Cold-adapted Influenza Virus Research Articles

Determination of cold-adapted influenza virus (Orthomyxoviridae: Alphainfluenzavirus) polymerase activity by the minigenome method with a fluorescent protein.

Polymerase proteins PB1 and PB2 determine the cold-adapted phenotype of the influenza virus A/Krasnodar/101/35/59 (H2N2), as was shown earlier. The development of the reporter construct to determine the activity of viral polymerase at 33 and 37 °C using the minigenome method. Co-transfection of Cos-1 cells with pHW2000 plasmids expressing viral polymerase proteins PB1, PB2, PA, NP (minigenome) and reporter construct. Based on segment 8, two reporter constructs were created that contain a direct or inverted NS1-GFP-NS2 sequence for the expression of NS2 and NS1 proteins translationally fused with green fluorescent protein (GFP), which allowed the evaluation the transcriptional and/or replicative activity of viral polymerase. Polymerase of virus A/Krasnodar/101/35/59 (H2N2) has higher replicative and transcriptional activity at 33 °C than at 37 °C. Its transcriptional activity is more temperature-dependent than its replicative activity. The replicative and transcriptional activity of polymerase A/Puerto Rico/8/34 virus (H1N1, Mount Sinai variant) have no significant differences and do not depend on temperature.

Influenza A virus-specific memory T cell persistence and functional recall are retained during pregnancy

Abstract Influenza A virus (IAV)-specific memory T cells induced by infection or through vaccination with live attenuated cold adapted influenza virus (LAIV) vaccine strains are well known to be able to confer protection against secondary homotypic as well as heterotypic IAV challenge. Whether IAV-specific memory T cell responses induced by LAIV vaccination can persist and retain their capacity to protect the maternal-fetal unit when generated prior to the altered physiological state of pregnancy, during which systemic levels of several hormones with putative immunomodulatory functions increase, is largely unknown. We show here that LAIV A/Alaska/6/77 CR-29 (H3N2)-vaccination prior to pregnancy induces IAV-specific memory CD4 and CD8 T cells that do indeed persist and functionally respond during recall in a manner similar to memory cells present in non-pregnant mice. This recall response can fully protect pregnant dams as well as their offspring from the adverse outcomes of lethal heterotypic infection with A/Puerto Rico/8/1934 (PR8-H1N1) virus. Using an adoptive transfer model and congenic HNT TcR transgenic CD4 T cells specific for PR8, we verify the observations observed with endogenous memory T cells that the recovery, surface marker phenotype, transcription factor profile, and TNF, IFN-g, and IL-2 multi-cytokine producing capacity of memory CD4 T cells responding to infection are largely unaltered by the pregnancy environment. These observations thus support that LAIV-vaccination prior to pregnancy results in the generation of IAV-specific memory T cells that have the potential to universally protect the maternal-fetal unit from the adverse outcomes of seasonal as well as pandemic strains of IAV. Supported by grants from NIH (R21HD093948), the University of Central Florida Foundation and the Florida Education Fund McKnight Doctoral Dissertation Fellowship.

Evaluation of influenza A and B cold-adapted reassortant virus reproduction in trivalent live influenza vaccines

BackgroundThe objective of the present study was to compare reproduction of trivalent LAIV vaccine strains in MDCK cells and to perform quantitative RT-PCR analysis of trivalent LAIV replication after inoculation in mice. MethodsWe applied a reverse transcriptase real-time PCR (rRT-PCR) analysis using TaqMan technique to evaluate the infectious titers of vaccine strains containing in trivalent live influenza vaccines (LAIVs). We confirmed the PCR data in ELISA using staining of MDCK monolayer with mouse monoclonal antibodies to hemagglutinin. ResultsThe viral load during the reproduction of mono-vaccines and trivalent LAIV in MDCK cells was similar at low dilutions. The content of vaccine viruses was evaluated using quantitative RT-PCR analysis in the nasal turbinate and lungs of CBA mice on day 3 after intranasal immunization. It was shown that despite the almost complete absence of reproduction of the A/H3N2 virus in mice, the immune response of A/H3N2-specific antibodies was formed at the same level as to other viruses. In MDCK cells, a decreased infectious titers of vaccine viruses in trivalent LAIV compared to mono-vaccines was demonstrated except for B/Yamagata virus. ConclusionRT-PCR analysis is applicable to assess the growth characteristics of cold-adapted reassortant influenza viruses in vitro and in mice. The interference of trivalent LAIV vaccine viruses in MDCK cells was minimal at low dilutions. In mice, decrease in infectious titers did not lead to a decline of the immunogenicity.

