Structures of complexes formed by H5 influenza hemagglutinin with a potent broadly neutralizing human monoclonal antibody.

H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer's formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion

Live, Attenuated Influenza A H5N1 Candidate Vaccines Provide Broad Cross-Protection in Mice and Ferrets

BACKGROUND: Recent outbreaks of highly pathogenic influenza A H5N1 viruses in humans and avian species that began in Asia and have spread to other continents underscore an urgent need to develop vaccines that would protect the human population in the event of a pandemic. METHODS AND FINDINGS: Live, attenuated candidate vaccines possessing genes encoding a modified H5 hemagglutinin (HA) and a wild-type (wt) N1 neuraminidase from influenza A H5N1 viruses isolated in Hong Kong and Vietnam in 1997, 2003, and 2004, and remaining gene segments derived from the cold-adapted (ca) influenza A vaccine donor strain, influenza A/Ann Arbor/6/60 ca (H2N2), were generated by reverse genetics. The H5N1 ca vaccine viruses required trypsin for efficient growth in vitro, as predicted by the modification engineered in the gene encoding the HA, and possessed the temperature-sensitive and attenuation phenotypes specified by the internal protein genes of the ca vaccine donor strain. More importantly, the candidate vaccines were immunogenic in mice. Four weeks after receiving a single dose of 10(6) 50% tissue culture infectious doses of intranasally administered vaccines, mice were fully protected from lethality following challenge with homologous and antigenically distinct heterologous wt H5N1 viruses from different genetic sublineages (clades 1, 2, and 3) that were isolated in Asia between 1997 and 2005. Four weeks after receiving two doses of the vaccines, mice and ferrets were fully protected against pulmonary replication of homologous and heterologous wt H5N1 viruses. CONCLUSIONS: The promising findings in these preclinical studies of safety, immunogenicity, and efficacy of the H5N1 ca vaccines against antigenically diverse H5N1 vaccines provide support for their careful evaluation in Phase 1 clinical trials in humans

Influenza H5 Hemagglutinin DNA Primes the Antibody Response Elicited by the Live Attenuated Influenza A/Vietnam/1203/2004 Vaccine in Ferrets

Priming immunization plays a key role in protecting individuals or populations to influenza viruses that are novel to humans. To identify the most promising vaccine priming strategy, we have evaluated different prime-boost regimens using inactivated, DNA and live attenuated vaccines in ferrets. Live attenuated influenza A/Vietnam/1203/2004 (H5N1) candidate vaccine (LAIV, VN04 ca) primed ferrets efficiently while inactivated H5N1 vaccine could not prime the immune response in seronegative ferrets unless an adjuvant was used. However, the H5 HA DNA vaccine alone was as successful as an adjuvanted inactivated VN04 vaccine in priming the immune response to VN04 ca virus. The serum antibody titers of ferrets primed with H5 HA DNA followed by intranasal vaccination of VN04 ca virus were comparable to that induced by two doses of VN04 ca virus. Both LAIV-LAIV and DNA-LAIV vaccine regimens could induce antibody responses that cross-neutralized antigenically distinct H5N1 virus isolates including A/HongKong/213/2003 (HK03) and prevented nasal infection of HK03 vaccine virus. Thus, H5 HA DNA vaccination may offer an alternative option for pandemic preparedness

Eurasian-Origin Gene Segments Contribute to the Transmissibility, Aerosol Release, and Morphology of the 2009 Pandemic H1N1 Influenza Virus

The epidemiological success of pandemic and epidemic influenza A viruses relies on the ability to transmit efficiently from person-to-person via respiratory droplets. Respiratory droplet (RD) transmission of influenza viruses requires efficient replication and release of infectious influenza particles into the air. The 2009 pandemic H1N1 (pH1N1) virus originated by reassortment of a North American triple reassortant swine (TRS) virus with a Eurasian swine virus that contributed the neuraminidase (NA) and M gene segments. Both the TRS and Eurasian swine viruses caused sporadic infections in humans, but failed to spread from person-to-person, unlike the pH1N1 virus. We evaluated the pH1N1 and its precursor viruses in a ferret model to determine the contribution of different viral gene segments on the release of influenza virus particles into the air and on the transmissibility of the pH1N1 virus. We found that the Eurasian-origin gene segments contributed to efficient RD transmission of the pH1N1 virus likely by modulating the release of influenza viral RNA-containing particles into the air. All viruses replicated well in the upper respiratory tract of infected ferrets, suggesting that factors other than viral replication are important for the release of influenza virus particles and transmission. Our studies demonstrate that the release of influenza viral RNA-containing particles into the air correlates with increased NA activity. Additionally, the pleomorphic phenotype of the pH1N1 virus is dependent upon the Eurasian-origin gene segments, suggesting a link between transmission and virus morphology. We have demonstrated that the viruses are released into exhaled air to varying degrees and a constellation of genes influences the transmissibility of the pH1N1 virus

Antigenic Fingerprinting of H5N1 Avian Influenza Using Convalescent Sera and Monoclonal Antibodies Reveals Potential Vaccine and Diagnostic Targets

