Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge

Influenza HA is the primary target of neutralizing antibodies during infection, and its sequence undergoes genetic drift and shift in response to immune pressure. The receptor binding HA1 subunit of HA shows much higher sequence variability relative to the metastable, fusion-active HA2 subunit, presumably because neutralizing antibodies are primarily targeted against the former in natural infection. We have designed an HA2-based immunogen using a protein minimization approach that incorporates designed mutations to destabilize the low pH conformation of HA2. The resulting construct (HA6) was expressed in Escherichia coli and refolded from inclusion bodies. Biophysical studies and mutational analysis of the protein indicate that it is folded into the desired neutral pH conformation competent to bind the broadly neutralizing HA2 directed monoclonal 12D1, not the low pH conformation observed in previous studies. HA6 was highly immunogenic in mice and the mice were protected against lethal challenge by the homologous A/HK/68 mouse-adapted virus. An HA6-like construct from another H3 strain (A/Phil/2/82) also protected mice against A/HK/68 challenge. Regions included in HA6 are highly conserved within a subtype and are fairly well conserved within a clade. Targeting the highly conserved HA2 subunit with a bacterially produced immunogen is a vaccine strategy that may aid in pandemic preparedness.

Innate Immune Recognition of RNA-virus infection in human macrophages

Innate immunity and host defence are rapidly evoked by structurally invariant molecular motifs common to microbial world, called pathogen associated molecular patterns (PAMPs). In addition to PAMPs, endogenous molecules released in response to inflammation and tissue damage, danger associated molecular patterns (DAMPs), are required for eliciting the response. The most important PAMPs of viruses are viral nucleic acids, their genome or its replication intermediates, whereas the identity and characteristics of virus infection-induced DAMPs are poorly defined. PAMPs and DAMPs engage a limited set of germ-line encoded pattern recognition receptors (PRRs) in immune and non-immune cells. Membrane-bound Toll-like receptors (TLRs), cytoplasmic retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) and nucleotide-binding oligomerization domain-like receptor (NLRs) are important PRRs involved in the recognition of the molecular signatures of viral infection, such as double-stranded ribonucleic acids (dsRNAs). Engagement of PRRs results in local and systemic innate immune responses which, when activated against viruses, evoke secretion of antiviral and pro-inflammatory cytokines, and programmed cell death i.e., apoptosis of the virus-infected cell. Macrophages are the central effector cells of innate immunity. They produce significant amounts of antiviral cytokines, called interferons (IFNs), and pro-inflammatory cytokines, such as interleukin (IL)-1β and IL-18. IL-1β and IL-18 are synthesized as inactive precursors, pro-IL-1β and pro-IL-18, that are processed by caspase-1 in a cytoplasmic multiprotein complex, called the inflammasome. After processing, these cytokines are biologically active and will be secreted. The signals and secretory routes that activate inflammasomes and the secretion of IL-1β and IL-18 during virus infections are poorly characterized. The main goal of this thesis was to characterize influenza A virus-induced innate immune responses and host-virus interactions in human primary macrophages during an infection. Methodologically, various techniques of cellular and molecular biology, as well as proteomic tools combined with bioinformatics, were utilized. Overall, the thesis provides interesting insights into inflammatory and antiviral innate immune responses, and has characterized host-virus interactions during influenza A virus-infection in human primary macrophages.

Hemagglutinin sequence conservation guided stem immunogen design from influenza A H3 subtype

Seasonal epidemics caused by influenza A (H1 and H3 subtypes) and B viruses are a major global health threat. The traditional, trivalent influenza vaccines have limited efficacy because of rapid antigenic evolution of the circulating viruses. This antigenic variability mediates viral escape from the host immune responses, necessitating annual vaccine updates. Influenza vaccines elicit a protective antibody response, primarily targeting the viral surface glycoprotein hemagglutinin (HA). However, the predominant humoral response is against the hypervariable head domain of HA, thereby restricting the breadth of protection. In contrast, the conserved, subdominant stem domain of HA is a potential ``universal'' vaccine candidate. We designed an HA stem-fragment immunogen from the 1968 pandemic H3N2 strain (A/Hong Kong/1/68) guided by a comprehensive H3 HA sequence conservation analysis. The biophysical properties of the designed immunogen were further improved by C-terminal fusion of a trimerization motif, ``isoleucine-zipper'', or ``foldon''. These immunogens elicited cross-reactive, antiviral antibodies and conferred partial protection against a lethal, homologous HK68 virus challenge in vivo. Furthermore, bacterial expression of these immunogens is economical and facilitates rapid scale-up.

