Self-amplifying mRNA seasonal influenza vaccines elicit mouse neutralizing antibody and cell-mediated immunity and protect ferrets.

Currently licensed influenza vaccines focus immune responses on viral hemagglutinin (HA), while the other major surface glycoprotein neuraminidase (NA) is not tightly controlled in inactivated vaccine formulations despite evidence that anti-NA antibodies reduce clinical disease. We utilized a bicistronic self-amplifying mRNA (sa-mRNA) platform encoding both HA and NA from four seasonal influenza strains, creating a quadrivalent influenza vaccine. sa-mRNA vaccines encoding an NA component induced the production of NA-inhibiting antibodies and CD4+ T-cell responses in both monovalent and quadrivalent formulations. Including NA in the vaccine enabled cross-neutralization against antigenically drifted strains and provided greater protection than HA alone upon A(H3N2) challenge in ferrets. These results demonstrate that next-generation bicistronic sa-mRNA vaccines expressing HA and NA induce potent antibodies against both viral coat proteins, as well as vaccine-specific cell-mediated immunity. When formulated as a quadrivalent seasonal influenza vaccine, the sa-mRNA platform provides an opportunity to increase the breadth of protection through cross-neutralizing anti-NA antibodies.

Erratum: Self-amplifying mRNA bicistronic influenza vaccines raise cross-reactive immune responses in mice and prevent infection in ferrets.

[This corrects the article DOI: 10.1016/j.omtm.2022.09.013.].

Self-amplifying mRNA bicistronic influenza vaccines raise cross-reactive immune responses in mice and prevent infection in ferrets.

Vaccines are the primary intervention against influenza. Currently licensed inactivated vaccines focus immunity on viral hemagglutinin (HA). Self-amplifying mRNA (sa-mRNA) vaccines offer an opportunity to generate immunity to multiple viral proteins, including additional neuraminidase (NA). This evaluation of a bicistronic approach for sa-mRNA vaccine development compared subgenomic promoter and internal ribosome entry site strategies and found consistent and balanced expression of both HA and NA proteins in transfected cells. In mice, sa-mRNA bicistronic A/H5N1 vaccines raised potent anti-HA and anti-NA neutralizing antibody responses and HA- or NA-specific CD4+ and CD8+ T cell responses. The addition of NA also boosted the cross-neutralizing response to heterologous A/H1N1. Similar immunogenicity results were obtained for bicistronic seasonal A/H3N2 and B/Yamagata vaccines. In ferrets, sa-mRNA bicistronic A/H1N1 vaccine fully protected lung from infection by homologous virus and showed significant reduction of viral load in upper respiratory tract, warranting further evaluation of sa-mRNA bicistronic vaccine in humans.

Self-amplifying mRNA SARS-CoV-2 vaccines raise cross-reactive immune response to variants and prevent infection in animal models.

The spike (S) protein of SARS-CoV-2 plays a crucial role in cell entry, and the nucleocapsid (N) protein is highly conserved among human coronavirus homologs. For potentially broad effectiveness against both original virus and emerging variants, we developed Alphavirus-based self-amplifying mRNA (sa-mRNA) SARS-CoV-2 vaccines: an sa-mRNA S encoding a full-length S protein stabilized in a prefusion conformation and an sa-mRNA S-N co-expressing S and N proteins for the original virus. We show that these sa-mRNA SARS-CoV-2 vaccines raised potent neutralizing antibody responses in mice against not only the original virus but also the Alpha, Beta, Gamma, and Delta variants. sa-mRNA S vaccines against the Alpha and Beta variants also raised robust cross-reactive neutralizing antibody responses against their homologous viruses and heterologous variants. sa-mRNA S and sa-mRNA S-N vaccines elicited Th1-dominant, antigen-specific CD4+ T cell responses to S and N proteins and robust and broad CD8+ T cell responses to S protein. Hamsters immunized with either vaccine were fully protected from lung infection and showed significant reduction of viral load in upper respiratory tract. Our findings demonstrate that sa-mRNA SARS-CoV-2 vaccines are potent in animal models with potential to be highly effective against SARS-CoV-2 infection in humans.