Assessment of trivalent live influenza vaccines in MDCK cell line

We applied a one-step reverse transcriptase real-time PCR (rRT-PCR) analysis using TaqMan technique to evaluate the infectious titers of vaccine strains containing in trivalent live influenza vaccines (LAIVs).The cold-adapted reassortant influenza viruses A/H1N1 pdm09, A/H3N2, B/Yamagata and B/Victoria, included in the composition of the LAIV in 2015-2016, 2017-2018 and 2018-2019 flu season were studied for reproductive activity in MDCK cells as part of a mono-vaccine and tri-vaccine. For this we have developed a set of specific primers and probes. Method validation was performed using ELISA-test after mouse monoclonal antibodies to hemagglutinin (HA) staining of MDCK monolayer.Influenza B viruses B/Yamagata and B/Victoria were studied in MDCK cells in mono-infection and coinfection with different multiplicity of infection (MOI) using quantitative rRT-PCR.•RT-PCR analysis was adjusted to assess the growth characteristics of cold-adapted reassortant influenza viruses in MDCK cell line. The greatest suppression in the composition of the tri-vaccine was exposed to the H1N1 pdm09 LAIV component.•Influenza B viruses are least suppressed in trivalent LAIV. Influenza viruses B/Yamagata and B/Victoria reproduced as part of a mixed preparation not lower, if not better than as a mono-preparation at an MOI of 0.1. At an MOI of 0.01, the reproduction of both B/Yamagata and B/Victoria in the mixture was reduced compared to mono-vaccine.•The interference of trivalent LAIV vaccine viruses in MDCK cells was minimal at low dilutions. This indicates that it is undesirable to reduce the titers of vaccine viruses, including at the stages of transportation and storage of LAIV

Immunogenicity and Protective Efficacy of Seasonal Human Live Attenuated Cold-Adapted Influenza Virus Vaccine in Pigs.

Influenza A virus infection is a global health threat to livestock and humans, causing substantial mortality and morbidity. As both pigs and humans are readily infected with influenza viruses of similar subtype, the pig is a robust and appropriate model for investigating swine and human disease. We evaluated the efficacy of the human cold-adapted 2017–2018 quadrivalent seasonal LAIV in pigs against H1N1pdm09 challenge. LAIV immunized animals showed significantly reduced viral load in nasal swabs. There was limited replication of the H1N1 component of the vaccine in the nose, a limited response to H1N1 in the lung lymph nodes and a low H1N1 serum neutralizing titer. In contrast there was better replication of the H3N2 component of the LAIV, accompanied by a stronger response to H3N2 in the tracheobronchial lymph nodes (TBLN). Our data demonstrates that a single administration of human quadrivalent LAIV shows limited replication in the nose and induces detectable responses to the H1N1 and H3N2 components. These data suggest that pigs may be a useful model for assessing LAIV against influenza A viruses.

Anti-caries vaccine based on clinical cold-adapted influenza vaccine: A promising alternative for scientific and public-health protection against dental caries

Dental caries remains one of the most pervasive infectious disease around the world. Protection against dental caries can be achieved experimentally by eliciting salivary IgA targeting surficial antigens of S. mutans, however, no such a vaccine has been launched for human use yet. Live vectored vaccines hold the greatest feasibility to induce potent and long-lasting immunity in the host. The FDA approved intranasal cold-adapted influenza vaccine has been used in clinical settings for many years. The vaccine can not only induce broad adaptive immune responses especially mucosal immunity, but the member strains can also circumvent existing immunity, presenting promising candidates for live vectored anti-caries vaccine. Moreover, the genetic techniques for modification of cold-adapted influenza viruses are well developed and widely applicable. Thus, we hypothesize that effective anti-caries vaccine can be developed with the backbone of cold-adapted influenza viruses by inserting specific antigenic identifier sequences of S. mutans into the viral genome, which is anticipated to protect against dental caries in humans with easy inoculation. The immune efficacies of recombinant cold-adapted influenza viruses expressing exogenous antigens have been verified by in vivo experiments for multiple infectious diseases, giving us great confidence to validate the safety properties and protection effect with this chimeric vaccine in animals or even humans. Existing data suggests that the live anti-caries vaccine may help improve public oral health by controlling the caries disease itself.