A host transcriptional signature for presymptomatic detection of infection in humans exposed to influenza H1N1 or H3N2.

There is great potential for host-based gene expression analysis to impact the early diagnosis of infectious diseases. In particular, the influenza pandemic of 2009 highlighted the challenges and limitations of traditional pathogen-based testing for suspected upper respiratory viral infection. We inoculated human volunteers with either influenza A (A/Brisbane/59/2007 (H1N1) or A/Wisconsin/67/2005 (H3N2)), and assayed the peripheral blood transcriptome every 8 hours for 7 days. Of 41 inoculated volunteers, 18 (44%) developed symptomatic infection. Using unbiased sparse latent factor regression analysis, we generated a gene signature (or factor) for symptomatic influenza capable of detecting 94% of infected cases. This gene signature is detectable as early as 29 hours post-exposure and achieves maximal accuracy on average 43 hours (p = 0.003, H1N1) and 38 hours (p-value = 0.005, H3N2) before peak clinical symptoms. In order to test the relevance of these findings in naturally acquired disease, a composite influenza A signature built from these challenge studies was applied to Emergency Department patients where it discriminates between swine-origin influenza A/H1N1 (2009) infected and non-infected individuals with 92% accuracy. The host genomic response to Influenza infection is robust and may provide the means for detection before typical clinical symptoms are apparent.

Temporal dynamics of host molecular responses differentiate symptomatic and asymptomatic influenza a infection.

Exposure to influenza viruses is necessary, but not sufficient, for healthy human hosts to develop symptomatic illness. The host response is an important determinant of disease progression. In order to delineate host molecular responses that differentiate symptomatic and asymptomatic Influenza A infection, we inoculated 17 healthy adults with live influenza (H3N2/Wisconsin) and examined changes in host peripheral blood gene expression at 16 timepoints over 132 hours. Here we present distinct transcriptional dynamics of host responses unique to asymptomatic and symptomatic infections. We show that symptomatic hosts invoke, simultaneously, multiple pattern recognition receptors-mediated antiviral and inflammatory responses that may relate to virus-induced oxidative stress. In contrast, asymptomatic subjects tightly regulate these responses and exhibit elevated expression of genes that function in antioxidant responses and cell-mediated responses. We reveal an ab initio molecular signature that strongly correlates to symptomatic clinical disease and biomarkers whose expression patterns best discriminate early from late phases of infection. Our results establish a temporal pattern of host molecular responses that differentiates symptomatic from asymptomatic infections and reveals an asymptomatic host-unique non-passive response signature, suggesting novel putative molecular targets for both prognostic assessment and ameliorative therapeutic intervention in seasonal and pandemic influenza.

Bayesian inference of the number of factors in gene-expression analysis: application to human virus challenge studies.

BACKGROUND: Nonparametric Bayesian techniques have been developed recently to extend the sophistication of factor models, allowing one to infer the number of appropriate factors from the observed data. We consider such techniques for sparse factor analysis, with application to gene-expression data from three virus challenge studies. Particular attention is placed on employing the Beta Process (BP), the Indian Buffet Process (IBP), and related sparseness-promoting techniques to infer a proper number of factors. The posterior density function on the model parameters is computed using Gibbs sampling and variational Bayesian (VB) analysis. RESULTS: Time-evolving gene-expression data are considered for respiratory syncytial virus (RSV), Rhino virus, and influenza, using blood samples from healthy human subjects. These data were acquired in three challenge studies, each executed after receiving institutional review board (IRB) approval from Duke University. Comparisons are made between several alternative means of per-forming nonparametric factor analysis on these data, with comparisons as well to sparse-PCA and Penalized Matrix Decomposition (PMD), closely related non-Bayesian approaches. CONCLUSIONS: Applying the Beta Process to the factor scores, or to the singular values of a pseudo-SVD construction, the proposed algorithms infer the number of factors in gene-expression data. For real data the "true" number of factors is unknown; in our simulations we consider a range of noise variances, and the proposed Bayesian models inferred the number of factors accurately relative to other methods in the literature, such as sparse-PCA and PMD. We have also identified a "pan-viral" factor of importance for each of the three viruses considered in this study. We have identified a set of genes associated with this pan-viral factor, of interest for early detection of such viruses based upon the host response, as quantified via gene-expression data.