Improved immunologic responses to heterologous influenza strains in children with low preexisting antibody response vaccinated with MF59-adjuvanted influenza vaccine.

Vaccination is the most effective approach to reduce the substantial morbidity and mortality caused by influenza infection. Vaccine efficacy is highly sensitive to antigenic changes causing differences between circulating and vaccine viruses. Adjuvants such as MF59 increase antibody-mediated cross-reactive immunity and therefore may provide broader seasonal protection. A recent clinical trial showed that an MF59-adjuvanted vaccine was more efficacious than a nonadjuvanted comparator in subjects < 2 years of age, although not in those ≥ 2 years, during influenza seasons in which the predominant circulating virus was an A/H3N2 strain that was antigenically different from the vaccine virus. This finding suggested that the increased efficacy of the adjuvanted vaccine in younger subjects may be mediated by strain cross-reactive antibodies. A subset of the trial population, representing subjects with distinct age and/or immunological history, was tested for antibody responses to the vaccine A/H3N2 strain as well as A/H3N2 drifted strains antigenically matching the viruses circulating during the trial seasons. The neutralizing tests showed that, compared with nonadjuvanted vaccine, the adjuvanted vaccine improved not only the neutralizing antibody response to the vaccine strain but also the cross-reactive antibody response to the drifted strains in subjects with lower preexisting antibody titers, regardless of their age or vaccine history. The results demonstrated an immunological benefit and suggested a potential efficacy benefit by adjuvanted vaccine in subjects with lower preexisting antibody responses.

Novel Surrogate Neutralizing Assay Supports Parvovirus B19 Vaccine Development for Children with Sickle Cell Disease.

Children with sickle cell disease (SCD) suffer life-threatening transient aplastic crisis (TAC) when infected with parvovirus B19. In utero, infection of healthy fetuses may result in anemia, hydrops, and death. Unfortunately, although promising vaccine candidates exist, no product has yet been licensed. One barrier to vaccine development has been the lack of a cost-effective, standardized parvovirus B19 neutralization assay. To fill this void, we evaluated the unique region of VP1 (VP1u), which contains prominent targets of neutralizing antibodies. We discovered an antigenic cross-reactivity between VP1 and VP2 that, at first, thwarted the development of a surrogate neutralization assay. We overcame the cross-reactivity by designing a mutated VP1u (VP1uAT) fragment. A new VP1uAT ELISA yielded results well correlated with neutralization (Spearman's correlation coefficient = 0.581; p = 0.001), superior to results from a standard clinical diagnostic ELISA or an ELISA with virus-like particles. Virus-specific antibodies from children with TAC, measured by the VP1uAT and neutralization assays, but not other assays, gradually increased from days 0 to 120 post-hospitalization. We propose that this novel and technically simple VP1uAT ELISA might now serve as a surrogate for the neutralization assay to support rapid development of a parvovirus B19 vaccine.

Limited Tryptic Digestion-Isotope Dilution Mass Spectrometry (LTD-IDMS): A Reagent-Free Analytical Assay To Quantify Hemagglutinin of A(H5N1) Vaccine Material.

