Bozena Jaentschke

Qualitative and quantitative analyses of virtually all subtypes of influenza A and B viral neuraminidases using antibodies targeting the universally conserved sequences.

Neuraminidase-induced immune responses are correlated with protection of humans and animals from influenza. However, the amounts of neuraminidase in influenza vaccines are yet to be standardized. Thus, a simple method capable of quantifying neuraminidase would be desirable. Here we identified two universally conserved sequences in all influenza A and B neuraminidases, one representing a novel finding of nearly 100% conservation near the enzymatically active site. Antibodies generated against the two highly conserved sequences bound to all nine subtypes of influenza A neuraminidase and demonstrated remarkable specificity against the viral neuraminidase sequences without any cross-reactivity with allantoic and cellular proteins. Importantly, employing these antibodies for the analyses of vaccines from eight manufacturers using the same vaccine seeds revealed marked variations of neuraminidase levels in addition to considerable differences between lots from the same producer. The reasons for the absence or low level of neuraminidase in vaccine preparations are complex and could be multi-factorial. The antibody-based assays reported here could be of practical value for better vaccine quality control.

A simple slot blot for the detection of virtually all subtypes of the influenza A viral hemagglutinins using universal antibodies targeting the fusion peptide

The fusion peptide of influenza virus hemagglutinin (HA) has a critical role in mediating the entry of the virus into the cells and is also the only universally conserved sequence in the HAs of all strains of influenza A and influenza B viruses. Therefore, it could be an attractive target for new vaccine development and a potency marker for existing influenza vaccines. The fusion peptide epitope is hidden inside the HA proteins, making it inaccessible for quantitative antibody binding. Our simple slot blot protocol highlights pre-treatment of HA samples with moderate concentrations of denaturant to maximally expose the fusion peptide on the protein surface, followed by detection using universal antibodies targeting the fusion peptide. The method is highly reliable, inexpensive and easy to follow. The entire procedure takes only 5 h and can be applied to the quantitative determination of virtually all influenza viral HAs using a single antibody targeting the fusion peptide.

Significant alterations of biodistribution and immune responses in Balb/c mice administered with adenovirus targeted to CD40(+) cells

CD40 ligation has been shown to promote antigen-presenting functions of dendritic cells, which express CD40 receptor. Here we reported significantly altered biodistribution and immune responses with the use of CD40-targeted adenovirus. Compared with unmodified adenovirus 5, the CD40-targeted adenovirus following intravenous administration (i.v.) resulted in increased transgene expressions in the lung and thymus, which normally do not take up significant amounts of adenovirus. Intradermal injection saw modified adenovirus being mainly processed in local draining lymph nodes and skin. Following intranasal administration (i.n.), neither unmodified nor targeted viruses were found to be in the liver or spleen, which predominantly took up the virus following i.v. administration. However, inadvertent infection of the brain was found with unmodified adenoviruses, with the second highest gene expression among 14 tissues examined. Importantly, such undesirable effects were largely ablated with the use of targeted vector. Moreover, the targeted adenovirus elicited more sustained antigen-specific cellular immune responses (up to 17-fold) at later time points (30 days post boosting), but also significantly hampered humoral responses irrespective of administration routes. Additional data suggest the skewed immune responses induced by the targeted adenoviruses were not due to the identity of the transgene but more likely a combination of overall transgene load and CD40 stimulation.

Quantitative analyses of all influenza type A viral hemagglutinins and neuraminidases using universal antibodies in simple slot blot assays.

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses(1,2). As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)(3). However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity(4), the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method(5). Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines. Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses(6-7). One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide(6), while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)(7). Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera(7,8). Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

CD40 Ligand Preferentially Modulates Immune Response and Enhances Protection against Influenza Virus

CD40L, a key regulator of the immune system, was studied as both a targeting ligand and a molecular adjuvant in nucleoprotein (NP)-based host defense against influenza in mouse models with different genetic backgrounds. Adenoviral vectors secreting NP-CD40L fusion protein (denoted as rAd-SNP40L) afforded full protection of immunocompetent and immunocompromised mice (CD40L−/− and CD4−/−) against lethal influenza infection. Mechanistically, rAd-SNP40L preferentially induced early and persistent B cell germinal center formation, and accelerated Ig isotype-switching and Th1-skewed, NP-specific Ab response. Moreover, it drastically augmented primary and memory NP-specific CTL activity and polyfunctional CD8+ T cells. The markedly enhanced nonneutralizing Abs and CTLs significantly reduced viral burdens in the lungs of mice upon lethal virus challenge. Data generated from CD40L−/− and CD4−/− mice revealed that the protection was indeed CD40L mediated but CD4+ T cell independent, demonstrating the viability of the fusion Ags in protecting immunodeficient hosts. Notably, a single dose of rAd-SNP40L completely protected mice from lethal viral challenge 4 mo after immunization, representing the first report, to our knowledge, on NP in conjunction with a molecular adjuvant inducing a robust and long-lasting memory immune response against influenza. This platform is characterized by an increased in vivo load of CD40-targeted Ag upon the secretion of the fusion protein from adenovirus-infected cells and may represent a promising strategy to enhance the breadth, durability, and potency of Ag-specific immune responses.

