Pre-existing immunity and vaccine history determine hemagglutinin-specific CD4 T cell and IgG response following seasonal influenza vaccination.

Effectiveness of seasonal influenza vaccination varies between individuals and might be affected by vaccination history among other factors. Here we show, by monitoring frequencies of CD4 T cells specific to the conserved hemagglutinin epitope HA118-132 and titres of IgG against the corresponding recombinant hemagglutinin protein, that antigen-specific CD4 T cell and antibody responses are closely linked to pre-existing immunity and vaccine history. Upon immunization, a strong early reaction is observed in all vaccine naïve participants and also in vaccine experienced individuals who have not received the respective seasonal vaccine in the previous year. This response is characterized by HA118-132 specific CD4 T cells with a follicular helper T cell phenotype and by ascending titers of hemagglutinin-specific antibodies from baseline to day 28 following vaccination. This trend was observed in only a proportion of those participants who received the seasonal vaccine the year preceding the study. Regardless of history, levels of pre-existing antibodies and CD127 expression on CD4 T cells at baseline were the strongest predictors of robust early response. Thus, both pre-existing immunity and vaccine history contribute to the response to seasonal influenza vaccines.

Hemagglutinin Nanoparticulate Vaccine with Controlled Photochemical Immunomodulation for Pathogenic Influenza-Specific Immunity.

Recently, viral infectious diseases, including COVID-19 and Influenza, are the subjects of major concerns worldwide. One strategy for addressing these concerns focuses on nasal vaccines, which have great potential for achieving successful immunization via safe, easy, and affordable approaches. However, conventional nasal vaccines have major limitations resulting from fast removal when pass through nasal mucosa and mucociliary clearance hindering their effectiveness. Herein a nanoparticulate vaccine (NanoVac) exhibiting photochemical immunomodulation and constituting a new self-assembled immunization system of a photoactivatable polymeric adjuvant with influenza virus hemagglutinin for efficient nasal delivery and antigen-specific immunity against pathogenic influenza viruses is described. NanoVac increases the residence period of antigens and further enhances by spatiotemporal photochemical modulation in the nasal cavity. As a consequence, photochemical immunomodulation of NanoVacs successfully induces humoral and cellular immune responses followed by stimulation of mature dendritic cells, plasma cells, memory B cells, and CD4+ and CD8+ T cells, resulting in secretion of antigen-specific immunoglobulins, cytokines, and CD8+ T cells. Notably, challenge with influenza virus after nasal immunization with NanoVacs demonstrates robust prevention of viral infection. Thus, this newly designed vaccine system can serve as a promising strategy for developing vaccines that are active against current hazardous pathogen outbreaks and pandemics.

A novel selection strategy for antibody producing hybridoma cells based on a new transgenic fusion cell line.

The use of monoclonal antibodies is ubiquitous in science and biomedicine but the generation and validation process of antibodies is nevertheless complicated and time-consuming. To address these issues we developed a novel selective technology based on an artificial cell surface construct by which secreted antibodies were connected to the corresponding hybridoma cell when they possess the desired antigen-specificity. Further the system enables the selection of desired isotypes and the screening for potential cross-reactivities in the same context. For the design of the construct we combined the transmembrane domain of the EGF-receptor with a hemagglutinin epitope and a biotin acceptor peptide and performed a transposon-mediated transfection of myeloma cell lines. The stably transfected myeloma cell line was used for the generation of hybridoma cells and an antigen- and isotype-specific screening method was established. The system has been validated for globular protein antigens as well as for haptens and enables a fast and early stage selection and validation of monoclonal antibodies in one step.

Isohemagglutinin titering performed on an automated solid-phase and hemagglutinin-based analyzer is comparable to results obtained by manual gel testing.