Avian influenza viruses, such as A(H5N1) and A(H7N9), are primary public health concerns due to their pandemic potential. Influenza vaccines represent the most effective response to this threat especially with timely provision. The current pandemic response timelines require a substantial period for strain-specific reference antigen and sera preparation for use with single-radial immunodiffusion (SRID), the accepted vaccine potency assay. To address this time lag, the isotope dilution mass spectrometry (IDMS) method was developed to quantify the absolute hemagglutinin (HA, the main influenza antigen) amount in the vaccine without the need for purified, inactivated, and calibrated virus reference antigens. However, an additional challenge in determining potency is to differentiate between vaccine antigens in their most potent form from other less potent, stressed antigen forms. The limited trypsin digestion (LTD) method has been developed and does not require strain-specific full-length reference antigens or antibodies; instead, stressed HA is selectively degraded, leaving the more potent form to be measured. LTD, followed by precipitation and IDMS, allows for efficient differentiation between potent and significantly less potent HA for vaccine release and potency testing across the vaccine's shelf life. In this study, we tested the LTD-IDMS assay on A(H5N1) vaccine material that had been stressed by low pH, heat, and multiple freeze-thaw cycles. The results showed that the LTD-IDMS method effectively quantified the potent HA in A(H5N1) vaccine material with results comparable to SRID. As such, it shows great promise to complement and potentially replace SRID in a pandemic when strain-specific reagents may not be readily available.

Reference antigen-free and antibody-free LTD-IDMS assay for influenza H7N9 vaccine in vitro potency determination.

Influenza vaccines are the most effective intervention to prevent the substantial public health burden of seasonal and pandemic influenza. Hemagglutinin (HA), as the main antigen in inactivated influenza vaccines (IIVs), elicits functional neutralizing antibodies and largely determines IIV effectiveness. HA potency has been evaluated by single-radial immunodiffusion (SRID), the standard in vitro potency assay for IIVs, to predict vaccine immunogenicity with a correlation to protective efficacy. We previously reported that limited trypsin digestion (LTD) selectively degraded stressed HA, so that an otherwise conformationally insensitive biophysical quantification technique could specifically quantify trypsin-resistant, immunologically active HA. Here, we demonstrate that isotope dilution mass spectrometry (IDMS), a method capable of quantifying the absolute HA concentration without reference antigen use, can be further expanded by adding LTD followed with precipitation to selectively quantify the active HA. We test the LTD-IDMS assay on H7N9 vaccines stressed by low pH, raised temperature, or freeze/thaw cycles. This method, unlike SRID, has no requirement for strain-specific reference antigens or antibodies and can generate potency values that correlate with SRID. Thus, LTD-IDMS is a promising alternative in vitro potency assay for influenza vaccines to complement and potentially replace SRID in a pandemic when strain specific reagents may not be readily available.

Intra-seasonal antibody repertoire analysis of a subject immunized with an MF59®-adjuvanted pandemic 2009 H1N1 vaccine.

During the height of the 2009 H1N1 swine-derived influenza pandemic, a clinical trial was conducted in which seven subjects were immunized using a monovalent, MF59®-adjuvanted vaccine, developed from an egg-passaged candidate vaccine virus (CVV), A/California/07/2009 X-181. Whole blood was collected prior to immunization and at 8, 22, and 202 days post-vaccination, and subjects' serological responses were evaluated. Here, we reconstruct and examine the longitudinal, influenza-specific circulating B cell repertoire of one subject in that study. Genotypic analysis of 390 total subject-derived antibodies (Abs) revealed a total of 29 germline genes in use among immunoglobulin heavy chain variable regions (IgHV), with the majority of those sequences isolated representing memory recall responses and two major lineages dominating the early response. In vitro phenotyping showed a diverse set of binding epitopes on the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), many of which are considered subdominant. Strong correlations were found between IgHV germline usage among non-related lineages and both binding epitope and neutralization breadth. Results here highlight the potential for Ab responses to be misdirected to egg-adaptive artifacts on CVVs while simultaneously stressing the ability to mount potent, broadly neutralizing responses to mostly novel antigens via recall of subdominant memory responses, as well as the need for evaluating alternative endpoint assays and anti-NA responses following clinical trials.

Inactivated influenza vaccine stress can affect in vitro potency assay relationship to immunogenicity.

