Amounts Of Neuraminidase Research Articles

Influenza a Neuraminidase-Based Bivalent mRNA Vaccine Induces Th1-Type Immune Response and Provides Protective Effects in Mice.

Vaccines are one of the most effective means of preventing influenza A, typically containing the hemagglutinin (HA) of the influenza A virus. However, antigenic drift and shift of the influenza A virus can lead to instability in vaccine efficacy. Compared to HA, the antigenic variation rate of neuraminidase (NA) is slower. In traditional inactivated influenza vaccines, although they contain a certain amount of NA, there are significant differences between different batches, which cannot consistently induce NA-based immune responses. Therefore, NA is often overlooked in vaccine development. In this study, we report an mRNA vaccine encoding the NA of two strains of influenza A virus. The experimental results demonstrated that when matched with the viral strain, this mRNA vaccine induced high levels of neutralizing antibodies, providing a protective effect to mice in viral challenge experiments, and this immune response was shown to be biased towards the Th1 type. In summary, this study demonstrates that NA is a promising potential antigen, providing new insights for the development of influenza A virus vaccines.

Broadly Protective Neuraminidase-Based Influenza Vaccines and Monoclonal Antibodies: Target Epitopes and Mechanisms of Action.

Neuraminidase (NA) is an important surface protein on influenza virions, playing an essential role in the viral life cycle and being a key target of the immune system. Despite the importance of NA-based immunity, current vaccines are focused on the hemagglutinin (HA) protein as the target for protective antibodies, and the amount of NA is not standardized in virion-based vaccines. Antibodies targeting NA are predominantly protective, reducing infection severity and viral shedding. Recently, NA-specific monoclonal antibodies have been characterized, and their target epitopes have been identified. This review summarizes the characteristics of NA, NA-specific antibodies, the mechanism of NA inhibition, and the recent efforts towards developing NA-based and NA-incorporating influenza vaccines.

A practical and rapid screening method for influenza virus neuraminidase inhibitors based on fluorescence detection.

A new analytical method for rapid screening of influenza virus neuraminidase inhibitors was established. The method is based on the principle that, given a certain amount of neuraminidase, the sample and the neuraminidase act in the microplate for a period of time, and the active neuraminidase that is not inhibited by the sample can generate a fluorescence value at a specific wavelength after binding to the substrate, and the rate of inhibition of neuraminidase by the sample can be calculated based on the actual detected fluorescence value. This newly developed method was used to screen and evaluate the in vitro anti-neuraminidase activity of 39 high-purity compounds contained in three traditional Chinese herbal medicines, and finally 25 compounds with strong activity were obtained. The newly established neuraminidase inhibitor analytical method has these advantages of practicality, rapidity, high sensitivity and low cost, and has a good value for promotion and application. This article newly establishes a rapid, sensitive, simple and practical screening method for influenza virus neuraminidase inhibitors, which is a great complement to the existing methods and has a good promotion and application value.

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 responses1,2. 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 immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. 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 analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, 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 antisera7,8. 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.

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.

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.

Virus–receptor interactions of human parainfluenza viruses types 1, 2 and 3

Human parainfluenza viruses types 1, 2 and 3 (HPF 1, 2 and 3) are important pathogens in children. While these viruses share common structures and replication strategies, they target different parts of the respiratory tract; the most common outcomes of infection with HPF3 are bronchiolitis and pneumonia, while HPF 1 and 2 are associated with croup. While the HPF3 fusion protein (F) is critical for membrane fusion, our previous work revealed that the receptor binding hemagglutinin–neuraminidase (HN) is also essential to the fusion process; interaction between HN and its sialic acid-containing receptor on cell surfaces is required for HPF3 mediated cell fusion. Using our understanding of HPF3 HN's functions in the cell-binding and viral entry process, we are investigating the ways in which these processes differ in HPF 1 and 2, in part by manipulating receptor availability. Three experimental treatments were used to compare the HN–receptor interaction of HPF 1, 2 and 3: infection at high multiplicity of infection (m.o.i.); bacterial neuraminidase treatment of cells infected at low m.o.i.; and viral neuraminidase treatment of cells infected at low m.o.i. (using Newcastle disease virus [NDV] neuraminidase or UV irradiated HPF3 as sources of neuraminidase). In cells infected with HPF3, we have shown that infection with high m.o.i. blocks fusion, by removing sialic acid receptors for the viral HN. However, in cells infected with HPF 1 and 2, infection with high m.o.i. did not block fusion; the fusion increases with increasing m.o.i.. In cells infected with HPF 1 and 2, neither bacterial nor NDV neuraminidase blocked cell fusion, using amounts of neuraminidase that completely block fusion of HPF3 infected cells. However, when inactivated HPF3 was used as a source of viral neuraminidase, the treatment inhibited fusion of cells infected with HPF 1 and 2 as well as 3. The differences found between these viruses in terms of their interaction with the cell, ability to modulate cell–cell fusion and reponse to exogenous neuraminidases of various specificities, may reflect salient differences in biological properties of the three viruses.