BACKGROUND: Isohemagglutinins (anti-A and anti-B) mediate hemolytic transfusion reactions, antibody-mediated rejection of solid-organ transplants, and delayed engraftment after stem cell transplant. However, quantification of isohemagglutinins is often labor intensive and operator dependent, limiting availability and interfacility comparisons. We evaluated an automated, solid-phase and agglutination-based antibody titer platform versus manual gel testing. STUDY DESIGN AND METHODS: Plasma samples were obtained from 54 randomly selected patients. Titers were determined by our laboratory's standard assay (manual dilution followed by manual gel testing) and were compared to results obtained on a fully automated blood bank analyzer (Galileo NEO, Immucor). The analyzer determined immunoglobulin G (IgG) antibodies using solid-phase and immunoglobulin M (IgM) antibodies by direct hemagglutination. RESULTS: Isohemagglutinin titers obtained by manual gel versus the automated assay generally (>80%) agreed within one doubling dilution, and always (100%) agreed within two dilutions. Among O samples, the gel titer and the highest titer obtained with the automated assay (either IgG or IgM) were similar in paired, nonparametric analysis (p = 0.06 for anti-A; p = 0.13 for anti-B). Gel titers from group A and group B patients were slightly higher than the highest titer obtained using the automated assay (p = 0.04 for group A; p = 0.009 for group B), although these differences were within the accepted error of measurement. CONCLUSION: Manual and automated methodologies yielded similar isohemagglutinin titers. Separate quantification of IgM and IgG isohemagglutinins via automated titration may yield additional insight into hemolysis, graft survival after ABO-incompatible transplantation, and red blood cell engraftment after ABO-incompatible stem cell transplant.

Hemagglutinin Stability Regulates H1N1 Influenza Virus Replication and Pathogenicity in Mice by Modulating Type I Interferon Responses in Dendritic Cells.

Hemagglutinin (HA) stability, or the pH at which HA is activated to cause membrane fusion, has been associated with the replication, pathogenicity, transmissibility, and interspecies adaptation of influenza A viruses. Here, we investigated the mechanisms by which a destabilizing HA mutation, Y17H (activation pH, 6.0), attenuates virus replication and pathogenicity in DBA/2 mice compared to wild-type (WT) virus (activation pH, 5.5). The extracellular lung pH was measured to be near neutral (pH 6.9 to 7.5). WT and Y17H viruses had similar environmental stability at pH 7.0; thus, extracellular inactivation was unlikely to attenuate the Y17H virus. The Y17H virus had accelerated replication kinetics in MDCK, A549, and RAW 264.7 cells when inoculated at a multiplicity of infection (MOI) of 3 PFU/cell. The destabilizing mutation also increased early infectivity and type I interferon (IFN) responses in mouse bone marrow-derived dendritic cells (DCs). In contrast, the HA-Y17H mutation reduced virus replication in murine airway murine nasal epithelial cell and murine tracheal epithelial cell cultures and attenuated virus replication, virus spread, the severity of infection, and cellular infiltration in the lungs of mice. Normalizing virus infection and weight loss in mice by inoculating them with Y17H virus at a dose 500-fold higher than that of WT virus revealed that the destabilized mutant virus triggered the upregulation of more host genes and increased type I IFN responses and cytokine expression in DBA/2 mouse lungs. Overall, HA destabilization decreased virulence in mice by boosting early infection in DCs, resulting in the greater activation of antiviral responses, including the type I IFN response. These studies reveal that HA stability may regulate pathogenicity by modulating IFN responses.IMPORTANCE Diverse influenza A viruses circulate in wild aquatic birds, occasionally infecting farm animals. Rarely, an avian- or swine-origin influenza virus adapts to humans and starts a pandemic. Seasonal and many universal influenza vaccines target the HA surface protein, which is a key component of pandemic influenza viruses. Understanding the HA properties needed for replication and pathogenicity in mammals may guide response efforts to control influenza. Some antiviral drugs and broadly reactive influenza vaccines that target the HA protein have suffered resistance due to destabilizing HA mutations that do not compromise replicative fitness in cell culture. Here, we show that despite not compromising fitness in standard cell cultures, a destabilizing H1N1 HA stalk mutation greatly diminishes viral replication and pathogenicity in vivo by modulating type I IFN responses. This encourages targeting the HA stalk with antiviral drugs and vaccines as well as reevaluating previous candidates that were susceptible to destabilizing resistance mutations.

Hemagglutinin head-specific responses dominate over stem-specific responses following prime boost with mismatched vaccines.