Influenza vaccines are the most effective intervention to prevent the substantial public health burden of seasonal and pandemic influenza. The capability of hemagglutinin (HA), the main antigen in inactivated influenza vaccines (IIVs), to elicit functional neutralizing antibodies determines IIV effectiveness. When HA is subjected to environmental stress during manufacturing or while stored prior to administration, such as low pH and temperature excursions, the HA immunological activity can be affected. Single-radial immunodiffusion (SRID), the standard in vitro potency assay for IIVs, is believed to specifically detect immunologically active HA and has been applied to evaluate HA stability against stress. Here we report that transient low pH treatment and freeze/thaw cycles with HA in PBS abolish SRID-quantified in vitro potency for all HAs of multiple influenza strains. Raised temperature substantially decreases in vitro potency with more extensive HA structural changes. Chemical stress and mechanical stress moderately change SRID in vitro potency values in a strain-dependent manner. Trypsin digestion, which selectively degrades stressed HA, followed by RP-HPLC quantification as a candidate alternative in vitro potency assay yields results comparable to SRID. Mouse immunogenicity studies confirm that HA stressed by transient low pH treatment does not elicit functional antibodies in vivo, nor does it have a measureable SRID value. However, HA stressed by raised temperature elicits high titers of functional antibodies in vivo despite substantial loss of SRID in vitro potency. This discrepancy between SRID in vitro potency and vaccine immunogenicity suggests that SRID may not reliably indicate IIV potency under all conditions. Further efforts to develop alternate potency assays that can better predict in vivo immunogenicity should continue along with additional studies exploring HA conformation, SRID values and consequent immunogenicity.

Conserved epitope on influenza-virus hemagglutinin head defined by a vaccine-induced antibody.

Circulating influenza viruses evade neutralization in their human hosts by acquiring escape mutations at epitopes of prevalent antibodies. A goal for next-generation influenza vaccines is to reduce escape likelihood by selectively eliciting antibodies recognizing conserved surfaces on the viral hemagglutinin (HA). The receptor-binding site (RBS) on the HA "head" and a region near the fusion peptide on the HA "stem" are two such sites. We describe here a human antibody clonal lineage, designated CL6649, members of which bind a third conserved site ("lateral patch") on the side of the H1-subtype, HA head. A crystal structure of HA with bound Fab6649 shows the conserved antibody footprint. The site was invariant in isolates from 1977 (seasonal) to 2012 (pdm2009); antibodies in CL6649 recognize HAs from the entire period. In 2013, human H1 viruses acquired mutations in this epitope that were retained in subsequent seasons, prompting modification of the H1 vaccine component in 2017. The mutations inhibit Fab6649 binding. We infer from the rapid spread of these mutations in circulating H1 influenza viruses that the previously subdominant, conserved lateral patch had become immunodominant for individuals with B-cell memory imprinted by earlier H1 exposure. We suggest that introduction of the pdm2009 H1 virus, to which most of the broadly prevalent, neutralizing antibodies did not bind, conferred a selective advantage in the immune systems of infected hosts to recall of memory B cells that recognized the lateral patch, the principal exposed epitope that did not change when pdm2009 displaced previous seasonal H1 viruses.

Saccharomyces cerevisiae-derived virus-like particle parvovirus B19 vaccine elicits binding and neutralizing antibodies in a mouse model for sickle cell disease.

Parvovirus B19 infections are typically mild in healthy individuals, but can be life threatening in individuals with sickle cell disease (SCD). A Saccharomyces cerevisiae-derived B19 VLP vaccine, now in pre-clinical development, is immunogenic in wild type mice when administered with the adjuvant MF59. Because SCD alters the immune response, we evaluated the efficacy of this vaccine in a mouse model for SCD. Vaccinated mice with SCD demonstrated similar binding and neutralizing antibody responses to those of heterozygous littermate controls following a prime-boost-boost regimen. Due to the lack of a mouse parvovirus B19 challenge model, we employed a natural mouse pathogen, Sendai virus, to evaluate SCD respiratory tract responses to infection. Normal mucosal and systemic antibody responses were observed in these mice. Results demonstrate that mice with SCD can respond to a VLP vaccine and to a respiratory virus challenge, encouraging rapid development of the B19 vaccine for patients with SCD.

