Jean-Michel Garcia

Kinetics of neutralizing antibodies in patients naturally infected by H5N1 virus

BACKGROUND Little is known about the kinetics of anti-H5 neutralizing antibodies in naturally H5N1-infected patients with severe clinical illness or asymptomatic infection. METHODS Using H5N1 microneutralisation (MN) and H5-pseudotype particle-based microneutralisation assays (H5pp) we analyzed sera sequentially obtained from 11 severely ill patients diagnosed by RT-PCR (follow-up range 1-139 weeks of disease onset) and 31 asymptomatically infected individuals detected in a sero-epidemiological study after exposure to H5N1 virus (follow-up range: 1-2 month-11 months after exposure). RESULTS Of 44 sera from 11 patients with H5N1 disease, 70% tested positive by MN (antibody titre > or = 80) after 2 weeks and 100% were positive by 3 weeks after disease onset. The geometric mean MN titers in severely ill patients were 540 at 1-2 months and 173 at 10-12 months and thus were higher than the titers from asymptomatic individuals (149 at 1-2 months, 62.2 at 10-12 months). Fractional polynomial regression analysis demonstrated that in all severely ill patients, positive titers persisted beyond 2 years of disease onset, while 10 of 23 sera collected 10-11 months after exposure in asymptomatically infected individuals tested negative. CONCLUSIONS Our results indicate that people with asymptomatic H5N1 infection have lower H5N1 antibody titres compared to those with severe illness and that in many asymptomatically infected patients the antibody titer decreased to levels below the threshold of positivity within one year. These data are essential for the design and interpretation of sero-epidemiological studies.

Avian Influenza H5-Containing Virus-Like Particles (VLPs): Host-Cell Receptor Specificity by STD NMR Spectroscopy†

H5N1 influenza virus is a highly contagious pathogen infecting poultry and other birds. The emergence of a human pandemic influenza virus from an avian progenitor appears to involve a switch in preferential binding of the influenza virus hemagglutinin (HA) from a(2,3)-linked N-acetylneuraminic acid containing glycans (the major form in the avian enteric tract) to a(2,6)-linked N-acetylneuraminic acid containing glycans (the major form in the human upper respiratory tract). Avian influenza viruses such as the H3 virus HA receptor typically contain the amino acids Gln226 and Gly228, which create a narrow binding pocket favoring a(2,3)-linked N-acetylneuraminic acid containing glycans. A number of other studies have also investigated HA–glycan recognition. Interestingly, the X-ray crystal structure of the reassembled HA of the 1918 Spanish influenza virus shows that its avianlike narrow binding pocket still allows highaffinity binding of a(2,6)-linked N-acetylneuraminic acid containing glycans. The mutation of only one amino acid (Asp190) within the HA binding site appears to determine the preference of the avianlike 1918 virus for human a(2,6)linked N-acetylneuraminic acid containing glycans. A mutation of Asp190 to Glu190 in the HA of the H5N1 strain could potentially switch its binding preference to a(2,6)linked glycans and consequently result in the emergence of a human pandemic virus. This fact emphasizes an urgent need to investigate the Nacetylneuraminic acid containing glycan recognition requirements by influenza virus HA that may lead to the development of novel antiinfluenza drugs that bind to the viral HA protein and consequently prevent the entry of the virus into human host cells. Rapid access to structural information would provide a detailed understanding of how virus particles interact with host cells on a molecular level and the determinants that prevent interspecies transmission of influenza viruses. Herein we present the first saturation transfer difference (STD) NMR spectroscopic study analyzing H5containing virus-like particles (VLPs) derived from the highly pathogenic avian H5N1 influenza strain in a complex with a(2,6)and a(2,3)-linked N-acetylneuraminides to mimic an in vitro or in vivo virus–host-cell interaction. We recently reported the production of H5 pseudotyped virus particles. To provide larger quantities of suitable VLPs for NMR studies, we have now successfully engineered heterologous H5 influenza VLPs by coexpression of pCDNA–synH5 coding for the hemagglutinin (H5) of H5N1 influenza virus and pCMV-dR8.91-expressing HIV-Gag-pol protein. Coexpression leads to spontaneous assembly of chimeric H5-VLPs, which contain the HA protein. The hemagglutinin, detected as uncleaved HA-0 precursor and HA-1/HA-2 cleaved mature forms, was incorporated on the surface of the viral particles at high levels. Expression of the viral HA protein was characterized by Western blot using specific C-terminal flag-tag M2 antibodies (Figure 1) and detection of VLPs by electron microscopy (Figure 2). In this study we investigated the capacity of H5-VLPs for competitive selection from a mixture of a(2,6)and a(2,3)sialyllactose (6’-SL and 3’-SL, respectively) of a preferred ligand (and therefore linkage) by means of STD NMR spectroscopy. It has been previously demonstrated that STD NMR spectroscopy can be utilized to investigate ligand interactions with whole virus particles, platelets, and intact cells. The large size of viruses and cells makes them attractive for studies with STD NMR spectroscopy because the inherently large line width enables saturation of the particle without affecting ligand signals. Additionally, the larger correlation time of bulky virus particles results in efficient spin diffusion and consequently stronger saturation transfer. To ensure the stability of the influenza H5-VLPs, NMR experiments were performed without prior purification, and 10% D2O was added for locking purposes. The H NMR spectrum of the influenza H5-VLPs (Figure 3a) shows the signals of the 20% sucrose cushion [*] Dr. T. Haselhorst, Dr. T. Islam, F. J. Rose, Prof. M. von Itzstein Institute for Glycomics, Griffith University, Gold Coast Campus Queensland, 4222 (Australia) Fax: (+61)7-555-29040 E-mail: m.vonitzstein@griffith.edu.au Homepage: http://www.griffith.edu.au/glycomics Dr. J.-M. Garcia, J. C. C. Lai, Prof. J. S. M. Peiris Hong Kong University Pasteur Research Centre Ltd., Dexter H.C. Man Building 8 Sassoon Road, Pokfulam, Hong Kong (China)