Maternal Effects of Respiratory Syncytial Virus Infection during Pregnancy.

Given the illness and deaths caused by respiratory syncytial virus (RSV) infection during the first year of life, preventing infant RSV infections through maternal vaccination is intriguing. However, little is known about the extent and maternal effects of RSV infection during pregnancy. We describe 3 cases of maternal RSV infection diagnosed at a US center during winter 2014. Case-patient 1 (26 years old, week 33 of gestation) received a diagnosis of RSV infection and required mechanical ventilation. Case-patient 2 (27 years old, week 34 of gestation) received a diagnosis of infection with influenza A(H1N1) virus and RSV and required mechanical ventilation. Case-patient 3 (21 years old, week 32 of gestation) received a diagnosis of group A streptococcus pharyngitis and RSV infection and was monitored as an outpatient. Clarifying the effects of maternal RSV infection could yield valuable insights into potential maternal and fetal benefits of an effective RSV vaccination program.

Sequence divergence of measles virus haemagglutinin during natural evolution and adaptation to cell culture

Phylogenetic analysis of the sequence of the H gene of 75 measles virus (MV) strains (32 published and 43 new sequences) was carried out. The lineage groups described from comparison of the nucleotide sequences encoding the C-terminal regions of the N protein of MV were the same as those derived from the H gene sequences in almost all cases. The databases document a number of distinct genotype switches that have occurred in Madrid (Spain). Well-documented is the complete replacement of lineage group C2, the common European genotype at that time, with that of group D3 around the autumn of 1993. No further isolations of group C2 took place in Madrid after this time. The rate of mutation of the H gene sequences of MV genotype D3 circulating in Madrid from 1993 to 1996 was very low (5 x 10(-4) per annum for a given nucleotide position). This is an order of magnitude lower than the rates of mutation observed in the HN genes of human influenza A viruses. The ratio of expressed over silent mutations indicated that the divergence was not driven by immune selection in this gene. Variations in amino acid 117 of the H protein (F or L) may be related to the ability of some strains to haemagglutinate only in the presence of salt. Adaptation of MV to different primate cell types was associated with very small numbers of mutations in the H gene. The changes could not be predicted when virus previously grown in human B cell lines was adapted to monkey Vero cells. In contrast, rodent brain-adapted viruses displayed a lot of amino acid sequence variation from normal MV strains. There was no convincing evidence for recombination between MV genotypes.

Respiratory syncytial virus, an ongoing medical dilemma: an expert commentary on respiratory syncytial virus prophylactic and therapeutic pharmaceuticals currently in clinical trials

As the most important viral cause of severe respiratory disease in infants and increasing recognition as important in the elderly and immunocompromised, respiratory syncytial virus (RSV) is responsible for a massive health burden worldwide. Prophylactic antibodies were successfully developed against RSV. However, their use is restricted to a small group of infants considered at high risk of severe RSV disease. There is still no specific therapeutics or vaccines to combat RSV. As such, it remains a major unmet medical need for most individuals. The World Health Organisations International Clinical Trials Registry Platform (WHO ICTRP) and PubMed were used to identify and review all RSV vaccine, prophylactic and therapeutic candidates currently in clinical trials. This review presents an expert commentary on all RSV-specific prophylactic and therapeutic candidates that have entered clinical trials since 2008.

Lyophilisation of influenza, rabies and Marburg lentiviral pseudotype viruses for the development and distribution of a neutralisation-assay based diagnostic kit

Pseudotype viruses (PVs) are chimeric, replication-deficient virions that mimic wild-type virus entry mechanisms and can be safely employed in neutralisation assays, bypassing the need for high biosafety requirements and performing comparably to established serological assays. However, PV supernatant necessitates -80°C long-term storage and cold-chain maintenance during transport, which limits the scope of dissemination and application throughout resource-limited laboratories. We therefore investigated the effects of lyophilisation on influenza, rabies and Marburg PV stability, with a view to developing a pseudotype virus neutralisation assay (PVNA) based kit suitable for affordable global distribution. Infectivity of each PV was calculated after lyophilisation and immediate reconstitution, as well as subsequent to incubation of freeze-dried pellets at varying temperatures, humidities and timepoints. Integrity of glycoprotein structure following treatment was also assessed by employing lyophilised PVs in downstream PVNAs. In the presence of 0.5M sucrose-PBS cryoprotectant, each freeze-dried pseudotype was stably stored for 4 weeks at up to 37°C and could be neutralised to the same potency as unlyophilised PVs when employed in PVNAs. These results confirm the viability of a freeze-dried PVNA-based kit, which could significantly facilitate low-cost serology for a wide portfolio of emerging infectious viruses.