Monoclonal antibody against N2 neuraminidase of cold adapted A/Leningrad/134/17/57 (H2N2) enables efficient generation of live attenuated influenza vaccines

Cold adapted influenza virus A/Leningrad/134/17/57 (H2N2) is a reliable master donor virus (Len/17-MDV) for preparing live attenuated influenza vaccines (LAIV). LAIVs are 6:2 reasortants that contain 6 segments of Len/17-MDV and the hemagglutinin (HA) and neuraminidase (NA) of contemporary circulating influenza A viruses. The problem with the classical reassortment procedure used to generate LAIVs is that there is limited selection pressure against NA of the Len/17-MDV resulting in 7:1 reassortants with desired HA only, which are not suitable LAIVs. The monoclonal antibodies (mAb) directed against the N2 of Len/17-MDV were generated. 10C4–8E7 mAb inhibits cell-to-cell spread of viruses containing the Len/17-MDV N2, but not viruses with the related N2 from contemporary H3N2 viruses. 10C4–8E7 antibody specifically inhibited the Len/17-MDV replication in vitro and in ovo but didn’t inhibit replication of H3N2 or H1N1pdm09 reassortants. Our data demonstrate that addition of 10C4–8E7 in the classical reassortment improves efficiency of LAIV production.

Enhanced CD103 Expression and Reduced Frequencies of Virus-Specific CD8+ T Cells Among Airway Lymphocytes After Influenza Vaccination of Mice Deficient in Vitamins A + D.

Previous research has evaluated antibody responses toward an influenza virus vaccine in the context of deficiencies for vitamins A and D (VAD+VDD). Results showed that antibodies and antibody-forming cells in the respiratory tract were reduced in VAD+VDD mice. However, effectors were recovered when oral supplements of vitamins A + D were delivered at the time of vaccination. Here we address the question of how vaccine-induced CD8+ T cell responses are affected by deficiencies for vitamins A + D. VAD+VDD and control mice were vaccinated with an intranasal, cold-adapted influenza virus A/Puerto Rico/8/34 vaccine, with or without oral supplements of vitamins A + D. Results showed that the percentages of vaccine-induced CD8+ T cell and total CD4+ T cell responses were low among lymphocytes in the airways of VAD+VDD animals compared to controls. The CD103 membrane marker, a protein that binds e-cadherin (expressed on respiratory tract epithelial cells), was unusually high on virus-specific T cells in VAD+VDD mice compared to controls. Interestingly, when T cells specific for the PA224-233/Db epitope were compared with T cells specific for the NP366-374/Db epitope, the former population was more strongly positive for CD103. Preliminary experiments revealed normal or above-normal percentages for vaccine-induced T cells in airways when VAD+VDD animals were supplemented with vitamins A + D at the time of vaccination and on days 3 and 7 after vaccination. Our results suggest that close attention should be paid to levels of vitamins A and D among vaccine recipients in the clinical arena, as low vitamin levels may render individuals poorly responsive to vaccines.

Comparative studies of infectivity, immunogenicity and cross-protective efficacy of live attenuated influenza vaccines containing nucleoprotein from cold-adapted or wild-type influenza virus in a mouse model

This study sought to improve an existing live attenuated influenza vaccine (LAIV) by including nucleoprotein (NP) from wild-type virus rather than master donor virus (MDV). H7N9 LAIV reassortants with 6:2 (NP from MDV) and 5:3 (NP from wild-type virus) genome compositions were compared with regard to their growth characteristics, induction of humoral and cellular immune responses in mice, and ability to protect mice against homologous and heterologous challenge viruses. Although, in general, the 6:2 reassortant induced greater cell-mediated immunity in C57BL6 mice than the 5:3 vaccine, mice immunized with the 5:3 LAIV were better protected against heterologous challenge. The 5:3 LAIV-induced CTLs also had better in vivo killing activity against target cells loaded with the NP366 epitope of recent influenza viruses. Modification of the genome of reassortant vaccine viruses by incorporating the NP gene from wild-type viruses represents a simple strategy to improve the immunogenicity and cross-protection of influenza vaccines.

