HA Antigen Research Articles

Correction: Split inactivated COBRA vaccine elicits protective antibodies against H1N1 and H3N2 influenza viruses.

[This corrects the article DOI: 10.1371/journal.pone.0204284.].

Safety and immunogenicity of influenza A(H5N1) vaccine stored up to twelve years in the National Pre-Pandemic Influenza Vaccine Stockpile (NPIVS)

BackgroundAs part of the U.S. Department of Health and Human Services (HHS) Pandemic Influenza Plan preparedness and response strategy, the National Pre-Pandemic Influenza Vaccine Stockpile (NPIVS) program was established by the Biomedical Advanced Research and Development Authority (BARDA) in 2005 with the goal of building and maintaining a stockpile of vaccines for influenza viruses with pandemic potential to vaccinate 20 million people in the critical workforce in the event of a pandemic. The NPIVS program continuously monitors the integrity of influenza vaccine antigens and adjuvants stored within the stockpile. In addition to monitoring physical and chemical properties in stability studies, it is important to regularly assess the safety and immunogenicity of stockpiled vaccines and adjuvants to maintain preparedness for use in the event of an influenza pandemic. MethodsBARDA conducted a randomized, double-blinded Phase 2 clinical study with the oldest stockpiled influenza A(H5N1) antigen, stored over the previous 10–12 years administered with or without MF59® adjuvant, stored over the previous 2–7 years at the time of vaccination. ResultsStockpiled vaccines were well-tolerated, adverse events were generally mild, and there was no drop in immunogenicity to the oldest stockpiled A(H5N1) vaccine. Compared to unadjuvanted vaccine, greater peak antibody responses were observed in subjects who were vaccinated with MF59-adjuvanted vaccines, regardless of antigen dose. Vaccination with the A(H5N1) vaccine antigen also results in cross-reactive antibody responses to contemporary circulating strains of A(H5) influenza viruses. ConclusionsThe frequency, type, and severity of AEs observed during this study are similar to historical clinical study data with A(H5N1) vaccines and MF59 adjuvant indicating that a stockpiled A(H5N1) vaccine appears to remain safe and tolerable. The vaccines were immunogenic when administered as a two-dose vaccine regimen in healthy adults, despite extended storage of HA antigen or MF59 adjuvant within the NPIVS. Trial registration:ClinicalTrials.gov: NCT02680002.

2490. A Phase 1, Randomized, Observer Blind, Antigen and Adjuvant Dosage Finding Study to Evaluate the Safety and Immunogenicity of an Adjuvanted, Trivalent Subunit Influenza Vaccine in Elderly Subjects ≥65 Years of Age

BackgroundInfluenza virus infection in the elderly remains one of the ten leading causes of death. One successful strategy to enhance the magnitude of their influenza vaccine immune response has been the addition of the adjuvant MF59®.Methods196 subjects ≥ 65 years of age were enrolled in a dose ranging study with seven treatment arms to assess the safety and immunogenicity of the current formulation of aTIV compared with aTIV-modified formulations in which the dosage of MF59 was doubled or tripled and/or the dosage of the three influenza virus strains (A/H1N1, A/H3N2, and B) was doubled. Vaccine was administered by single or bilateral deltoid inoculations. The antibody responses to all three influenza virus vaccine strains were compared 21 days after a dose or doses of aTIV or aTIV-modified formulations, as measured by hemagglutination inhibition (HI) assay and microneutralization (MN) assay.ResultsIn general, HI and MN titers at Day 22 increased to a greater degree with the dosage of MF59 compared with that of HA (HI presented in Figure 1). This was evident when comparing the HI and MN titers where antigen content was a constant 45 μg, but MF59 dose ranged from 9.75, 19.5 to 29.25 mg in a single vaccine dose (Group 1, 2 and 6, respectively). Generally, the highest titers against all strains were evident with the highest MF59 dose (29.25 mg). The relationship of antigen content and immunogenicity of the vaccine was less apparent when comparing titers between groups in which HA antigen content doubled from 45 to 90 μg. Administering the dose of MF59 (19.5 mg) and TIV (90 μg) into either a single arm or dividing between two arms resulted in comparable titers. The incidence of solicited AEs tended to increase with the dose of MF59 and to a lesser degree, antigen. The majority of solicited AEs were mild to moderate in severity. The number of unsolicited AEs were similar across the different dosages used in this trial.ConclusionIn elderly subjects ≥65 years of age, increase in MF59 dose is associated with increased immunogenicity against all 3 components of seasonal influenza vaccine.Figure 1.Geometric mean titer (HI) against influenza A/H3N2/Texas/2012 according to dose of MF59 and HA.Disclosures G. Otten, Seqirus: Employee, Salary. V. Matassa, Seqirus: Employee, Salary. M. Ciarlet, Novartis Vaccines: Employee, Salary. B. Leav, Seqirus: Employee and Shareholder, Salary.