Influenza immunization elicits antibodies specific for an egg-adapted vaccine strain.

For broad protection against infection by viruses such as influenza or HIV, vaccines should elicit antibodies that bind conserved viral epitopes, such as the receptor-binding site (RBS). RBS-directed antibodies have been described for both HIV and influenza virus, and the design of immunogens to elicit them is a goal of vaccine research in both fields. Residues in the RBS of influenza virus hemagglutinin (HA) determine a preference for the avian or human receptor, α-2,3-linked sialic acid and α-2,6-linked sialic acid, respectively. Transmission of an avian-origin virus between humans generally requires one or more mutations in the sequences encoding the influenza virus RBS to change the preferred receptor from avian to human, but passage of a human-derived vaccine candidate in chicken eggs can select for reversion to avian receptor preference. For example, the X-181 strain of the 2009 new pandemic H1N1 influenza virus, derived from the A/California/07/2009 isolate and used in essentially all vaccines since 2009, has arginine at position 226, a residue known to confer preference for an α-2,3 linkage in H1 subtype viruses; the wild-type A/California/07/2009 isolate, like most circulating human H1N1 viruses, has glutamine at position 226. We describe, from three different individuals, RBS-directed antibodies that recognize the avian-adapted H1 strain in current influenza vaccines but not the circulating new pandemic 2009 virus; Arg226 in the vaccine-strain RBS accounts for the restriction. The polyclonal sera of the three donors also reflect this preference. Therefore, when vaccines produced from strains that are never passaged in avian cells become widely available, they may prove more capable of eliciting RBS-directed, broadly neutralizing antibodies than those produced from egg-adapted viruses, extending the established benefits of current seasonal influenza immunizations.

Antigenic characterization of influenza viruses produced using synthetic DNA and novel backbones.

The global system for manufacturing seasonal influenza vaccines has been developed to respond to the natural evolution of influenza viruses, but the problem of antigenic mismatch continues to be a challenge in certain years. In some years, mismatches arise naturally due to the antigenic drift of circulating viruses after vaccine strain selection has already been made. In other years, antigenic differences between the vaccine virus and circulating viruses are introduced as part of the current system, which relies on the use of egg-adapted isolates as a starting material for candidate vaccine viruses (CVVs). Improving the current process for making vaccine viruses can provide great value. We have previously established a synthetic approach for rapidly generating influenza viruses in a vaccine-approved Madin Darby canine kidney (MDCK) cell line using novel, high-growth backbones that increase virus rescue efficiency and antigen yield. This technology also has the potential to produce viruses that maintain antigenic similarity to the intended reference viruses, depending on the hemagglutinin (HA) and neuraminidase (NA) sequences used for gene synthesis. To demonstrate this utility, we generated a panel of synthetic viruses using HA and NA sequences from recent isolates and showed by hemagglutination inhibition (HI) tests that all synthetic viruses were antigenically-like their conventional egg- or cell-propagated reference strains and there was no impact of the novel backbones on antigenicity. This synthetic approach can be used for the efficient production of CVVs that may be more representative of circulating viruses and may be used for both egg- and cell-based vaccine manufacturing platforms. When combined with mammalian cell culture technology for antigen production, synthetic viruses generated using HA and NA sequences from a non-egg-adapted prototype can help to reduce the potential impact of antigenic differences between vaccine virus and circulating viruses on vaccine effectiveness.

Trypsin pre-treatment corrects SRID over-estimation of immunologically active, pre-fusion HA caused by mixed immunoprecipitin rings.