The Universal Epitope of Influenza A Viral Neuraminidase Fundamentally Contributes to Enzyme Activity and Viral Replication

Background: The influenza viral neuraminidase has only one universally conserved peptide sequence with unknown function. Results: Sequence alterations in this region decrease substrate binding, enzymatic activity, protein stability, and viral growth. Conclusion: The universal epitope is indispensable for maximal enzymatic function and robust viral propagation. Significance: The universally conserved NA sequence is an attractive target for antiviral intervention and vaccine development. The only universally conserved sequence among all influenza A viral neuraminidases is located between amino acids 222 and 230. However, the potential roles of these amino acids remain largely unknown. Through an array of experimental approaches including mutagenesis, reverse genetics, and growth kinetics, we found that this sequence could markedly affect viral replication. Additional experiments revealed that enzymes with mutations in this region demonstrated substantially decreased catalytic activity, substrate binding, and thermostability. Consistent with viral replication analyses and enzymatic studies, protein modeling suggests that these amino acids could either directly bind to the substrate or contribute to the formation of the active site in the enzyme. Collectively, these findings reveal the essential role of this unique region in enzyme function and viral growth, which provides the basis for evaluating the validity of this sequence as a potential target for antiviral intervention and vaccine development.

Subcutaneous immunization with recombinant adenovirus expressing influenza A nucleoprotein protects mice against lethal viral challenge

Current influenza vaccines mainly induce strain-specific neutralizing antibodies and need to be updated each year, resulting in significant burdens on vaccine manufacturers and regulatory agencies. Genetic immunization strategies based on the highly conserved nucleoprotein (NP) of influenza have attracted great attention as NP could induce heterosubtypic immunity. It is unclear, however, whether different forms of vectors and/or vaccination regimens could have contributed to the previously reported discrepancies in the magnitude of protection of NP-based genetic vaccinations. Here, we evaluated a plasmid DNA vector (pNP) and a recombinant adenovirus vector (rAd-NP) containing the NP gene through various combinations of immunization regimens in mice. We found that pNP afforded only partial protection even after 4 injections, with full protection against lethal challenge achieved only with the fourth boost using rAd-NP. Alternatively, only two doses of rAd-NP delivered subcutaneously were needed to induce an enhanced immune response and completely protect the animals, a finding which, to our knowledge, has not been reported before.

SARS coronavirus: Unusual lability of the nucleocapsid protein

Abstract The severe acute respiratory syndrome (SARS) is a contagious disease that killed hundreds and sickened thousands of people worldwide between November 2002 and July 2003. The nucleocapsid (N) protein of the coronavirus responsible for this disease plays a critical role in viral assembly and maturation and is of particular interest because of its potential as an antiviral target or vaccine candidate. Refolding of SARS N-protein during production and purification showed the presence of two additional protein bands by SDS–PAGE. Mass spectroscopy (MALDI, SELDI, and LC/MS) confirmed that the bands are proteolytic products of N-protein and the cleavage sites are four SR motifs in the serine–arginine-rich region—sites not suggestive of any known protease. Furthermore, results of subsequent testing for contaminating protease(s) were negative: cleavage appears to be due to inherent instability and/or autolysis. The importance of N-protein proteolysis to viral life cycle and thus to possible treatment directions are discussed.

Development and applications of universal H7 subtype-specific antibodies for the analysis of influenza H7N9 vaccines

H7N9 is a newly emerged avian influenza virus with a relatively high mortality rate in humans. At this time, there is no licensed vaccine for human protection. Development of analytical tools for H7N9 vaccine could facilitate vaccine development. Here, a universally conserved epitope in all H7 hemagglutinin (HA) sequences was identified through comprehensive bioinformatics analyses. The peptide epitope, RSGSSFYAEMK, (aa positions 149 to 159), is located on the head of the HA molecule. Antibodies generated against this universal H7 epitope were remarkably specific against H7 viral sequence with no detectable cross-reactivity to other HA subtypes. A new immunoblotting assay based on the universal H7 antibody was developed and compared with the traditional single radial immunodiffusion assay (SRID) for potency analyses of candidate H7N9 vaccines. This new assay was more sensitive and rapid compared to SRID. In addition to statistically acceptable precision and reproducibility, the new assay differs from many other alternative potency assays for influenza vaccine in that it is potentially stability-indicating, which is an important requirement for industry vaccine stability studies analyses. Furthermore, the robustness of this new assay was demonstrated by the quantitative determination of HA content in four H7N9 vaccines (split or inactivated) from different manufacturers.

Cytotoxic Doses of Ketoconazole Affect Expression of a Subset of Hepatic Genes

Ketoconazole is a widely prescribed antifungal drug, which has also been investigated as an anticancer therapy in both clinical and pre-clinical settings. However, severe hepatic injuries were reported to be associated with the use of ketoconazole, even in patients routinely monitored for their liver functions. Several questions concerning ketoconazole-induced hepatic injury remain unanswered, including (1) does ketoconazole alter cytochrome P450 expression at the transcriptional level?, (2) what types of gene products responsible for cytotoxicity are induced by ketoconazole?, and (3) what role do the major metabolites of ketoconazole play in this pathophysiologic process? A mouse model was employed to investigate hepatic gene expression following hepatotoxic doses of ketoconazole. Hepatic gene expression was analyzed using a toxicogenomic microarray platform, which is comprised of cDNA probes generated from livers exposed to various hepatoxicants. These hepatoxicants fall into five well-studied toxicological categories: peroxisome proliferators, aryl hydrocarbon receptor agonists, noncoplanar polychlorinated biphenyls, inflammatory agents, and hypoxia-inducing agents. Nine genes encoding enzymes involved in Phase I metabolism and one Phase II enzyme (glutathione S-transferase) were found to be upregulated. Serum amyloid A (SAA1/2) and hepcidin were the only genes that were downregulated among the 2364 genes assessed. In vitro cytotoxicity and transcription analyses revealed that SAA and hepcidin are associated with the general toxicity of ketoconazole, and might be usefully explored as generalized surrogate markers of xenobiotic-induced hepatic injury. Finally, it was shown that the primary metabolite of ketoconazole (de-N-acetyl ketoconazole) is largely responsible for the hepatoxicity and the downregulation of SAA and hepcidin.