Broadly neutralizing Abs targeting the HA stem can provide broad protection against different influenza subtypes, raising the question of how best to elicit such Abs. We have previously demonstrated that vaccination with pandemic live-attenuated influenza vaccine (pLAIV) establishes immune memory for HA head-specific Abs. Here, we determine the extent to which matched versus mismatched LAIV-inactivated subunit vaccine (IIV) prime-boost vaccination elicits stem-specific memory B cells and Abs. We vaccinated African green monkeys with H5N1 pLAIV-pIIV or H5N1 pLAIV followed by seasonal IIV (sIIV) or with H5N1 pLAIV alone and measured Abs and HA-specific B cell responses. While we observed an increase in stem-specific memory B cells, head-specific memory B cell responses were substantially higher than stem-specific responses and were dominant even following boost with mismatched IIV. Neutralizing Abs against heterologous influenza viruses were undetectable. Head-specific B cells from draining lymph nodes exhibited germinal center markers, while stem-specific B cells found in the spleen and peripheral blood did not. Thus, although mismatched prime-boost generated a pool of stem-specific memory B cells, head-specific B cells and serum Abs substantially dominated the immune response. These findings have implications for including full-length native HA in prime-boost strategies intended to induce stem-specific Abs for universal influenza vaccination.

Analysis of the prevalence of systemic de novo thrombotic microangiopathy after ABO-incompatible kidney transplantation and the associated risk factors.

OBJECTIVES: To analyze the prevalence of systemic de novo thrombotic microangiopathy in ABO-incompatible kidney transplantation and risk factors associated with this condition. METHODS: A total of 201 patients who received living-donor kidney transplantation (114 patients with ABO-identical kidney transplantation and 87 patients with ABO-incompatible kidney transplantation) were retrospectively analyzed. Systemic de novo thrombotic microangiopathy was diagnosed clinically according to the presence of thrombocytopenia with microangiopathic hemolytic anemia and pathological findings of thrombotic microangiopathy. Anti-A and anti-B antibodies were purified from human plasma, and these antibodies' bindings to human kidney were investigated in vitro. RESULTS: ABO-incompatible kidney transplantation was a significant risk factor of systemic de novo thrombotic microangiopathy (odds ratio 55.9, 95% CI 1.8-8.9, P < 0.001) after transplantation. Multivariate logistic regression analysis showed that non-use of mycophenolate mofetil, pretreatment immunoglobulin G antibody titer ≥64-fold and pretransplant immunoglobulin M antibody titer ≥16-fold were significant risk factors for systemic de novo thrombotic microangiopathy in ABO-incompatible kidney transplantation. Microvascular inflammation of 1-h post-transplant biopsy could be observed more frequently in thrombotic microangiopathy patients than in non-thrombotic microangiopathy patients. Anti-A and anti-B antibodies purified from human plasma showed a strong in vitro reaction against human kidney when the antibody titer was ≥16-fold. CONCLUSIONS: Antibody titer should be decreased to ≤16-fold until the day of ABO-incompatible kidney transplantation by desensitization therapy including mycophenolate mofetil. The 1-h biopsy results might help to diagnose systemic de novo thrombotic microangiopathy.

Vaccination With Viral Vectors Expressing Chimeric Hemagglutinin, NP and M1 Antigens Protects Ferrets Against Influenza Virus Challenge.