Influenza vaccines are the primary intervention to prevent the substantial health burden of seasonal and pandemic influenza. Subunit and split influenza vaccines are formulated, released for clinical use, and tested for stability based on their content of immunologically active (capable of eliciting functional antibodies) hemagglutinin (HA). Single-radial immunodiffusion (SRID), the standard in vitro potency assay in the field, is believed to specifically detect immunologically active HA. We confirmed that, with conformationally homogeneous HA preparations, SRID specifically detected native, pre-fusion HA, which elicited influenza neutralizing and hemagglutination inhibiting (HI) antibodies in mice, and it did not detect low-pH stressed, post-fusion HA, which was selectively removed from the SRID gel during a blotting step and was significantly less immunologically active. This selective detection was due to the SRID format, not a conformational specificity of the sheep antiserum used in the SRID, as the same antiserum detected non-stressed and low-pH stressed HA similarly when used in an ELISA format. However, when low-pH stressed HA was mixed with non-stressed HA, SRID detected both forms in mixed immunoprecipitin rings, leading to over-quantification of pre-fusion HA. We previously reported that trypsin digestion of antigen samples selectively degrade stressed HA, so that an otherwise conformationally insensitive biophysical quantification technique, reversed-phase high pressure liquid chromatography (RP-HPLC), can specifically quantify trypsin-resistant, immunologically active, pre-fusion HA. Here, we report that trypsin digestion can also improve the specificity of SRID so that it can quantify immunologically active, pre-fusion HA when it is mixed with less immunologically active, post-fusion HA.

Antigenic Characterization of the HCMV gH/gL/gO and Pentamer Cell Entry Complexes Reveals Binding Sites for Potently Neutralizing Human Antibodies.

Human Cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and in fetuses following congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are required for HCMV entry in fibroblasts and endothelial/epithelial cells, respectively, and are targeted by potently neutralizing antibodies in the infected host. Using purified soluble forms of gH/gL/gO and Pentamer as well as a panel of naturally elicited human monoclonal antibodies, we determined the location of key neutralizing epitopes on the gH/gL/gO and Pentamer surfaces. Mass Spectrometry (MS) coupled to Chemical Crosslinking or to Hydrogen Deuterium Exchange was used to define residues that are either in proximity or part of neutralizing epitopes on the glycoprotein complexes. We also determined the molecular architecture of the gH/gL/gO- and Pentamer-antibody complexes by Electron Microscopy (EM) and 3D reconstructions. The EM analysis revealed that the Pentamer specific neutralizing antibodies bind to two opposite surfaces of the complex, suggesting that they may neutralize infection by different mechanisms. Together, our data identify the location of neutralizing antibodies binding sites on the gH/gL/gO and Pentamer complexes and provide a framework for the development of antibodies and vaccines against HCMV.

Structure of HCMV glycoprotein B in the postfusion conformation bound to a neutralizing human antibody.

Human cytomegalovirus (HCMV) poses a significant threat to immunocompromised individuals and neonates infected in utero. Glycoprotein B (gB), the herpesvirus fusion protein, is a target for neutralizing antibodies and a vaccine candidate due to its indispensable role in infection. Here we show the crystal structure of the HCMV gB ectodomain bound to the Fab fragment of 1G2, a neutralizing human monoclonal antibody isolated from a seropositive subject. The gB/1G2 interaction is dominated by aromatic residues in the 1G2 heavy chain CDR3 protruding into a hydrophobic cleft in the gB antigenic domain 5 (AD-5). Structural analysis and comparison with HSV gB suggest the location of additional neutralizing antibody binding sites on HCMV gB. Finally, immunoprecipitation experiments reveal that 1G2 can bind to HCMV virion gB suggesting that its epitope is exposed and accessible on the virus surface. Our data will support the development of vaccines and therapeutic antibodies against HCMV infection.

Conformationally selective biophysical assay for influenza vaccine potency determination.

