaccine safety profiles. CONCLUSION: BRV-TV was immunogenic but did not meet immunogenic non-inferiority criteria to RV5 when administered concomitantly with routine pediatric antigens in infants.

Two residues in the hemagglutinin of A/Fujian/411/02-like influenza viruses are responsible for antigenic drift from A/Panama/2007/99.

The H3N2 vaccine strain (A/Panama/2007/99) for the 2003-2004 influenza season did not antigenically match the circulating A/Fujian/411/02-like H3N2 viruses and had reduced effectiveness against influenza outbreaks. A/Wyoming/03/2003, an A/Fujian-like virus, was recommended as the vaccine strain for the 2004-2005 season. A/Wyoming differed from A/Panama by 16 amino acids in the HA1 molecule. Reverse genetics was used to determine the minimal amino acid changes that were responsible for the antigenic drift from A/Panama to A/Wyoming. After substitutions of 2 of the 16 amino acids in the HA (H155T, Q156H), the A/Panama HA variant was antigenically equivalent to A/Wyoming as determined by hemagglutination inhibition and microneutralization assays using ferret postinfection antisera. Conversely, A/Wyoming containing the His-155 and Gln-156 residues from A/Panama was antigenically equivalent to A/Panama. These results indicated that only these two HA residues specified the antigenic drift from A/Panama to A/Wyoming; other amino acid differences between these two H3N2 viruses had minimal impact on virus antigenicity but impacted virus replication efficiency in eggs.

Measuring antibody responses to a live attenuated influenza vaccine in children.

BACKGROUND: Hemagglutination inhibition (HAI) assay is the standard method for evaluating inactivated influenza vaccines, but no standard assay has been established for evaluating live attenuated influenza vaccines (LAIV). LAIV containing A/Beijing/262/95(H1N1) induced low serum HAI antibody responses to the antigenic variant, A/New Caledonia/20/99(H1N1) in a serologic study but provided protection against the A/New Caledonia-like viruses in a community study. Neutralization and HAI assays were compared by measuring H1N1 cross-reactive antibody responses to the LAIV in children. METHODS: Sera were collected from 50 children 1-8 years of age before vaccination and 4-6 weeks after each dose of the LAIV. Antibody titers to the 3 vaccine viruses were measured by the HAI assay, whereas antibody titers against the H1N1 vaccine virus (A/Beijing/262/95) and 2 H1N1 antigenic variants (A/Shenzhen/227/95 and A/New Caledonia/20/99) were measured by the HAI and neutralization assays. RESULTS: Initially seronegative participants were more likely to develop HAI seroconversion responses to the 3 vaccine viruses than the baseline seropositive participants (77% versus 14% for H1N1, 100% versus 20% for H3N2, 100% versus 19% for B, P < 0.01, Fisher's exact test). For the H1N1 cross-reactive antibody responses, seroconversion rates measured by the neutralization assay were significantly higher than those measured by the HAI assay (95% versus 78%, P = 0.0485 for A/Beijing/262/95; 75% versus 24%, P < 0.0001 for A/Shenzhen/227/95; 51% versus 5%, P < 0.0001 for A/New Caledonia/20/99). CONCLUSIONS: The neutralization assay was more sensitive than the HAI assay for measuring H1N1 antibody responses after vaccination of children with the LAIV and may provide a better correlate of clinical protection provided by the LAIV.

Role of transmembrane domain and cytoplasmic tail amino acid sequences of influenza a virus neuraminidase in raft association and virus budding.