Split inactivated COBRA vaccine elicits protective antibodies against H1N1 and H3N2 influenza viruses.

Development of broadly reactive or universal influenza vaccines will be a paradigm shifting event for the influenza vaccine field. These next generation vaccines could replace the current standard of care with vaccines that elicit broadly cross-protective immune responses. However, a variety of in vitro and in vivo models are necessary to make the best assessments of these vaccine formulations to determine their mechanisms of action, and allow for downselection of candidates prior to human clinical trials. Our group has developed the computationally optimized broadly reactive antigen (COBRA) technology to develop HA head-based strategies to elicit antibodies against H1, H3, and H5 influenza strains. These vaccines elicit broadly reactive antibody responses that neutralize not only historical and contemporary vaccine strains, but also co-circulating variants in mice. In this study, we used H1 and H3 HA antigens in a split, inactivated vaccine (IIV) formulation in combination with the AF03 squalene-in-water emulsion adjuvant in ferrets immunologically naïve to influenza virus. The H3 COBRA IIV vaccine T11 elicited antibodies with HAI activity against more H3N2 influenza strains compared to IIV expressing wild-type H3 HA antigens, except for IIV vaccines expressing the HA from A/Texas/50/2012 (Tx/12) virus. H1 COBRA IIV vaccines, P1 and X6, elicited antibodies that recognized a similar number of H1N1 viruses as those antibodies elicited by IIV expressing the A/California/07/2009 (CA/09) HA. Ferrets vaccinated with the P1 or X6 COBRA IIV were protected against CA/09 challege and cleared virus from the lungs of the ferrets, similar to ferrets vaccinated with the CA/09 IIV.

Generation of a broadly reactive influenza H1 antigen using a consensus HA sequence

H1N1, one of the most prevalent influenza A virus subtypes affecting the human population, can cause infections varying from mild respiratory syndrome to severe pneumonia. The current H1N1 vaccine needs to be updated annually and does not protect against future outbreaks. Here, we downloaded 2,656 HA protein sequences of human H1N1 viruses from the NCBI influenza database (up to the date of Aug. 2012) and constructed a phylogenetic tree of these H1 proteins via the neighbor-joining method using MEGA 5.0 software. A consensus H1 protein (CH1) was generated and was further modified with published conserved T-cell and B-cell epitopes. Interestingly, this CH1 protein is genetically similar to an H1 isolate obtained during the 1980s (A/Memphis/7/1980), indicating that a universal HA antigen may exist in nature. Vaccination with a DNA vaccine expressing CH1 elicited broadly reactive T-cell and B-cell responses to heterologous H1N1 viruses, though this vaccine did not successfully neutralize pdm09 H1N1 viruses. A combination of CH1 and pdm09 HA in a DNA vaccination neutralized pdm09 H1N1 viruses and protected mice from lethal infections by all representative H1N1 viruses. Moreover, a recombinant chimeric PR8-CH1 virus carrying HA sequence of the consensus H1 and all other seven genes from the PR8 strain was highly attenuated in mice, with a lethal dose (LD50) of more than 106 pfu. Vaccination with PR8-CH1 virus provided complete protection against infections by heterologous H1N1 strains. Taken together, a universal H1 antigen, CH1, was developed by constructing a consensus HA sequence, and the PR8-CH1 virus containing this consensus sequence elicited broadly protective immunity against heterologous H1N1 viruses.