A checkpoint requiring cognate antigen recognition at the effector stage is necessary for protective memory CD4 T cell generation.

Abstract Effective vaccination depends on the generation of memory T cells, B cells, and long-lived plasma cells. However, the mechanisms that govern memory CD4 T cell formation are not well understood. Antigen (Ag) recognition during priming drives effector generation, but it is unclear how Ag recognition at the effector stage impacts T cell fate. Using an influenza infection model, we find that effector CD4 T cells must re-encounter Ag at the effector phase to avoid default apoptosis and become memory. Cognate interaction at this defined “memory checkpoint” drives continued proliferation, enhances survival, upregulates memory markers CD25, CD127, CXCR3, and Bcl-6, and drives IL-2 production, resulting in the formation of long-lived memory cells with enhanced production of multiple cytokines and increased protective function. A cold-adapted influenza virus, like those used in live-attenuated influenza vaccines, fails to present Ag at this critical effector time point and fails to induce substantial CD4 memory. However, memory CD4 T cell formation was rescued by adding short-lived Ag-pulsed antigen presenting cells during the checkpoint. Importantly, the Ag alone without infection-induced inflammation, was sufficient to restore memory formation. Our findings imply that in order for vaccines to promote optimal memory CD4 T cell formation, they must provide Ag presentation long enough that effector CD4 T cells are effectively restimulated during the “memory checkpoint”.

MECHANISMS OF ATTENUATION OF COLD-ADAPTED STRAIN A/KRASNODAR/101/35/59 (H2N2)

Study of mechanisms of attenuation of cold-adapted (ca) influenza virus strain A/ Krasnodar/101/35/59 (H2N2), associated with disruption of NS1 protein functions. Study of interferonogenic activity of ca strain A/Krasnodar/101/35/59 (H2N2), its parent variant A/Krasnodar/101/59 (H2N2), virulent strain A/WSN/33 (H1N1) and a number of single gene and multiple gene reassortants between these strains, obtained using reverse genetics, was carried out. Study of dynamics of IFNβ gene expression was carried out by using a methodical approach of RT-PCR in real time mode. Inclusion of PB-1 gene of ca strain A/ Krasnodar/101/35/59 (H2N2) with reversion to wild type into genome composition of virulent strain A/WSN/33 (H1N1) does not result in a sharp change of interferonogenic activity of the reassortant. At the same time, similar inclusion of PB-1 gene of ca strain resulted in an incredible growth of interferonogenic activity of the reassortant. On the other hand, inclusion of NP-gene of wild type strain A/Krasnodar/101/59 (H2N2) into genome composition of the wild type strain A/WSN/33 did not differ by effect on interferonogenicity of the reassortant from inclusion of NP-gene of ca strain. Both constellations of genes of parent variants and mutations localized in these genes could affect formation of attenuation phenotype of reassortants. The data obtained allow to assume possible mechanisms of attenuation of ca strains, associated with disruption.of NS gene function.

Vitamin Supplementation at the Time of Immunization with a Cold-Adapted Influenza Virus Vaccine Corrects Poor Mucosal Antibody Responses in Mice Deficient for Vitamins A and D.