Influenza virus neuraminidase (NA), a type II transmembrane glycoprotein, possesses receptor-destroying activity and thereby facilitates virus release from the cell surface. Among the influenza A viruses, both the cytoplasmic tail (CT) and transmembrane domain (TMD) amino acid sequences of NA are highly conserved, yet their function(s) in virus biology remains unknown. To investigate the role of amino acid sequences of the CT and TMD on the virus life cycle, we systematically mutagenized the entire CT and TMD of NA by converting two to five contiguous amino acids to alanine. In addition, we also made two chimeric NA by replacing the CT proximal one-third amino acids of the NA TMD [NA(1T2N)NA] and the entire NA TMD (NATRNA) with that of human transferrin receptor (TR) (a type II transmembrane glycoprotein). We rescued transfectant mutant viruses by reverse genetics and examined their phenotypes. Our results show that all mutated and chimeric NAs could be rescued into transfectant viruses. Different mutants showed pleiotropic effects on virus growth and replication. Some mutants (NA2A5, NA3A7, and NA4A10) had little effect on virus growth while others (NA3A2, NA5A27, and NA5A31) produced about 50- to 100-fold-less infectious virus and still some others (NA5A14, NA4A19, and NA4A23) exhibited an intermediate phenotype. In general, mutations towards the ectodomain-proximal sequences of TMD progressively caused reduction in NA enzyme activity, affected lipid raft association, and attenuated virus growth. Electron microscopic analysis showed that these mutant viruses remained aggregated and bound to infected cell surfaces and could be released from the infected cells by bacterial NA treatment. Moreover, viruses containing mutations in the extreme N terminus of the CT (NA3A2) as well as chimeric NA containing the TMD replaced partially [NA(1T2N)NA] or fully (NATRNA) with TR TMD caused reduction in virus growth and exhibited the morphological phenotype of elongated particles. These results show that although the sequences of NA CT and TMD per se are not absolutely essential for the virus life cycle, specific amino acid sequences play a critical role in providing structural stability, enzyme activity, and lipid raft association of NA. In addition, aberrant morphogenesis including elongated particle formation of some mutant viruses indicates the involvement of NA in virus morphogenesis and budding.

Influenza A virus hemagglutinin containing basolateral localization signal does not alter the apical budding of a recombinant influenza A virus in polarized MDCK cells.

Morphogenesis of influenza virus is a complex multistep process involving transport of all viral components as either individual or subviral components to the specified assembly site and interaction among the viral components in an ordered fashion to initiate the budding process. Envelope glycoprotein(s) is believed to be the major determinant in selecting the viral budding site since the majority of the viral glycoproteins are directed to the budding site independent of other viral components. Influenza viruses bud from the apical surface of polarized epithelial cells and all three envelope proteins, hemagglutinin (HA), neuraminidase (NA), and M2, are also targeted independently to the apical surface. Since HA is the major viral envelope protein, we decided to test whether basolaterally expressed HA can make the virus bud from the basolateral surface. Accordingly, we introduced the tyrosine-based basolateral-sorting signal to the cytoplasmic tail of HA by changing Cys561 --> Tyr561 and generated a transfectant virus by reverse genetics. Compared to the parent WSN virus, the mutant virus (HAtyr virus) contained less HA on its envelope. While the wild-type (wt) HA was >95% apical, the mutated HA (HAtyr) was approximately 60% basolateral in both transfected and virus-infected polarized MDCK cells. Also, HAtyr protein exhibited a much higher rate of endocytosis than the wt HA, in both apical and basolateral surface of transfected as well as virus-infected cells. However, the HAtyr virus, similar to wt WSN virus, was seen to bud almost exclusively (>99%) from the apical side of polarized MDCK cells. This finding was confirmed by using neuraminidase to facilitate virus release, by treating the collected virus particles with trypsin to cleave HA0 --> HA1 and HA2, by protein analysis of released virus particles, and finally, by electron microscopy. Therefore HA, the major glycoprotein alone, does not determine the budding site, and other factor(s), possibly both viral and host, is responsible for selecting the budding site of influenza virus.

Influenza infection promotes macrophage traffic into arteries of mice that is prevented by D-4F, an apolipoprotein A-I mimetic peptide.

BACKGROUND: We reported that HDL loses its antiinflammatory properties during acute influenza A infection in mice, and we hypothesized that these changes might be associated with increased trafficking of macrophages into the artery wall. The present study tested this hypothesis. METHODS AND RESULTS: D-4F, an apolipoprotein A-I mimetic peptide, or vehicle in which it was dissolved (PBS) was administered daily to LDL receptor-null mice after a Western diet and after influenza infection. D-4F treatment increased plasma HDL cholesterol and paraoxonase activity compared with PBS and inhibited increases in LDL cholesterol and peak levels of interleukin-6 after infection. Lung viral titers were reduced by 50% in mice receiving D-4F. Injection of female mice with male macrophages, which were detected with real-time polymerase chain reaction to measure the male Sry gene, revealed a marked increase in macrophage traffic into the aortic arch and innominate arteries after infection that was prevented by administration of D-4F. CONCLUSIONS: We conclude that loss of antiinflammatory properties of HDL after influenza infection in mice is associated with increased arterial macrophage traffic that can be prevented by administration of D-4F.