Multigenic DNA vaccine induces protective cross-reactive T cell responses against heterologous influenza virus in nonhuman primates.

Recent avian and swine-origin influenza virus outbreaks illustrate the ongoing threat of influenza pandemics. We investigated immunogenicity and protective efficacy of a multi-antigen (MA) universal influenza DNA vaccine consisting of HA, M2, and NP antigens in cynomolgus macaques. Following challenge with a heterologous pandemic H1N1 strain, vaccinated animals exhibited significantly lower viral loads and more rapid viral clearance when compared to unvaccinated controls. The MA DNA vaccine induced robust serum and mucosal antibody responses but these high antibody titers were not broadly neutralizing. In contrast, the vaccine induced broadly-reactive NP specific T cell responses that cross-reacted with the challenge virus and inversely correlated with lower viral loads and inflammation. These results demonstrate that a MA DNA vaccine that induces strong cross-reactive T cell responses can, independent of neutralizing antibody, mediate significant cross-protection in a nonhuman primate model and further supports development as an effective approach to induce broad protection against circulating and emerging influenza strains.

Developing a platform system for gene delivery: amplifying virus-like particles (AVLP) as an influenza vaccine

Delivery of a gene of interest to target cells is highly desirable for translational medicine, such as gene therapy, regenerative medicine, vaccine development, and studies of gene function. Parainfluenza virus 5 (PIV5), a paramyxovirus with a negative-sense RNA genome, normally infects cells without causing obvious cytopathic effect, and it can infect many cell types. To exploit these features of PIV5, we established a system generating self-amplifying, virus-like particles (AVLP). Using enhanced green fluorescent protein (EGFP) as a reporter, AVLP encoding EGFP (AVLP–EGFP) successfully delivered and expressed the EGFP gene in primary human cells, including stem cells, airway epithelial cells, monocytes, and T cells. To demonstrate the application of this system for vaccine development, we generated AVLPs to express the HA and M1 antigens from the influenza A virus strain H5N1 (AVLP–H5 and AVLP–M1H5). Immunization of mice with AVLP–H5 and AVLP–M1H5 generated robust antibody and cellular immune responses. Vaccination with a single dose of AVLP–H5 and M1H5 completely protected mice against lethal H5N1 challenge, suggesting that the AVLP-based system is a promising platform for delivery of desirable genes.

Broad cross-protective anti-hemagglutination responses elicited by influenza microconsensus DNA vaccine

Despite the routine development and distribution of seasonal influenza vaccines, influenza remains an important pathogen contributing to significant human morbidity as well as mortality each year. The seasonal variability of influenza creates a significant issue for vaccine development of seasonal strains that can afford protection from infection or disease based on serotype matching. It is appreciated that the globular head of the HA antigen contained in the vaccines generates antibodies that result in HAI activity that are a major correlates of the protection against a particular strain. Due to seasonal genetic changes in the HA protein, however, new vaccine strains are needed to be developed continually to match the new HA antigen of that seasons virus. A distinct advantage in seasonal vaccine development would be if a small group of antigens could be developed that could span many seasons without needed to be replaced due to this genetic drift. Here we report on a synthetic microconsensus approach that relies on a small collection of 4 synthetic H1HA DNA antigens which together induce broad protective HAI immunity spanning decades of H1 influenza viruses in mice, guinea pigs and non-human primates. The protective HAI titers induced by microconsensus immunogens are fully functional in vivo as immunized ferrets were completely protected from A/Mexico/InDRE4487/2009 virus infection and morbidity associated with lethal challenge. These results are encouraging that a limited easy-to-formulate collection of invariant antigens can be developed which can span seasonal vaccine changes allowing for continued immune protection.

A structural explanation for the low effectiveness of the seasonal influenza H3N2 vaccine.