Vitamin A and D deficiencies and insufficiencies are prevalent worldwide in developed and developing countries. Vitamin metabolites are functionally intertwined in that they are high-affinity ligands for related receptors of the nuclear receptor superfamily. The effects of vitamin A deficiencies (VAD) on antibody responses to respiratory virus vaccines have already been demonstrated. Of particular concern was the reduction in IgA, a first line of defense against pathogens in the respiratory tract. Here, we describe the individual and combined effects of vitamin A and D deficiencies in mice immunized with an attenuated influenza virus vaccine. Relative to VAD, vitamin D deficiency (VDD) had a limited effect, but double deficiencies for vitamins A and D (VAD+VDD) further reduced antibody responses in the respiratory tract. The administration of supplemental vitamins A and D to VAD+VDD mice at the time of vaccination restored responses in a dose-dependent manner. Results suggest that vitamin supplementation programs may be beneficial in a clinical setting to promote healthy immune responses to respiratory virus vaccines in vitamin-deficient individuals.

A new safe and effective cold-adapted modified live equine influenza virus vaccine that enables the differentiation of infected from vaccinated animals

ABSTRACT An analysis of the main advantages and shortcomings of the existing inactivated and live vaccines against the equine influenza viruses (EIVs) is given in this paper. For the first time, the most important information, concerning the development of a new live modified cold-adapted (ca) equine influenza virus vaccine based on the A/HK/Otar/6:2/2010 strain is summarized. We discuss a number of unique features of the developed vaccine that have not previously been reported, and compare the new vaccine with the existing equine influenza vaccines. The properties of the developed equine vaccine include: long-lasting (12 months or more) protective immunity after a single immunization; sterile immunity after double vaccination; cross-protection against the heterologous virus at 12 months after double vaccination and the differentiation of infected from vaccinated animals.

Новая безопасная и эффективная живая модифицированная холодоадаптированная вирусная вакцина против гриппа лошадей, позволяющая дифференцировать инфицированных животных от вакцинированных

В настоящей работе дается анализ основных преимуществ и недостатков применяемых на практике инактивированных и живых вакцин против гриппа лошадей. Впервые обобщены наиболее значимые сведения, касающиеся разработки новой живой модифицированной холодоадаптированной вирусной вакцины против гриппа лошадей на основе штамма А/HK/Otar/6:2/2010. Обсуждается ряд уникальных свойств разработанной вакцины, о которых ранее не сообщалось, в сравнении с аналогичными вакцинами против гриппа лошадей. К числу подобных свойств вакцины относятся: продолжительный (не менее 12 месяцев) протективный иммунитет после однократной вакцинации; стерильный иммунитет после двукратной вакцинации; перекрестная защита от гетерологичного вируса через 12 месяцев после двукратной вакцинации; дифференциация инфицированных животных от вакцинированных.

Разработка холодоадаптированного реассортантного вируса гриппа А/H6N1 на основе донора аттенуации A/Ленинград/134/17/57 (H2N2) и его генотипирование методом анализа кривых плавления высокого разрешения (HRM-анализ)

Холодоадаптированный реассортантный штамм вируса гриппа А/17/серебристая чайка/Сарма/2006/887 (H6N1) получен на основе донора аттенуации А/Ленинград/134/17/57 (H2N2) методом генетической реассортации в куриных эмбрионах. Структура генома реассортанта была проанализирована методом ПЦР в реальном времени с последующим анализом кривых плавления высокого разрешения (high resolution melting, HRM-анализ) при использовании интеркалирующего флуоресцентного красителя EvaGreen. Анализ кривых плавления ДНК копий РНК фрагментов (PB2, PB1, PA, NP, М, NS) показал, что реассортантный вирус гриппа А/17/серебристая чайка/Сарма/2006/887 (H6N1) унаследовал гены внутренних и неструктурных белков от донора аттенуации. По антигенным свойствам реассортант соответствовал вирусу гриппа птиц А/серебристая чайка/Сарма/51с/2006 (H6N1). Изучение фенотипических свойств реассортанта показало, что вирус А/17/ серебристая чайка/Сарма/2006/887 (H6N1) характеризуется температурочувствительностью и холодоадаптированностью в куриных эмбрионах и аттенуирован для мышей при интраназальном введении. Данный реассортант может быть рекомендован кандидатом в вакцинные штаммы для живой гриппозной вакцины для людей.