Enhancing pnemonia surveillance in acute care facilities in post SARS era : a Canadian hospital surveillance study, 2003-2004 influenza season

A retrospective study of patients hospitalized with influenza and/or pneumonia in a Niagara area community hospital for the influenza season 2003-04 was designed with the main goal of enhancing pneumonia surveillance in acute care facilities and the following specific objectives: 1) identify etiologies, factors, and clinical presentation associated with pneumonia; 2) assess the ODIN score on ICU patients to predict outcomes of severe pneumonia; 3) identify the frequency of pneumonia and influenza in a hospital setting; and 4) develop a hospital pneumonia electronic surveillance tool. A total of 172 patients' charts (50% females) were reviewed and classified into two groups: those with diagnosis of pneumonia (n=132) and those without pneumonia (n=40). The latter group consisted mainly of patients with influenza (85%). Most patients were young (<10yrs) or elderly (>71yrs). Presenting body temperature <38°C, cough symptoms, respiratory and cardiac precomorbidities were common in both groups. Pneumonia was more frequent in males (p= .032) and more likely community-acquired (98%) than nosocomial (2%). No evidence of ventilator-associated pneumonia was found. Microbiology testing in 72% of cases detected 19 different pathogens. In pneumonia patients the most common organisms were Streptococcus pneumoniae (3%), Respiratory syncytial virus (4%), and Influenza A virus (2%). Conversely, Influenza A virus was identified in 73% of non-pneumonia patients. Community-acquired influenza was more common (80%) than nosocomial influenza (20%). The ODIN score was a good predictor of mortality and the new electronic surveillance tool was an effective prototype to monitor patients in acute care, especially during influenza season. The results of this study provided baseline data on respiratory illness surveillance and demonstrated that future research, including prospective studies, is warranted in acute care facilities.

Étude sur les déterminants psychosociaux de la vaccination contre le virus A(H1N1) auprès des parents d’enfants qui fréquentent des services de garde éducatifs de Montréal

Introduction En juin 2009, l’Organisation Mondiale de la Santé (OMS) a déclaré l’état de pandémie pour le nouveau virus influenza A(H1N1). Malgré les recommandations des autorités de santé publique, lors de la mise en place de la campagne de vaccination de masse au Québec contre ce virus pandémique, certains groupes de la population ont été plus enclins à être vaccinés que d’autres groupes. Ceci souligne l’importance des déterminants psychosociaux du comportement humain, sujet qui a donné cadre à notre étude. Objectifs Le but de cette étude a été de documenter les attitudes, les connaissances et les influences sociales des parents dont les enfants fréquentent des services de garde éducatifs (SGE) vis-à-vis la vaccination contre la pandémie ainsi que la couverture vaccinale. Méthodologie Un questionnaire auto-administré et anonyme basé sur la théorie des comportements interpersonnels de Triandis a été distribué aux parents d’enfants âgés de 0-59 mois de neuf centres de la petite enfance sur l’île de Montréal. Résultats Le taux de réponse de l’enquête a été de 32,0% (N=185). Le taux de vaccination des enfants s’est retrouvé à 83,4%; ceci représente une couverture plus élevée que la moyenne régionale et nationale. Toutefois, à une question sur l’intention des parents face à une autre pandémie, seuls 46% des parents feraient vacciner leur enfant. Les facteurs les plus significatifs associés à la vaccination de leur enfant ont été les croyances personnelles positives, de bonnes habitudes vaccinales et l’influence des média, tous mesurés par plusieurs indicateurs (RC respectifs de 7,7, 3,1 et 4,2, p<0,05). Conclusion Pour la grippe A(H1N1), plusieurs facteurs contextuels ont joué en faveur des taux de vaccination acceptables chez les enfants. Toutefois, la mise en place d’une campagne de la vaccination par les instances gouvernementales et de santé publique en utilisant divers média pourraient contribuer à un taux de vaccination encore plus élevé en cas d’épidémie ou de pandémie.