Seasonal influenza viruses cause significant morbidity and mortality in the global population every year. Although seasonal vaccination limits disease, mismatches between the circulating strain and the vaccine strain can severely impair vaccine effectiveness. Because of this, there is an urgent need for a universal vaccine that induces broad protection against drifted seasonal and emerging pandemic influenza viruses. Targeting the conserved stalk region of the influenza virus hemagglutinin (HA), the major glycoprotein on the surface of the virus, results in the production of broadly protective antibody responses. Furthermore, replication deficient viral vectors based on Chimpanzee Adenovirus Oxford 1 (ChAdOx1) and modified vaccinia Ankara (MVA) virus expressing the influenza virus internal antigens, the nucleoprotein (NP) and matrix 1 (M1) protein, can induce strong heterosubtypic influenza virus-specific T cell responses in vaccinated individuals. Here, we combine these two platforms to evaluate the efficacy of a viral vectored vaccination regimen in protecting ferrets from H3N2 influenza virus infection. We observed that viral vectored vaccines expressing both stalk-targeting, chimeric HA constructs, and the NP+M1 fusion protein, in a prime-boost regimen resulted in the production of antibodies toward group 2 HAs, the HA stalk, NP and M1, as well as in induction of influenza virus-specific-IFNγ responses. The immune response induced by this vaccination regime ultimately reduced viral titers in the respiratory tract of influenza virus infected ferrets. Overall, these results improve our understanding of vaccination platforms capable of harnessing both cellular and humoral immunity with the goal of developing a universal influenza virus vaccine.

Haemagglutinin stability was not the primary cause of the reduced effectiveness of live attenuated influenza vaccine against A/H1N1pdm09 viruses in the 2013-2014 and 2015-2016 seasons.

During the 2013-2014 influenza season, the quadrivalent live attenuated influenza vaccine (QLAIV), had lower than expected vaccine effectiveness (VE) against circulating A/H1N1pdm09 viruses in the USA. The underlying reason proposed for this was that the A/H1N1pdm09 vaccine strain, A/California/07/2009 (A/CA09), had a thermally unstable haemagglutinin (HA) protein. Consequently, a new A/H1N1pdm09 candidate strain, A/Bolivia/559/2013 (A/BOL13), was developed for inclusion in the 2015-2016 QLAIV. A key parameter for selection of A/BOL13 was its more thermostable HA phenotype compared with A/CA09. During the 2015-2016 season, QLAIV containing A/BOL13 was found in some studies to have improved, but still with suboptimal, VE against circulating A/H1N1pdm09 viruses and was not recommended for use by the CDC in the US market in the 2016-2017 influenza season. This suggested that improved HA thermostability had not entirely resolved the reduced VE observed. One hypothesis for this was that, by improving thermostability, the A/BOL13 HA protein had been over-stabilised, compromising its activation at the low endosomal pH required for successful viral entry. Here we demonstrate that, while the A/BOL13 HA protein is more stable than that of A/CA09, its thermal and pH stability were comparable with historically efficacious LAIV strains, suggesting that the HA had not been over-stabilised. Furthermore, studies simulating potential heat exposure during distribution by exposing QLAIV nasal sprayers to 33 °C for 4 h showed that, while remaining within product specification, A/CA09 viral potency was statistically decreased after 12 weeks at 2-8 °C. These data suggest that although unfavourable HA protein stability may have contributed to the reduced VE of A/CA09 in 2013-2014, it was unlikely to have affected A/BOL13 in 2015-2016. We conclude that HA stability was not the primary cause of the reduced effectiveness of LAIV against A/H1N1pdm09 viruses in the 2013-2014 and 2015-2016 seasons.

Universal influenza virus vaccines: what needs to happen next?

INTRODUCTION: Influenza occurs worldwide and causes significant disease burden in terms of morbidity, associated complications, hospitalizations, and deaths. Vaccination constitutes the primary approach for controlling influenza. Current influenza vaccines elicit a strain-specific response yet occasionally exhibit suboptimal effectiveness. This review describes the limits of available immunization tools and the future prospects and potentiality of universal influenza vaccines. AREAS COVERED: New 'universal' vaccines, which are presently under development, are expected to overcome the problems related to the high variability of influenza viruses, such as the need for seasonal vaccine updates and re-vaccination. Here, we explore vaccines based on the highly conserved epitopes of the HA, NA, or extracellular domain of the influenza M2 protein, along with those based on the internal proteins such as NP and M1. EXPERT OPINION: The development of a universal influenza vaccine that confers protection against homologous, drifted, and shifted influenza virus strains could obviate the need for annual reformulation and mitigate disease burden. The scientific community has long been awaiting the advent of universal influenza vaccines; these are currently under development in laboratories worldwide. If such vaccines are immunogenic, efficacious, and able to confer long-lasting immunity, they might be integrated with or supplant traditional influenza vaccines.