Optimization and evaluation of an influenza A (H5) pseudotyped lentiviral particle-based serological assay

BACKGROUND Novel serological methods provide alternative options for sero-diagnosis, sero-epidemiology and for determining evidence of naturally acquired or vaccine induced immunity. Micro-neutralization tests are currently the gold standard for serological studies of highly pathogenic avian influenza in mammalian species but require handling live virus in a biosafety level (BSL) 3 environment. We previously reported the use of H5 pseudotyped lentiviral particles (H5pp) as an alternative to micro-neutralization tests in a BSL-2 setting (Nefkens et al., 2007). OBJECTIVE To optimize and evaluate this newly developed H5pp assay on relevant clinical specimens. STUDY DESIGN We optimise and evaluate the performance of the H5pp assay using well-characterized sera from humans with confirmed H5N1 disease or controls. RESULTS The H5pp assay is a reliable serological method for the detection and quantification of neutralizing antibody to H5-viruses. CONCLUSION H5pp provide a reliable and safe alternative for sero-diagnosis and sero-epidemiology of H5N1 infections in a BSL-2 setting.

Prevalence of antibodies against avian influenza A (H5N1) virus among cullers and poultry workers in Ho Chi Minh City, 2005.

Background Between 2003 and 2005, highly pathogenic avian influenza A (H5N1) viruses caused large scale outbreaks in poultry in the Ho Chi Minh City area in Vietnam. We studied the prevalence of antibodies against H5N1 in poultry workers and cullers who were active in the program in Ho Chi Minh City in 2004 and 2005. Methodology/Principal Findings Single sera from 500 poultry workers and poultry cullers exposed to infected birds were tested for antibodies to avian influenza H5N1, using microneutralization assays and hemagglutination inhibition assay with horse blood. All sera tested negative using microneutralization tests. Three samples showed a 1∶80 titer in the hemagglutination inhibition assay. Conclusions/Significance This study provides additional support for the low transmissibility of clade 1 H5N1 to humans, but limited transmission to highly exposed persons cannot be excluded given the presence of low antibody titers in some individuals.

Production of influenza pseudotyped lentiviral particles and their use in influenza research and diagnosis: an update.

Pseudotyped viral particles are being used as safe surrogates to mimic the structure and surface of many viruses, including highly pathogenic viruses such as avian influenza H5N1, to investigate biological functions mediated by the envelope proteins derived from these viruses. The first part of this article evaluates and discusses the differences in the production and characterization of influenza pseudoparticles. The second part focuses on the applications that such a flexible tool can provide in modern influenza research, in particular in the fields of drug discovery, molecular biology and diagnosis.

Transmission of pandemic influenza H1N1 (2009) in Vietnamese swine in 2009–2010

Please cite this paper as: Trevennec et al. (2012) Transmission of pandemic influenza H1N1 (2009) in Vietnamese swine in 2009–2010. Influenza and Other Respiratory Viruses 6(5), 348–357.

A Secondary Sialic Acid Binding Site on Influenza Virus Neuraminidase: Fact or Fiction?

One flu over the cuckoos nest: The biological significance of a secondary sialic acid binding site on influenza virus neuraminidase remains elusive. On blocking the active site influenza-virus-containing virus-like particles with oseltamivir carboxylate, binding to a(2,3)-sialyllactose is still detected. Thus the sialyllactose must bind at a secondary sialic acid binding site (see structures: docking study of a(2,3)-sialyllactose in the secondary binding site of avian flu neuraminidase).

Investigation of the binding and cleavage characteristics of N1 neuraminidases from avian, seasonal, and pandemic influenza viruses using saturation transfer difference nuclear magnetic resonance

The main function of influenza neuraminidase (NA) involves enzymatic cleavage of sialic acid from the surface of host cells resulting in the release of the newly produced virions from infected cells, as well as aiding the movement of virions through sialylated mucus present in the respiratory tract. However, there has previously been little information on the binding affinity of different forms of sialylated glycan with NA. Our objectives were then to investigate both sialic acid binding and cleavage of neuraminidase at an atomic resolution level.

Investigating the Interaction Between Influenza and Sialic Acid: Making and Breaking the Link

Since the early 1940s sialic acid (Sia) has been regarded as the primary receptor for influenza virus. This Sia is usually bound to an adjacent galactose (Gal) in an α2-3 or α2-6 configuration. This led to a concept about an interspecies barrier as avian viruses preferentially bind to Sia α2-3 linked to Gal, whereas human viruses have a preference for the Sia α2-6 linked to Gal and that transmission from one species to another would preferentially occur only in a host species in which both types of Sia were present. The viral haemagglutinin binds to Sia to facilitate cellular entry. To release progeny viral particles the second main component of the influenza viral envelope – neuraminidase, cleaves Sia. The viral-receptor interaction was initially investigated using agglutination of red blood cells and later using lectin histochemistry. Recent techniques investigating the HA-Sia/NA-Sia link have employed the use of glycan arrays and virus-like pseudoparticles with STD-NMR.