A sensitive and specific ELISA immunocapture assay for rapid quantitation of influenza A/H3N2 neuraminidase protein

Both HA and NA proteins elicit antibodies which have been shown to be capable of altering the course of infection. Nevertheless, while influenza virus vaccine standardization involves hemagglutinin (HA) and neuraminidase (NA) in terms of antigenic characterization, only HA protein quantitation is undertaken. An immunocapture ELISA (EIA) is described for N2 NA quantitation, based on the use of a highly specific monoclonal antibody (MAb) for capturing NA and an anti-NA antiserum for antigen detection. The amounts of NA in samples were deduced from the standard curve established by using purified NA. The NA-EIA is specific and detects as a little as 7 ng/ml. The capture and detector antibodies directed against A/Beijing/32/92 NA were shown to react with H3N2 prototype strains used in current influenza vaccines, provided that an antigenically matched reference NA is used as standard.

Immunotherapy of neoplastic diseases with neuraminidase: Contradictions, new aspects, and revised concepts

The divergent experimental results in immunotherapy of spontaneous, chemically induced or virus-induced solid tumors or leukemias with neuraminidase are reviewed and analyzed under the various aspects of the possible modes and conditions of action of the enzyme: Immunocompetence of the host, animal residual tumor volume, enzymatic activity of the neuraminidase, and identity of the antigenic specificity within the tumor system are well-known prerequisites for an effective tumor immunotherapy. In addition, there seems to be evidence that the number of tumor cells used for vaccination and the dose of enzymatically active VCN, whether bound to VCN-treated tumor cells or injected intratumorally, may be decisive in the negative or positive outcome. Moreover, there are indications that a preexistent sensitization against the so-called Thomsen-Friedenreich antigen, which seems to be unmasked after VCN treatment of cells, may influence the tumor therapeutic success. The effect of nonspecific immunostimulators given in addition to neuraminidase or to neuraminidase-treated cells is controversial. Thus, this combination cannot be recommended unless it is fully explored. To overcome the problem of the dependence of the tumor therapeutic effect on the dose of cells and the amount of neuraminidase with respect to different tumors and different adjuvant treatments, a new immunization concept, named ‘chessboard vaccination’, has been proposed. The data obtained so far in vitro and in vivo with this chessboard vaccination are briefly reviewed. They show that chessboard vaccination might be of diagnostic as well as of therapeutic interest.

<i>In vivo </i>Behaviour of Neuraminidase-Treated Rabbit Erythrocytes and Reticulocytes

51Cr rabbit erythrocytes were treated with different amount of neuraminidase and reinjected into the animal. The survival curves after the removal of more than 50% membrane sialic acid show a characteristic behaviour: after a rapid decrease, blood radioactivity increases again reaching a maximum level 50-80 h after reinjection, then tends to decrease with a slope similar to that of control curves. Liver radioactivity determined before the rise of blood radioactivity is evidently higher than the value determined after radioactivity elevation. Similar results were obtained with phenylhydrazine-induced young erythrocytes.

Isolation and characterization of influenza virus recombinants with high and low neuraminidase activity: Use of 2-(3′-methoxyphenyl)- N-acetylneuraminic acid to identify cloned populations

Two antigenically identical HON2 influenza virus recombinants derived from A/England/42/72 (H3N2) and A/PR8/34 (HON1) were separated from a mixed population by the use of a staining procedure specific for neuraminidase. This procedure employing an artificial neuraminidase substrate (2-(3′-methoxyphenyl)- N-acetylneuraminic acid, MPN) permitted the identification of deeply stained (MPN +) and poorly stained (MPN −) clones of virus in clone 1-5C-4 cells. Upon isolation and purification of the 2 variants, they were found to have eightfold differences in the ratios of neuraminidase/hemagglutination activity. Analysis by polyacrylamide gel electrophoresis demonstrated that the differences in neuraminidase activity reflected differences in the amount of neuraminidase per virion. The MPN − variant which had low neuraminidase content replicated to equal titer in eggs and formed larger plaques than MPN + variant on clone 1-5C-4 cells. Attempts to isolate clones of MPN − virus from wild type A/England/42/72 virus have not been successful so far.