Entry Inhibition and Modulation of Pro-Inflammatory Immune Response Against Influenza A Virus by a Recombinant Truncated Surfactant Protein D.

Surfactant protein D (SP-D) is expressed in the mucosal secretion of the lung and contributes to the innate host defense against a variety of pathogens, including influenza A virus (IAV). SP-D can inhibit hemagglutination and infectivity of IAV, in addition to reducing neuraminidase (NA) activity via its carbohydrate recognition domain (CRD) binding to carbohydrate patterns (N-linked mannosylated) on NA and hemagglutinin (HA) of IAV. Here, we demonstrate that a recombinant fragment of human SP-D (rfhSP-D), containing homotrimeric neck and CRD regions, acts as an entry inhibitor of IAV and downregulates M1 expression considerably in A549 cells challenged with IAV of H1N1 and H3N2 subtypes at 2 h treatment. In addition, rfhSP-D downregulated mRNA levels of TNF-α, IFN-α, IFN-ß, IL-6, and RANTES, particularly during the initial stage of IAV infection of A549 cell line. rfhSP-D also interfered with IAV infection of Madin Darby canine kidney (MDCK) cells through HA binding. Furthermore, rfhSP-D was found to reduce luciferase reporter activity in MDCK cells transduced with H1+N1 pseudotyped lentiviral particles, where 50% of reduction was observed with 10 µg/ml rfhSP-D, suggestive of a critical role of rfhSP-D as an entry inhibitor against IAV infectivity. Multiplex cytokine array revealed that rfhSP-D treatment of IAV challenged A549 cells led to a dramatic suppression of key pro-inflammatory cytokines and chemokines. In the case of pH1N1, TNF-α, IFN-α, IL-10, IL-12 (p40), VEGF, GM-CSF, and eotaxin were considerably suppressed by rfhSP-D treatment at 24 h. However, these suppressive effects on IL-10, VEGF, eotaxin and IL-12 (p40) were not so evident in the case of H3N2 subtype, with the exception of TNF-α, IFN-α, and GM-CSF. These data seem to suggest that the extent of immunomodulatory effect of SP-D on host cells can vary considerably in a IAV subtype-specific manner. Thus, rfhSP-D treatment can downregulate pro-inflammatory milieu encouraged by IAV that otherwise causes aberrant inflammatory cell recruitment leading to cell death and lung damage.

Cutting Edge: Check Your Mice-A Point Mutation in the Ncr1 Locus Identified in CD45.1 Congenic Mice with Consequences in Mouse Susceptibility to Infection.

B6.SJL-Ptprca Pepcb /Boy (CD45.1) mice have been used in hundreds of congenic competitive transplants, with the presumption that they differ from C57BL/6 mice only at the CD45 locus. In this study, we describe a point mutation in the natural cytotoxicity receptor 1 (Ncr1) locus fortuitously identified in the CD45.1 strain. This point mutation was mapped at the 40th nucleotide of the Ncr1 locus causing a single amino acid mutation from cysteine to arginine at position 14 from the start codon, resulting in loss of NCR1 expression. We found that these mice were more resistant to CMV due to a hyper innate IFN-γ response in the absence of NCR1. In contrast, loss of NCR1 increased susceptibility to influenza virus, a result that is consistent with the role of NCR1 in the recognition of influenza Ag, hemagglutinin. This work sheds light on potential confounding experimental interpretation when this congenic strain is used as a tool for tracking lymphocyte development.

Accessibility and contribution to glucan masking of natural and genetically tagged versions of yeast wall protein 1 of Candida albicans.

