Carolyn Nicolson

A sensitive retroviral pseudotype assay for influenza H5N1-neutralizing antibodies

Background  The World Health Organisation (WHO) recommended the development of simple, safe, sensitive and specific neutralization assays for avian influenza antibodies. We have used retroviral pseudotypes bearing influenza H5 hemagglutinin (HA) as safe, surrogate viruses for influenza neutralization assays which can be carried out at Biosafety Level 2.

Plasmid DNA encoding influenza virus haemagglutinin induces Th1 cells and protection against respiratory infection despite its limited ability to generate antibody responses

Direct intramuscular injection of plasmid DNA can generate immune responses against encoded antigens. However, the relative ability of DNA vaccines to induce cellular and humoral immunity after a single or booster immunization and the persistence of this response have not been fully elucidated. In this study, induction and maintenance of antibody and T cell subtypes with different doses of naked DNA encoding the haemagglutinin (HA) gene of influenza virus were examined and compared to the immune responses and protection induced by respiratory tract infection and immunization with a killed virus vaccine. Like natural infection, immunization with HA DNA induced potent Th1 responses. Spleen cells from mice immunized once with HA DNA in the dose range 10 ng to 100 microgram secreted significant levels of IFN-gamma, but low or undetectable IL-5, in response to influenza virus in vitro. Furthermore, CD4(+) HA-specific Th1 clones were generated from spleens of immunized mice. Although T cell responses waned 12 weeks after a single immunization, antigen-specific Th1 cells persisted in the spleen for at least 6 months after two booster immunizations. In contrast, influenza virus-specific ELISA IgG titres were low after a single immunization and required two booster immunizations to reach significant levels. Furthermore, haemagglutination inhibition (HI) antibodies were weak or undetectable after two immunizations. Nevertheless, two doses of HA DNA conferred almost complete protection against respiratory challenge with live virus. Thus, despite the limited ability to induce antibodies, DNA vaccines confer protective immunity against influenza virus infection, which appears to be mediated by Th1 cells.

The role of amniotic passage in the egg-adaptation of human influenza virus is revealed by haemagglutinin sequence analyses.

Obtaining an isolate of a human influenza virus in the allantoic cavity of the embryonated hens egg is more efficient if the clinical sample is initially passaged in the amniotic cavity. To investigate the extent to which the variants present after allantoic propagation are also selected by amniotic passage, clinical virus passaged once in the amnion has been subjected to extensive genetic and antigenic analyses. The data indicate that the natural virus can replicate unrestrictedly within the amnion. However, exposure of amniotic virus to the allantois during the incubation period, which will occur through the hole between the amniotic and allantoic cavities caused by the inoculating needle, allows for the possibility of an egg-adapted variant establishing replication within the allantois and returning to the amnion. These observations illustrate why prior passage in the amnion increases the probability of a variant successfully establishing itself during a subsequent allantoic passage.

Improved haemagglutinin antigen content in H5N1 candidate vaccine viruses with chimeric haemagglutinin molecules

The candidate vaccine virus NIBRG-14 was derived by reverse genetics and comprises the haemagglutinin (HA) and neuraminidase (NA) genes derived from the clade 1 virus A/Viet Nam/1194/2004 on an A/Puerto Rico/8/34 (PR8) backbone. The HA gene was modified to remove the multibasic cleavage site motif associated with high pathogenicity. Reports from manufacturers, confirmed by data generated in this laboratory, have shown that this virus yields a low amount of HA antigen. We have generated a panel of new viruses using reverse genetics in which each virus consists of the PR8 backbone, the NA gene from A/Viet Nam/1194/2004 and a chimeric HA gene with sequences from both PR8 and A/Viet Nam/1194/2004. Here we show that a number of these viruses have improved HA antigen content and yield and are therefore better candidate vaccine viruses for use in production of H5N1 vaccine.

High growth reassortant influenza vaccine viruses: New approaches to their control

When a new strain of an influenza virus is required to be incorporated into influenza vaccine, attempts are made to recombine such strains with laboratory adapted viruses, which will grow to high titre in order to improve the yield of the vaccine strain. It is important that such high growth reassortant vaccine strains are not contaminated with genes coding for the antigenic determinants of the high growth laboratory strain. We describe the characterization of two recent high growth reassortants and the application of the polymerase chain reaction to ensure their genetic identity and purity.

Preparation of vaccines against H5N1 influenza

In response to the pandemic warning provided by the highly pathogenic H5N1 influenza virus infections in Hong Kong, there were world-wide attempts to develop vaccines. Three strategies were followed and although each was associated with some success, there were also some problems. Pre-clinical vaccine efficacy results are presented from one such strategy, that of using an apathogenic H5N3 avian strain for vaccine production.

An additional oligosaccharide moiety in the HA of a pandemic influenza H1N1 candidate vaccine virus confers increased antigen yield in eggs.