Generation of candidate human influenza vaccine strains in cell culture: rehearsing the European response to an H7N1 pandemic threat

Background: Although H5N1 avian influenza viruses pose the most obvious imminent pandemic threat, there have been several recent zoonotic incidents involving transmission of H7 viruses to humans. Vaccines are the primary public health defense against pandemics, but reliance on embryonated chickens eggs to propagate vaccine and logistic problems posed by the use of new technology may slow our ability to respond rapidly in a pandemic situation. Objectives: We sought to generate an H7 candidate vaccine virus suitable for administration to humans whose generation and amplification avoided the use of eggs. Methods: We generated a suitable H7 vaccine virus by reverse genetics. This virus, known as RD3, comprises the internal genes of A/Puerto Rico/8/34 with surface antigens of the highly pathogenic avian strain A/Chicken/Italy/13474/99 (H7N1). The multi-basic amino acid site in the HA gene, associated with high pathogenicity in chickens, was removed. Results: The HA modification did not alter the antigenicity of the virus and the resultant single basic motif was stably retained following several passages in Vero and PER. C6 cells. RD3 was attenuated for growth in embryonated eggs, chickens, and ferrets. RD3 induced an antibody response in infected animals reactive against both the homologous virus and other H7 influenza viruses associated with recent infection by H7 viruses in humans. Conclusions: This is the first report of a candidate H7 vaccine virus for use in humans generated by reverse genetics and propagated entirely in mammalian tissue culture. The vaccine has potential use against a wide range of H7 strains.

Variation in the ability of human influenza A viruses to induce and inhibit the IFN-beta pathway

We investigated the ability of a selection of human influenza A viruses, including recent clinical isolates, to induce IFN-beta production in cultured cell lines. In contrast to the well-characterized laboratory strain A/PR/8/34, several, but not all, recent isolates of H3N2 viruses resulted in moderate IFN-beta stimulation. Through the generation of recombinant viruses, we were able to show that this is not due to a loss of the ability of the NS1 genes to suppress IFN-beta induction; indeed, the NS1 genes behaved similarly with respect to their abilities to block dsRNA signaling. Interestingly, replication of A/Sydney/5/97 virus was less Susceptible to pre-treatment with IFN-alpha than the other viruses. In contrast to the universal effect on dsRNA signaling, we noted differences in the effect of NS1 proteins on expression of interferon stimulated genes and also genes induced by a distinct pathway. The majority of NS1 proteins blocked expression From both IFN-dependent and TNF-dependent promoters by an apparent post-transcriptional mechanism. The NS1 gene of A/PR/8/34 NS1 did not confer these blocks. We noted striking differences in the Cellular localization of different influenza A virus NS1 proteins during infection, which might explain differences in biological activity. (C) 2005 Elsevier Inc. All rights reserved.

Characterization of recombinant influenza B viruses with key neuraminidase inhibitor resistance mutations

Objectives and methods: An influenza B virus plasmid-based rescue system was used to introduce site-specific mutations, previously observed in neuraminidase (NA) inhibitor-resistant viruses, into the NA protein of six recombinant viruses. Three mutations observed only among in vitro selected zanamivir-resistant influenza A mutants were introduced into the B/Beijing/1/87 virus NA protein, to change residue E116 to glycine, alanine or aspartic acid. Residue E116 was also mutated to valine, a mutation found in the clinic among oseltamivir-resistant viruses. An arginine to lysine change at position 291 (292 N2 numbering) mimicked that seen frequently in influenza A N2 clinical isolates resistant to oseltamivir. Similarly, an arginine to lysine change at position 149 (152 in N2 numbering) was made to reproduce the change found in the only reported zanamivir-resistant clinical isolate of influenza B virus. In vitro selection and prolonged treatment in the clinic leads to resistance pathways that require compensatory mutations in the haemagglutinin gene, but these appear not to be important for mutants isolated from immunocompetent patients. The reverse genetics system was therefore used to generate mutants containing only the NA mutation. Results and conclusions: With the exception of a virus containing the E116G mutation, mutant viruses were attenuated to different levels in comparison with wild-type virus. This attenuation was a result of altered NA activity or stability depending on the introduced mutation. Mutant viruses displayed increased resistance to zanamivir, oseltamivir and peramivir, with certain viruses displaying cross-resistance to all three drugs.