Using whole-genome-fragment phage display libraries, Hana Golding and colleagues identify the viral epitopes recognized by serum antibodies in humans who have recovered from infection with H5N1 avian influenza

Genetic Polymorphisms of Mannose-Binding Lectin Do Not Influence Placental Malaria but Are Associated with Preterm Deliveries▿

During pregnancy, Plasmodium falciparum-infected erythrocytes (IE) sequester in the placenta where they induce pathology and increase the risk of low-birth-weight (LBW) babies. The innate immune mediator, mannose-binding lectin (MBL), enhances phagocytosis of pathogens. Since MBL is reported to bind to IE, we hypothesized that it might aid in clearance of IE from the placenta, thereby reducing the risk of LBW babies. To test this hypothesis, molecular genotyping was used to detect polymorphisms at codon 57 (A/C) in exon 1 of MBL2 in 401 pregnant Cameroonian women, with or without placental malaria, who had LBW and normal-weight babies. Polymorphisms in the promoter region at positions −550 (H/L), −221 (X/Y), and +4 (P/Q) were also determined, and plasma MBL levels were measured during pregnancy and at delivery. The expected correlation between genotype and plasma MBL levels was confirmed. However, asymptomatic infections were not associated with an increase in MBL levels in the peripheral blood, and MBL levels were similar in the placental and cord blood of women with or without placental malaria at delivery. There was no evidence that MBL levels at delivery were associated with malaria-related poor pregnancy outcomes. Women with the LXPA haplotype, however, were more likely to have LBW babies, but the risk was not related to malaria. These results do not support the hypothesis that MBL aids in the clearance of parasites from the placenta but suggest that Cameroonian women with LXPA are at risk of having LBW babies due to other causes

Glycosylation at 158N of the Hemagglutinin Protein and Receptor Binding Specificity Synergistically Affect the Antigenicity and Immunogenicity of a Live Attenuated H5N1 A/Vietnam/1203/2004 Vaccine Virus in Ferrets ▿

A live attenuated influenza A/Vietnam/1203/2004 (H5N1) vaccine virus (VN04 ca) has receptor binding specificity to α2,3-linked sialosides (α2,3SAL), and a single dose induces a minimal serum antibody response in mice and ferrets. In contrast, A/Hong Kong/213/2003 (H5N1) vaccine virus (HK03 ca) binds to both α2,6SAL and α2,3SAL and generates a stronger serum antibody response in animals. Among the 9 amino acids that differed between the two H5 HA1 proteins, several HK03-specific residues enabled the VN04 ca virus to bind to both α2,3SAL and α2,6SAL receptors, but only the removal of the 158N glycosylation, together with an S227N change, resulted in more-efficient viral replication in the upper respiratory tract of ferrets and an increased serum antibody response. However, the antibody response was HK03 strain specific and did not significantly cross-neutralize VN04 virus. A second approach was taken to adapt the H5N1 VN04 ca virus in MDCK cells to select HA variants with larger plaque morphology. Although a number of large-plaque-size HA variants with amino acid changes in the HA receptor binding region were identified, none of these mutations affected virus receptor binding preference and immunogenicity. In addition, the known receptor binding site changes, Q226L and G228S, were introduced into the HA protein of the VN04 ca virus. Only in conjunction with the removal of the 158N glycosylation did the virus replicate efficiently in the upper respiratory tract of ferrets and became more immunogenic, yet the response was also HK03 specific. Thus, the mask of the antigenic epitopes by 158N glycosylation at the HA globular head and its α2,3SAL binding preference of VN04 ca virus affect virus antigenicity and replication in the host, resulting in a lower antibody response

Generation of Live Attenuated Novel Influenza Virus A/California/7/09 (H1N1) Vaccines with High Yield in Embryonated Chicken Eggs ▿

Several live attenuated influenza virus A/California/7/09 (H1N1) (CA09) candidate vaccine variants that possess the hemagglutinin (HA) and neuraminidase (NA) gene segments from the CA09 virus and six internal protein gene segments from the cold-adapted influenza virus A/Ann Arbor/6/60 (H2N2) virus were generated by reverse genetics. The reassortant viruses replicated relatively poorly in embryonated chicken eggs. To improve virus growth in eggs, reassortants expressing the HA and NA of CA09 were passaged in MDCK cells and variants exhibiting large-plaque morphology were isolated. These variants replicated at levels approximately 10-fold higher than the rate of replication of the parental strains in embryonated chicken eggs. Sequence analysis indicated that single amino acid changes at positions 119, 153, 154, and 186 were responsible for the improved growth properties in MDCK cells and eggs. In addition, the introduction of a mutation at residue 155 that was previously shown to enhance the replication of a 1976 swine influenza virus also significantly improved the replication of the CA09 virus in eggs. Each variant was further evaluated for receptor binding preference, antigenicity, attenuation phenotype, and immunogenicity. Mutations at residues 153, 154, and 155 drastically reduced viral antigenicity, which made these mutants unsuitable as vaccine candidates. However, changes at residues 119 and 186 did not affect virus antigenicity or immunogenicity, justifying their inclusion in live attenuated vaccine candidates to protect against the currently circulating 2009 swine origin H1N1 viruses