The effectiveness of the annual influenza vaccine has declined in recent years, especially for the H3N2 component, and is a concern for global public health. A major cause for this lack in effectiveness has been attributed to the egg-based vaccine production process. Substitutions on the hemagglutinin glycoprotein (HA) often arise during virus passaging that change its antigenicity and hence vaccine effectiveness. Here, we characterize the effect of a prevalent substitution, L194P, in egg-passaged H3N2 viruses. X-ray structural analysis reveals that this substitution surprisingly increases the mobility of the 190-helix and neighboring regions in antigenic site B, which forms one side of the receptor binding site (RBS) and is immunodominant in recent human H3N2 viruses. Importantly, the L194P substitution decreases binding and neutralization by an RBS-targeted broadly neutralizing antibody by three orders of magnitude and significantly changes the HA antigenicity as measured by binding of human serum antibodies. The receptor binding mode and specificity are also altered to adapt to avian receptors during egg passaging. Overall, these findings help explain the low effectiveness of the seasonal vaccine against H3N2 viruses, and suggest that alternative approaches should be accelerated for producing influenza vaccines as well as isolating clinical isolates.

A sandwich ELISA for the detection of neuraminidase of avian influenza A(H7N9) virus

Although exhibiting no or low virulence in poultry, avian influenza virus H7N9 has caused around 1400 confirmed human infections in China with a case-fatality rate of 30% since 2013. A highly pathogenic H7N9 virus (HP-H7), with the HA antigenicity distinct from the previous, were recently detected in patients and poultry. Therefore, convenient rapid diagnosis with reliability will allow early antiviral use and management for H7N9 infection. Here, a sandwich ELISA targeting the conserved viral antigen, neuraminidase (NA) was developed. The immunoassay employed mouse monoclonal antibody (mAb) 3C1 to specifically capture the N9 and 3E9 for the detection. Its limit of detection is 6.25ng/ml for N9 protein of A/Anhui/1/2013(H7N9, AH1/2013) and 0.125HAU/50μL for live virus, AH1/2013 and A/Environment/Jiangxi/28/2009 (H11N9), respectively. When applied to test the five clinic throat swabs from H7N9 patients confirmed by nuclear acid testing (NAT) using quantitative reverse-transcriptase polymerase chain reaction (Q-PCR), two samples showed positive result in sandwich ELISA while all were negative using commercial Flu A and H7 subtype rapid antigen tests (RAT). The ELISA using anti-N9 mAbs provided a valuable approach to detect H7N9 virus and quantify the N9 protein.

Chimeric virus-like particles containing influenza HA antigen and GPI-CCL28 induce long-lasting mucosal immunity against H3N2 viruses

Influenza virus is a significant cause of morbidity and mortality, with worldwide seasonal epidemics. The duration and quality of humoral immunity and generation of immunological memory to vaccines is critical for protective immunity. In the current study, we examined the long-lasting protective efficacy of chimeric VLPs (cVLPs) containing influenza HA and GPI-anchored CCL28 as antigen and mucosal adjuvant, respectively, when immunized intranasally in mice. We report that the cVLPs induced significantly higher and sustainable levels of virus-specific antibody responses, especially IgA levels and hemagglutination inhibition (HAI) titers, more than 8-month post-vaccination compared to influenza VLPs without CCL28 or influenza VLPs physically mixed with sCCL28 (soluble) in mice. After challenging the vaccinated animals at month 8 with H3N2 viruses, the cVLP group also demonstrated strong recall responses. On day 4 post-challenge, we measured increased antibody levels, ASCs and HAI titers with reduced viral load and inflammatory responses in the cVLP group. The animals vaccinated with the cVLP showed 20% cross-protection against drifted (Philippines) and 60% protection against homologous (Aichi) H3N2 viruses. Thus, the results suggest that the GPI-anchored CCL28 induces significantly higher mucosal antibody responses, involved in providing long-term cross-protection against H3N2 influenza virus when compared to other vaccination groups.

Humoral and Cellular Immunogenicity Induced by Avian Influenza A (H7N9) DNA Vaccine in Mice.