The H1N1 influenza pandemic in 2009 highlighted the need for the rapid generation of candidate vaccine viruses (CVVs) against an A/California/7/2009-like virus. The first available CVVs gave low protein yields in eggs but improved yields were achieved for second generation CVVs which contained amino acid substitutions compared to their precursor viruses. In this study, we investigated the basis for the increased virus protein yield of CVV NIBRG-121xp and whether the improved yield characteristics could be transferred between this virus and two other CVVs, NYMC X-179A and NYMC X-181. We generated variant viruses by reverse genetics to contain combinations of amino acid substitutions found in high yielding NIBRG-121xp and NYMC X-181. We found that the increase in total protein yield and functional HA yield of NIBRG-121xp in eggs is attributable to the single amino acid substitution K119N in the HA. We also found that the glycosylation of position 119 is essential for the improved virus protein yield in eggs. However, the K119N yield-enhancing effect was not transferable between viruses, nor was the N129D change found in high yielding NYMC X-181. However, position 119 may be a useful locus to monitor in future for viruses and CVVs with potentially high yield.

Genic amplification of the entire coding region of the HEF RNA segment of influenza C virus

Abstract In order to provide an easy and powerful analysis of influenza C viral HEF RNA segment of a recent strain, a combination of reverse transcription and the polymerase chain reaction was used. We amplified the entire coding region of the HEF gene of a laboratory strain of virus called C/Johannesburg/1/66, widely used for binding and esterase activity studies as well as that of a strain isolated in 1991 (C/Paris/145/91) from a patient suffering from severe flu syndrome. The sequences we amplified were about 2 kilobases long. In this work, we show that the forward ‘universal primer’ Unil, which has been used for influenza A and B viruses cDNA syntheses can also be used for influenza C virus. The PCR primers were designed to contain restriction sites to make the PCR products ready to be used for further purposes. A restriction analysis of the PCR products combined with analyses of all the human influenza C virus HEF gene sequences published so far permitted the design of sets of oligonucleotides which can prime PCR on cDNA of unknown influenza C virus for cloning.

Permissible Variation in the 3′ Non-Coding Region of the Haemagglutinin Genome Segment of the H5N1 Candidate Influenza Vaccine Cirus NIBRG-14

The candidate H5N1 vaccine virus NIBRG-14 was created in response to a call from the World Health Organisation in 2004 to prepare candidate vaccine viruses (CVVs) to combat the threat of an H5N1 pandemic. NIBRG-14 was created by reverse genetics and is composed of the neuraminidase (NA) and modified haemagglutinin (HA) genes from A/Vietnam/1194/2004 and the internal genes of PR8, a high growing laboratory adapted influenza A(H1N1) strain. Due to time constraints, the non-coding regions (NCRs) of A/Vietnam/1194/2004 HA were not determined prior to creating NIBRG-14. Consequently, the sequence of the primers used to clone the modified A/Vietnam/1194/2004 HA was based upon previous experience of cloning H5N1 viruses. We report here that the HA 3′ NCR sequence of NIBRG-14 is different to that of the parental wild type virus A/Vietnam/1194/2004; however this does not appear to impact on its growth or antigen yield. We introduced additional small changes into the 3′NCR of NIBRG-14; these had only minor effects on viral growth and antigen content. These findings may serve to assure the influenza vaccine community that generation of CVVs using best-guess NCR sequences, based on sequence alignments, are likely to produce robust viruses.

The Ability of a Non-Egg Adapted (Cell-Like) A(H1N1)pdm09 Virus to Egg-Adapt at HA Loci Other than 222 and 223 and Its Effect on the Yield of Viral Protein.

Previous studies on influenza A(H1N1)pdm09 candidate vaccine viruses (CVVs) that had adapted to growth in embryonated chicken eggs by the acquisition of amino acid substitutions at HA positions 222 or 223 showed that improved protein yield could be conferred by additional amino acid substitutions in the haemagglutinin (HA) that arose naturally during passaging of the virus in eggs. In this study we investigated, by means of reverse genetics, the ability of a non-egg adapted (cell-like) A(H1N1)pdm09 virus to egg-adapt at HA loci other than 222/223, introducing amino acid substitutions previously identified as egg adaptations in pre-H1N1pdm09 H1N1 viruses and assessing their effect on protein yield and antigenicity. We also investigated the effect on the protein yield of these substitutions in viruses that had A(H1N1)pdm09 internal genes rather than the traditional PR8 internal genes of a CVV. The data show that a cell-like A/Christchurch/16/2010 can be egg-adapted via amino acid substitutions in at least three alternative HA loci (187, 190 and 216), in viruses with either PR8 or A/California/7/2009 internal genes, but that the effects on protein yield vary depending on the amino acid substitution and the internal genes of the virus. Since CVVs need to produce high protein yields to be suitable for vaccine manufacture, the findings of this study will assist in the future characterisation of both wild type viruses and lab-derived CVVs for vaccine use.