Yeast wall protein 1 (Ywp1) is an abundant glycoprotein of the cell wall of the yeast form of Candida albicans, the most prevalent fungal pathogen of humans. Antibodies that bind to the polypeptide backbone of isolated Ywp1 show little binding to intact yeast cells, presumably because the Ywp1 epitopes are masked by the polysaccharides of the mannoproteins that form the outer layer of the cell wall. Rare cells do exhibit much greater anti-Ywp1 binding, however, and one of these was isolated and characterized. No differences were seen in its Ywp1, but it exhibited greater adhesiveness, sensitivity to wall perturbing agents, and exposure of its underlying ß-1,3-glucan layer to external antibodies. The molecular basis for this greater epitope accessibility has not been determined, but has facilitated exploration of how these properties change as a function of cell growth and morphology. In addition, previously engineered strains with reduced quantities of Ywp1 in their cell walls were also found to have greater ß-1,3-glucan exposure, indicating that Ywp1 itself contributes to the masking of wall epitopes, which may be important for understanding the anti-adhesive effect of Ywp1. Ectopic production of Ywp1 by hyphae, which reduces the adhesivity of these filamentous forms of C. albicans, was similarly found to reduce exposure of the ß-1,3-glucan in their walls. To monitor Ywp1 in the cell wall irrespective of its accessibility, green fluorescent protein (Gfp) was genetically inserted into wall-anchored Ywp1 using a bifunctional cassette that also allowed production from a single transfection of a soluble, anchor-free version. The wall-anchored Ywp1-Gfp-Ywp1 accumulated in the wall of the yeast forms but not hyphae, and appeared to have properties similar to native Ywp1, including its adhesion-inhibiting effect. Some pseudohyphal walls also detectably accumulated this probe. Strains of C. albicans with tandem hemagglutinin (HA) epitopes inserted into wall-anchored Ywp1 were previously created by others, and were further explored here. As above, rare cells with much greater accessibility of the HA epitopes were isolated, and also found to exhibit greater exposure of Ywp1 and ß-1,3-glucan. The placement of the HA cassette inhibited the normal N-glycosylation and propeptide cleavage of Ywp1, but the wall-anchored Ywp1-HA-Ywp1 still accumulated in the cell wall of yeast forms. Bifunctional transformation cassettes were used to additionally tag these molecules with Gfp, generating soluble Ywp1-HA-Gfp and wall-anchored Ywp1-HA-Gfp-Ywp1 molecules. The former revealed unexpected electrophoretic properties caused by the HA insertion, while the latter further highlighted differences between the presence of a tagged Ywp1 molecule (as revealed by Gfp fluorescence) and its accessibility in the cell wall to externally applied antibodies specific for HA, Gfp and Ywp1, with accessibility being greatest in the rapidly expanding walls of budding daughter cells. These strains and results increase our understanding of cell wall properties and how C. albicans masks itself from recognition by the human immune system.

Immunoprotective effects of a hemin-binding peptide derived from hemagglutinin-2 against infection with Porphyromonas gingivalis.

The principal etiologic agent in periodontal disease, Porphyromonas gingivalis, generates cysteine proteases that bind heme with domains such as hemagglutinin-2 (HA2). High-affinity HA2-hemin binding supplies the porphyrin and ferric iron needed for growth and virulence. The DHYAVMISK peptide, recently identified at the hemin-binding site of HA2, inhibits hemin binding. We now evaluate the protective effect of vaccination with DGFPGDHYAVMISK (termed DK) against P. gingivalis using a rat infection model. Rats immunized with DK generated anti-peptide serum IgGs and salivary sIgAs (as measured by ELISA). In a subcutaneous abscess model, the protective effect of immunization was then investigated by measuring abscess size following subcutaneous injection with P. gingivalis. In an oral infection model, a ligature inoculated with P. gingivalis was used to induce periodontitis. The degree of bone erosion, ordinarily provoked by infection, was then evaluated by micro-computed tomography. We found that anti-peptide antibody titers of serum IgGs and salivary sIgAs for rats immunized with DK and adjuvant were significantly higher than for sham-immunized rats (injected with adjuvant/PBS alone; P < .05). In the subcutaneous abscess model, the DK + adjuvant-vaccinated rats recovered faster than sham-vaccinated animals, with their abscess sizes significantly smaller (P < .05). Further, in the experimental periodontitis model, bone loss at the molar palatal side for DK + adjuvant-vaccinated rats was significantly lower than for sham-vaccinated animals (P < .05). Collectively, these data demonstrate the potential of (DK) peptide immunization in terms of eliciting an immunoprotective effect against infection with P. gingivalis.

Comparative evaluation of three capripoxvirus-vectored peste des petits ruminants vaccines.