BackgroundIn March 2013, human infection with avian influenza A (H7N9) virus emerged in China, causing serious public health concerns and raising the possibility of avian-source pandemic influenza. Thus, the development of an effective vaccine for preventing and rapidly controlling avian influenza A (H7N9) virus is needed. In this study, we evaluated the immunogenicity of a synthetic DNA vaccine against H7 HA antigens in mice.Materials and MethodsThe synthetic consensus H7 HA DNA vaccine (25 or 50 µg) was administered to BALB/c mice at 0, 14, and 28 days by intramuscular injection followed by electroporation. Humoral and cellular immune responses were analyzed in a hemagglutination inhibition test and interferon-gamma enzyme-linked immunospot (ELISpot) assay, respectively.ResultsH7 HA-vaccinated mice showed 100% seroprotection and seroconversion rate against H7N9 reassortant influenza virus after both second and third immunizations. The geometric mean titer by the hemagglutination inhibition test increased with an increasing number of immunizations. However, there was no significant difference in geometric titer between the two groups injected with 25 and 50 µg of H7 HA DNA vaccine after two (79.98 vs. 107.65, P = 0.39) and three (159.96 vs. 215.28, P = 0.18) doses. In addition, the ELISpot assay revealed that administration of H7 HA DNA vaccine induced potent interferon-gamma production from mouse splenocytes.ConclusionsThis study demonstrated the humoral and cellular immunogenicity of synthetic consensus H7 HA DNA vaccine in mice. This work demonstrates the potential of the H7 HA DNA vaccine as an efficient tool for the rapid control of emerging influenza A (H7N9) virus.

Immunogenicity of Virus Like Particle Forming Baculoviral DNA Vaccine against Pandemic Influenza H1N1.

An outbreak of influenza H1N1 in 2009, representing the first influenza pandemic of the 21st century, was transmitted to over a million individuals and claimed 18,449 lives. The current status in many countries is to prepare influenza vaccine using cell-based or egg-based killed vaccine. However, traditional influenza vaccine platforms have several limitations. To overcome these limitations, many researchers have tried various approaches to develop alternative production platforms. One of the alternative approach, we reported the efficacy of influenza HA vaccination using a baculoviral DNA vaccine (AcHERV-HA). However, the immune response elicited by the AcHERV-HA vaccine, which only targets the HA antigen, was lower than that of the commercial killed vaccine. To overcome the limitations of this previous vaccine, we constructed a human endogenous retrovirus (HERV) envelope-coated, baculovirus-based, virus-like-particle (VLP)–forming DNA vaccine (termed AcHERV-VLP) against pandemic influenza A/California/04/2009 (pH1N1). BALB/c mice immunized with AcHERV-VLP (1×107 FFU AcHERV-VLP, i.m.) and compared with mice immunized with the killed vaccine or mice immunized with AcHERV-HA. As a result, AcHERV-VLP immunization produced a greater humoral immune response and exhibited neutralizing activity with an intrasubgroup H1 strain (PR8), elicited neutralizing antibody production, a high level of interferon-γ secretion in splenocytes, and diminished virus shedding in the lung after challenge with a lethal dose of influenza virus. In conclusion, VLP-forming baculovirus DNA vaccine could be a potential vaccine candidate capable of efficiently delivering DNA to the vaccinee and VLP forming DNA eliciting stronger immunogenicity than egg-based killed vaccines.

Combined nanoemulsion and RIG-I agonist adjuvants-activation of diverse pathways for a broad spectrum influenza vaccine.