Influenza vaccines are the primary intervention for reducing the substantial health burden from pandemic and seasonal influenza. Hemagglutinin (HA) is the most important influenza vaccine antigen. Subunit and split influenza vaccines are formulated, released for clinical use, and tested for stability based on an in vitro potency assay, single-radial immunodiffusion (SRID), which selectively detects HA that is immunologically active (capable of eliciting neutralizing or hemagglutination inhibiting antibodies in an immunized subject). The time consuming generation of strain-specific sheep antisera and calibrated antigen standards for SRID can delay vaccine release. The limitation in generating SRID reagents was evident during the early days of the 2009 pandemic, prompting efforts to develop more practical, alternative, quantitative assays for immunologically active HA. Here we demonstrate that, under native conditions, trypsin selectively digests HA produced from egg or mammalian cell in monovalent vaccines that is altered by stress conditions such as reduced pH, elevated temperature, or deamidation, leaving native, pre-fusion HA, intact. Subsequent reverse-phase high pressure liquid chromatography (RP-HPLC) can separate trypsin-resistant HA from the digested HA. Integration of the resulting RP-HPLC peak yields HA quantities that match well the values obtained by SRID. Therefore, trypsin digestion, to pre-select immunologically active HA, followed by quantification by RP-HPLC is a promising alternative in vitro potency assay for influenza vaccines.

An Innovative Pseudotypes-Based Enzyme-Linked Lectin Assay for the Measurement of Functional Anti-Neuraminidase Antibodies.

Antibodies (Ab) to neuraminidase (NA) play a role in limiting influenza infection and might help reduce the disease impact. The most widely used serological assay to measure functional anti-NA immune responses is the Enzyme-Linked Lectin Assay (ELLA) which relies on hemagglutinin (HA) mismatched virus reassortants, or detergent treated viruses as the NA source to overcome interference associated with steric hindrance of anti-HA Ab present in sera. The difficulty in producing and handling these reagents, which are not easily adapted for screening large numbers of samples, limits the routine analysis of functional anti-NA Ab in clinical trials. In this study, we produced influenza lentiviral pseudoparticles (PPs) containing only the NA antigen (NA-PPs) with a simple two-plasmid co-transfection system. NA-PPs were characterized and tested as an innovative source of NA in the NA inhibition (NI) assay. Both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) N1s within NA-PPs retained their sialidase activity and were specifically inhibited by homologous and N1 subtype-specific, heterologous sheep sera. Moreover, A/California/07/2009 N1-PPs were a better source of NA compared to whole live and detergent treated H1N1 viruses in ELLA, likely due to lack of interference by anti-HA Ab, and absence of possible structural modifications caused by treatment with detergent. This innovative assay is safer and applicable to all NAs. Taken together, these results highlight the potential of NA-PPs-based NI assays to be developed as sensitive, flexible, easy to handle and scalable serological tests for routine NA immune response analysis.

Expression and Characterization of Recombinant, Tetrameric and Enzymatically Active Influenza Neuraminidase for the Setup of an Enzyme-Linked Lectin-Based Assay.

Developing a universal influenza vaccine that induces broad spectrum and longer-term immunity has become an important potentially achievable target in influenza vaccine research and development. Hemagglutinin (HA) and neuraminidase (NA) are the two major influenza virus antigens. Although antibody responses against influenza virus are mainly directed toward HA, NA is reported to be more genetically stable; hence NA-based vaccines have the potential to be effective for longer time periods. NA-specific immunity has been shown to limit the spread of influenza virus, thus reducing disease symptoms and providing cross-protection against heterosubtypic viruses in mouse challenge experiments. The production of large quantities of highly pure and stable NA could be beneficial for the development of new antivirals, subunit-based vaccines, and novel diagnostic tools. In this study, recombinant NA (rNA) was produced in mammalian cells at high levels from both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) influenza viruses. Biochemical, structural, and immunological characterizations revealed that the soluble rNAs produced are tetrameric, enzymatically active and immunogenic, and finally they represent good alternatives to conventionally used sources of NA in the Enzyme-Linked Lectin Assay (ELLA).