Sheep and goat pox (SGP) with peste des petits ruminants (PPR) are transboundary viral diseases of small ruminants that cause huge economic losses. Recombinant vaccines that can protect from both infections have been reported as a promising solution for the future. SGP was used as a vector to express two structural proteins hemagglutinin or the fusion protein of PPRV. We compared immunity conferred by recombinant capripoxvirus vaccines expressing H or F or both HF. Safety and efficacy were evaluated in goats and sheep. Two vaccine doses were tested in sheep, 104.5TCDI50 in 1ml dose was retained for the further experiment. Results showed that the recombinant HF confers an earlier and stronger immunity against both SGP and PPR. This recombinant vaccine protect also against the disease in exposed and unexposed sheep. The potential Differentiating Infected from Vaccinated Animals of recombinant vaccines is of great advantage in any eradication program.

The Magnitude and IgG Subclass of Antibodies Elicited by Targeted DNA Vaccines Are Influenced by Specificity for APC Surface Molecules.

Upon APC-targeted DNA vaccination, transfected cells secrete fusion proteins with targeting units specific for surface molecules on APC. In this study, we have tested several different targeting units for their ability to influence the magnitude and subclass of Ab responses to hemagglutinin from influenza A virus. The experiments employed bivalent homodimeric Ig-based molecules (vaccibodies). The overall efficiency in BALB/c mice depended on the targeting units in the following order: αMHC class II > αCD11c > αCD40 > Xcl-1 = MIP-1α > FliC > GM-CSF > Flt-3L > αDEC205. GM-CSF induced mainly IgG1, whereas Xcl1, MIP-1α, αCD40, and αDEC205 induced predominantly IgG2a. A more balanced mixture of IgG1 and IgG2a was observed with αCD11c, αMHC class II, Flt-3L, and FliC. Similar results of IgG subclass-skewing were obtained in Th1-prone C57BL/6 mice with a more limited panel of vaccines. IgG1 responses in BALB/c occurred early after immunization but declined relatively rapidly over time. IgG2a responses appeared later but lasted longer (>252 d) than IgG1 responses. The most efficient targeting units elicited short- and long-term protection against PR8 influenza (H1N1) virus in BALB/c mice. The results suggest that targeting of Xcr1+ conventional type 1 dendritic cells preferentially induces IgG2a responses, whereas simultaneous targeting of several dendritic cell subtypes also induces IgG1 responses. The induction of distinct subclass profiles by different surface molecules supports the APC-B cell synapse hypothesis. The results may contribute to generation of more potent DNA vaccines that elicit high levels of Abs with desired biologic effector functions.

Neutralizing immune responses induced by oligomeric H5N1-hemagglutinins from plants.

Plant-based transient expression is an alternative platform to produce hemagglutinin-based subunit vaccines. This production system provides not only fast and effective response in the context of a pandemic but also enables the supply of big volume vaccines at low cost. Crude plant extracts containing influenza hemagglutinin are considered to use as vaccine sources because of avoidance of related purification steps resulting in low cost production allowing veterinary applications. Highly immunogenic influenza hemagglutinins are urgently required to meet these pre-conditions. Here, we present a new and innovative way to generate functional H5 oligomers from avian flu hemagglutinin in planta by the specific interaction of S·Tag and S·Protein. A S·Tag was fused to H5 trimers and this construct was transiently co-expressed in planta with S·Protein-TPs which was multimerized by disulfide bonds via cysteine residues in tailpiece sequences (TP) of IgM antibody. Multimerized S·Protein-TPs serve as bridges/molecular docks to combine S·Tag-fused hemagglutinin trimers to form very large hemagglutinin H5 oligomers. H5 oligomers in the plant crude extract were highly active in hemagglutination resulting in high titers. Immunization of mice with two doses of plant crude extracts containing H5 oligomers after storage for 1 week at 4 °C caused strong immune responses and induced neutralizing specific humoral immune responses in mice. These results allow for the development of cheap influenza vaccines for veterinary application in future.

Effect of temperature post viral vector inoculation on the amount of hemagglutinin transiently expressed in Nicotiana benthamiana leaves.