Development of the A/H6N1 influenza vaccine candidate based on A/Leningrad/134/17/57 (H2N2) master donor virus and the genome composition analysis using high resolution melting (HRM)

ABSTRACT The avian influenza viruses of H6N1subtype present a potential danger for humans. The cold-adapted (ca) reassortant influenza virus A/17/herring gull/Sarma/2006/887 (H6N1) was obtained in chicken embryos by the genetic reassortment based on the cold-adapted A/Leningrad/134/17/57 (H2N2) master strain. The genome composition of the obtained reassortant was analyzed by means of real-time PCR with the high resolution melting (HRM) analysis using the intercalating fluorescent dye EvaGreen. Analysis of the gene segments showed that the reassortant A/17/herring gull/Sarma/2006/887 (H6N1) contains the internal proteins coding genes (PB2, PB1, PA, NP, M, and NS) of the master donor virus and the surface antigens coding genes of the A/herring gull/Sarma/51c/2006 (H6N1) avian influenza virus. The study of the phenotypic properties showed that the virus A/17/herring gull/Sarma/2006/887 (H6N1) is temperature sensitive (ts), ca in chicken embryos, and attenuated in mice when administered intranasally. This reassortant can be recommended as a live influenza vaccine candidate for humans.

Replication of live attenuated cold-adapted H2N2 influenza virus vaccine candidates in non human primates

The development of an H2N2 vaccine is a priority in pandemic preparedness planning. We previously showed that a single dose of a cold-adapted (ca) H2N2 live attenuated influenza vaccine (LAIV) based on the influenza A/Ann Arbor/6/60 (AA ca) virus was immunogenic and efficacious in mice and ferrets. However, in a Phase I clinical trial, viral replication was restricted and immunogenicity was poor. In this study, we compared the replication of four H2N2 LAIV candidate viruses, AA ca, A/Tecumseh/3/67 (TEC67 ca), and two variants of A/Japan/305/57 (JAP57 ca) in three non-human primate (NHP) species: African green monkeys (AGM), cynomolgus macaques (CM) and rhesus macaques (RM). One JAP57 ca virus had glutamine and glycine at HA amino acid positions 226 and 228 (Q–G) that binds to α2-3 linked sialic acids, and one had leucine and serine that binds to α2-3 and α2-6 linked residues (L–S). The replication of all ca viruses was restricted, with low titers detected in the upper respiratory tract of all NHP species, however replication was detected in significantly more CMs than AGMs. The JAP57 ca Q–G and TEC67 ca viruses replicated in a significantly higher percentage of NHPs than the AA ca virus, with the TEC67 ca virus recovered from the greatest percentage of animals. Altering the receptor specificity of the JAP57 ca virus from α2-3 to both α2-3 and α2-6 linked sialic acid residues did not significantly increase the number of animals infected or the titer to which the virus replicated. Taken together, our data show that in NHPs the AA ca virus more closely reflects the human experience than mice or ferret studies. We suggest that CMs and RMs may be the preferred species for evaluating H2N2 LAIV viruses, and the TEC67 ca virus may be the most promising H2N2 LAIV candidate for further evaluation.

Development of a High-Yield Live Attenuated H7N9 Influenza Virus Vaccine That Provides Protection against Homologous and Heterologous H7 Wild-Type Viruses in Ferrets

Live attenuated H7N9 influenza vaccine viruses that possess the hemagglutinin (HA) and neuraminidase (NA) gene segments from the newly emerged wild-type (wt) A/Anhui/1/2013 (H7N9) and six internal protein gene segments from the cold-adapted influenza virus A/Ann Arbor/6/60 (AA ca) were generated by reverse genetics. The reassortant virus containing the original wt A/Anhui/1/2013 HA and NA sequences replicated poorly in eggs. Multiple variants with amino acid substitutions in the HA head domain that improved viral growth were identified by viral passage in eggs and MDCK cells. The selected vaccine virus containing two amino acid changes (N133D/G198E) in the HA improved viral titer by more than 10-fold (reached a titer of 10(8.6) fluorescent focus units/ml) without affecting viral antigenicity. Introduction of these amino acid changes into an H7N9 PR8 reassortant virus also significantly improved viral titers and HA protein yield in eggs. The H7N9 ca vaccine virus was immunogenic in ferrets. A single dose of vaccine conferred complete protection of ferrets from homologous wt A/Anhui/1/2013 (H7N9) and nearly complete protection from heterologous wt A/Netherlands/219/2003 (H7N7) challenge infection. Therefore, this H7N9 live attenuated influenza vaccine (LAIV) candidate has been selected for vaccine manufacture and clinical evaluation to protect humans from wt H7N9 virus infection. In response to the recent avian H7N9 influenza virus infection in humans, we developed a live attenuated H7N9 influenza vaccine (LAIV) with two amino acid substitutions in the viral HA protein that improved vaccine yield by 10-fold in chicken embryonated eggs, the substrate for vaccine manufacture. The two amino acids also improved the antigen yield for inactivated H7N9 vaccines, demonstrating that this finding could great facilitate the efficiency of H7N9 vaccine manufacture. The candidate H7N9 LAIV was immunogenic and protected ferrets against homologous and heterologous wild-type H7 virus challenge, making it suitable for use in protecting humans from H7 infection.