Abstract Current influenza vaccines are limited by low immunogenicity, short duration of protection, and narrow cross-strain efficacy. Broad-based cellular immune responses and mucosal immunity are key to an effective vaccine. However, most single adjuvants exhibit only some of these qualities. Here we combine two adjuvants, an oil-in-water nanoemulsion (NE), and a synthetic RNA from Sendai virus (IVT DI), each individually shown to elicit protective immunity to influenza via distinct mechanisms. Intranasal (IN) immunization with NE and influenza hemagglutinin induces mucosal and systemic antibodies, activates NF-κB via TLR signaling, and induces a balanced TH1/TH2/TH17 cellular response. NE is also an antigen carrier, facilitating cellular uptake and processing. IVT DI activates the RIG-I receptor inducing type-I interferon and TH1 immunity. We sought to determine if combining the complementary properties of NE and IVT DI improves the strength of the immune response and affords new activities not seen with the single adjuvants. Combined adjuvant was evaluated with inactivated H1N1 virus (PR8) as antigen. While IN coadmin of NE and IVT DI did not improve humoral responses in mice, cellular recall responses were much enhanced relative to either adjuvant alone. Separate sequential admin of NE and IVT DI (2 wks apart) resulted in very high humoral and mucosal antibody responses and increased cytokine production. Interestingly, the cytokine pattern for sequential vs. coadmin of NE and IVT DI was distinct. Notably, both methods elicited a TH17 response not seen with IVT DI alone. Combining complementary adjuvants may be a promising approach for generating a broader, more optimal immune response for influenza when used with diverse HA antigens.

A clinical phase I study of an EB66 cell-derived H5N1 pandemic vaccine adjuvanted with AS03

BackgroundWe conducted a phase I clinical trial of a cell culture-derived AS03-adjuvanted influenza vaccine containing HA antigen (A/Indonesia/05/2005(H5N1)/PR8-IBCDC-RG2) derived from EB66 cells (KD-295). MethodsHealthy male adult volunteers (20–40 years old, N=60) enrolled in the study were divided into 3 groups, the MA group (3.8μg of HA+AS03), HA group (7.5μg of HA+AS03), and 1/2 MA group (half the volume of the MA group), and received KD-295 intramuscularly twice with a 21-day interval. After administration of KD-295, adverse events, clinical laboratory parameters, and immune response to the vaccine strain and heterologous virus strains were evaluated. ResultsNo severe adverse events leading to discontinuation of vaccine administration occurred. The vaccine was well-tolerated. There was no dose dependency in the rate, timing, or duration of the adverse events. Immunogenicity of the vaccines was evaluated by HI (hemagglutination inhibition) assay, which confirmed that the antibody response to the vaccine strain and heterologous strain in all groups met the three criteria for immunogenicity described in the Japanese guidelines for development of a pandemic prototype vaccine. We also measured the neutralizing antibody titers against several virus strains, and confirmed a significant rise in antibody levels to both the vaccine strain and heterologous strains. ConclusionThe EB66-derived H5N1 influenza vaccine adjuvanted with AS03 elicited a broad cross-reactive antibody response among H5N1 strains with acceptable reactogenicity. Therefore, KD-295 can be considered a useful pandemic and pre-pandemic influenza vaccine candidate.

Synthesis and immunogenicity evaluating of a discontinuous B-cell epitope predicted from influenza virus A/H5N1 HA antigen

To develop vaccine with stable efficiency against easily transforming virus such as influenza A/H5N1 virus, bioinformatic tools have been used to predict conserved epitopes from viral antigens to be used as materials for the development of polyvalent vaccine against this virus. Using this approach, we have successfully predicted one discontinuous B-cell epitope, a peptide of 14 aminoacids on conserved domains of HA antigen from H5N1 influenza A virus. In this study, we report results of the preparation of this discontinuous B-cell epitope as the recombinant fusion form with H:1,2 flagellin from Salmonella typhimurium to increase the immunogenicity of this epitope using genetic manipulating techniques with Escherichia coli as host cell. The immunogenicity of the chemically synthesized predicted epitope and the recombinant epitope was examined by immunization of each of these epitopes to mice. Using hemagglutination inhibition (HI) method, we successfully demonstrated that both anti-sera from mice immunized either with chemically synthesized peptide or with recombinant flagellin H:1,2 fused epitope could recognize and inhibit the agglutination of inactivated influenza H5N1 virus strains, such as A/H5N1/ Chicken13/ Vietnam/LA/2006 and A/Vietnam/CL26/2004 (H5N1) isolated from infected chicken and human in Vietnam.