Transient gene expression in whole plants by using viral vectors is promising as a rapid, mass production system for biopharmaceutical proteins. Recent studies have indicated that plant growth conditions such as air temperature markedly influence the accumulation levels of target proteins. Here, we investigated time course of the amount of recombinant hemagglutinin (HA), a vaccine antigen of influenza virus, in leaves of Nicotiana benthamiana plants grown at 20°C or 25°C post viral vector inoculation. The HA content per unit of leaf biomass increased and decreased from 4 to 6 days post inoculation at 20°C and 25°C, respectively, irrespective of the subcellular localization of HA. The overall HA contents were higher when HA was targeted to the endoplasmic reticulum (ER) rather than the apoplast. Necrosis of leaf tissues was specifically observed in plants inoculated with the ER-targeting vector and grown at 25°C. With the ER-targeting vector, the maximum HA contents at 20°C and 25°C were recorded at 6 and 4 days post inoculation, respectively, and were comparable to each other. HA contents thereafter decreased at both temperatures; the rate of reduction appeared faster at 25°C than at 20°C. From a practical point of view, our results indicate that the strategy of targeting HA to the ER, growing plants at a lower temperature of 20°C, and harvesting leaves at around a week after vector inoculation should be implemented to obtain a high HA yield stably and efficiently.

Encoded Silicon-Chip-Based Platform for Combinatorial Synthesis and Screening.

Solid-supported chemical libraries have proven useful for the rapid and cost-effective discovery of bioactive compounds. However, traditional on-bead screening involves time-intensive chemical characterization of hit compounds and high false positive rates. Herein, we report a new platform for encoded chemical synthesis and solid-supported screening using p-Chips, microsized silicon microtransponders capable of storing and emitting unique numerical identifiers (IDs). By encoding the structures of library members using p-Chip IDs, we can track compound identities throughout both split-and-pool synthesis and protein binding assays without destructive cleavage. Thanks to the numerical IDs, our p-Chip platform can provide binding constants for library members simply by stripping and reprobing with different protein concentrations, unlike traditional on-bead assays. To showcase these features, we synthesized a library of 108 hemagglutinin (HA) peptide variants using split-and-pool approach, and measured EC50s for each variant directly on p-Chips. On-chip EC50s obtained from these studies showed excellent correlation (80%) with those obtained using traditional ELISA methods. Our screen also yielded a false positive rate of 14%, markedly superior to that reported for conventional bead-based binding studies (66-96%).1-9 On the basis of these results, we believe the p-Chip platform has the potential to improve the effectiveness of solid-supported high-throughput screening by a significant margin.

Characterization of mAb6-9-1 monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and its engineered derivative, single-chain variable fragment antibody.

Hemagglutinin (HA), as a major surface antigen of influenza virus, is widely used as a target for production of neutralizing antibodies. Monoclonal antibody, mAb6-9-1, directed against HA of highly pathogenic avian influenza virus A/swan/Poland/305-135V08/2006(H5N1) was purified from mouse hybridoma cells culture and characterized. The antigenic specificity of mAb6-9-1 was verified by testing its cross-reactivity with several variants of HA. The mimotopes recognized by mAb6-9-1 were selected from two types of phage display peptide libraries. The comparative structural model of the HA variant used for antibody generation was developed to further facilitate epitope mapping. Based on the sequences of the affinity- selected polypeptides and the structural model of HA the epitope was located to the region near the receptor binding site (RBS). Such localization of the epitope recognized by mAb6-9-1 is in concordance with its moderate hemagglutination inhibiting activity and its antigenic specificity. Additionally, total RNA isolated from the hybridoma cell line secreting mAb6-9-1 was used for obtaining two variants of cDNA encoding recombinant single-chain variable fragment (scFv) antibody. To ensure high production level and solubility in bacterial expression system, the scFv fragments were produced as chimeric proteins in fusion with thioredoxin or displayed on a phage surface after cloning into the phagemid vector. Specificity and affinity of the recombinant soluble and phage-bound scFv were assayed by suitable variants of ELISA test. The observed differences in specificity were discussed.