Live attenuated influenza viruses produced in a suspension process with avian AGE1.CR.pIX cells.

BackgroundCurrent influenza vaccines are trivalent or quadrivalent inactivated split or subunit vaccines administered intramuscularly, or live attenuated influenza vaccines (LAIV) adapted to replicate at temperatures below body temperature and administered intranasally. Both vaccines are considered safe and efficient, but due to differences in specific properties may complement each other to ensure reliable vaccine coverage. By now, licensed LAIV are produced in embryonated chicken eggs. In the near future influenza vaccines for human use will also be available from adherent MDCK or Vero cell cultures, but a scalable suspension process may facilitate production and supply with vaccines.ResultsWe evaluated the production of cold-adapted human influenza virus strains in the duck suspension cell line AGE1.CR.pIX using a chemically-defined medium. One cold-adapted A (H1N1) and one cold-adapted B virus strain was tested, as well as the reference strain A/PR/8/34 (H1N1). It is shown that a medium exchange is not required for infection and that maximum virus titers are obtained for 1 × 10-6 trypsin units per cell. 1 L bioreactor cultivations showed that 4 × 106 cells/mL can be infected without a cell density effect achieving titers of 1 × 108 virions/mL after 24 h.ConclusionsOverall, this study demonstrates that AGE1.CR.pIX cells support replication of LAIV strains in a chemically-defined medium using a simple process without medium exchanges. Moreover, the process is fast with peak titers obtained 24 h post infection and easily scalable to industrial volumes as neither microcarriers nor medium replacements are required.

Vitamin A Deficiency Disrupts Vaccine-Induced Antibody-Forming Cells and the Balance of IgA/IgG Isotypes in the Upper and Lower Respiratory Tract

Vaccination by intranasal (IN) inoculation with a replication-competent virus forms the basis of licensed and novel candidate respiratory viral vaccines (e.g., the cold-adapted influenza virus vaccine). A positive global impact of vaccination depends on vaccine efficacy in developing countries where dietary deficiencies are commonplace. The current study was designed using Sendai virus (SeV) as a model respiratory viral vaccine to test antibody-forming cell (AFC) residence and isotype expression in the context of a vitamin A deficiency (VAD). Samples were taken 1 mo after vaccination when AFCs generally reach their peak in healthy animals. In control animals on a healthy diet, SeV induced an antibody response with a relative bias toward IgA in the upper respiratory tract (URT, as sampled by nasal wash), and IgG in the lower respiratory tract (LRT, as sampled by bronchoalveolar lavage [BAL]). In the context of VAD, the SeV-specific IgA antibodies in the nasal wash were significantly reduced in favor of enhanced IgG antibodies in the BAL. When AFCs were examined in diffuse nasal-associated lymphoid tissues (d-NALT), lungs, cervical lymph nodes (CLN), and mediastinal lymph nodes (MLN), a similar pattern emerged. AFCs were most frequent in the d-NALT and most expressed IgA in control mice. In the context of VAD, these IgA-producing AFCs were significantly reduced in number, skewing the natural balance of IgA and IgG. Taken together, the results show that the VAD diet, which is well known for its association with immune defects in the gut, significantly alters AFC induction and isotype expression in the respiratory tract.