Relationship of the quaternary structure of human secretory IgA to neutralization of influenza virus

Secretory IgA (S-IgA) antibodies, the major contributors to humoral mucosal immunity to influenza virus infection, are polymeric Igs present in many external secretions. In the present study, the quaternary structures of human S-IgA induced in nasal mucosa after administration of intranasal inactivated influenza vaccines were characterized in relation to neutralization potency against influenza A viruses. Human nasal IgA antibodies have been shown to contain at least five quaternary structures. Direct and real-time visualization of S-IgA using high-speed atomic force microscopy (AFM) demonstrated that trimeric and tetrameric S-IgA had six and eight antigen-binding sites, respectively, and that these structures exhibited large-scale asynchronous conformational changes while capturing influenza HA antigens in solution. Furthermore, trimeric, tetrameric, and larger polymeric structures, which are minor fractions in human nasal IgA, displayed increased neutralizing potency against influenza A viruses compared with dimeric S-IgA, suggesting that the larger polymeric than dimeric forms of S-IgA play some important roles in protection against influenza A virus infection in the human upper respiratory tract.

Rational design of medium supplementation strategy for improved influenza viruses production based on analyzing nutritional requirements of MDCK Cells

Influenza vaccine production using cell culture technology has become popular nowadays. However, to meet the ever increasing demand of influenza vaccine, it is prerequisite to improve the yield of influenza virus in cells. To achieve this, in the present study, the nutritional requirements of MDCK cells in the virus production process were analyzed and a nutrient-feeding strategy was developed accordingly. Based on the consumption rates and corresponding concentration optimization, glucose and fast metabolized amino acids were supplemented into the maintaining medium at the time of infection. Compared with the non-supplemented culture, the average cell specific death rate during 0–48h post-infection was 0.013h−1, which was 40.91% lower in the nutrient-supplemented culture. Total virus titer, HA antigen protein concentration and cell-specific virus yield were (1.88±0.23)×103HAunits/50μL, 11.70±0.22μg/mL and (10.06±1.16)×103virions/cell, respectively, which were 84.04±22.50%, 31.46±2.87% and 86.64±25.81% higher than those in the control, respectively. These data showed that the appropriate supplementation of nutrients during virus production process could reduce cell death, and improve cell-specific virus yield and total influenza virus output. This study laid foundation for the development of cell culture technology for influenza vaccine production.

Reverse genetic engineering of the human rhinovirus serotype 16 genome to introduce an antibody-detectable tag.

The ability to accurately detect viral proteins during infection is essential for virology research, and the lack of specific antibodies can make this detection difficult. Reverse genetic engineering of virus genomes to alter the wild-type genome is a powerful technique to introduce a detectable tag onto a viral protein. Here we outline a method to incorporate an influenza hemagglutinin epitope tag onto the 2A protease of HRV16. The method uses site-directed mutagenesis PCR to introduce the sequence for the HA antigen onto either the C or N termini of 2A protease while keeping the relevant internal cleavage sites intact. The new viral product is then cloned into a wild-type HRV16 plasmid and transfected into Ohio Hela cells to produce recombinant virus.

Optimal designs of an HA-based DNA vaccine against H7 subtype influenza viruses

The outbreak of a novel H7N9 influenza virus in 2013 has raised serious concerns for the potential of another avian-source pandemic influenza. Effective vaccines against H7N9 virus are important in the prevention and control of any major outbreak. Novel vaccination technologies are useful additions to existing approaches. In the current report, DNA vaccine studies were conducted to identify the optimal design of an H7 HA antigen using the HA gene from a previously reported H7N7 virus that is lethal in humans as the model antigen. New Zealand White rabbits were immunized with DNA vaccines expressing 1 of 3 forms of H7 HA antigen inserts encoding the HA gene from the same H7N7 virus. High-level H7 HA-specific IgG was detected by ELISA, and functional antibodies were confirmed by hemagglutination inhibition assay and pseudotyped virus-based neutralization assay against viruses expressing HA antigens from either the previous H7N7 virus or the novel H7N9 virus. HA antigen design under the tissue plasminogen activator leader (tPA) was the most immunogenic. The data presented in the current report confirm the immunogenicity of the H7 HA antigen and provide useful guidance to prepare for an optimized H7 HA DNA vaccine to help to control the emerging H7N9 virus if and when it is needed.