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Preclinical evaluation of a replication-deficient intranasal DeltaNS1 H5N1 influenza vaccine.

ABSTRACT: BACKGROUND:We developed a novel intranasal influenza vaccine approach that is based on the construction of replication-deficient vaccine viruses that lack the entire NS1 gene (DeltaNS1 virus). We previously showed that these viruses undergo abortive replication in the respiratory tract of animals. The local release of type I interferons and other cytokines and chemokines in the upper respiratory tract may have a "self-adjuvant effect", in turn increasing vaccine immunogenicity. As a result, DeltaNS1 viruses elicit strong B- and T- cell mediated immune responses. METHODOLOGY/PRINCIPAL FINDINGS:We applied this technology to the development of a pandemic H5N1 vaccine candidate. The vaccine virus was constructed by reverse genetics in Vero cells, as a 5:3 reassortant, encoding four proteins HA, NA, M1, and M2 of the A/Vietnam/1203/04 virus while the remaining genes were derived from IVR-116. The HA cleavage site was modified in a trypsin dependent manner, serving as the second attenuation factor in addition to the deleted NS1 gene. The vaccine candidate was able to grow in the Vero cells that were cultivated in a serum free medium to titers exceeding 8 log(10) TCID(50)/ml. The vaccine virus was replication deficient in interferon competent cells and did not lead to viral shedding in the vaccinated animals. The studies performed in three animal models confirmed the safety and immunogenicity of the vaccine. Intranasal immunization protected ferrets and mice from being infected with influenza H5 viruses of different clades. In a primate model (Macaca mulatta), one dose of vaccine delivered intranasally was sufficient for the induction of antibodies against homologous A/Vietnam/1203/04 and heterologous A/Indonesia/5/05 H5N1 strains. CONCLUSION/SIGNIFICANCE:Our findings show that intranasal immunization with the replication deficient H5N1 DeltaNS1 vaccine candidate is sufficient to induce a protective immune response against H5N1 viruses. This approach might be attractive as an alternative to conventional influenza vaccines. Clinical evaluation of DeltaNS1 pandemic and seasonal influenza vaccine candidates are currently in progress.

SUBMITTER: Romanova J

PROVIDER: S-EPMC2694350 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Preclinical evaluation of a replication-deficient intranasal DeltaNS1 H5N1 influenza vaccine.

Romanova Julia J Krenn Brigitte M BM Wolschek Markus M Ferko Boris B Romanovskaja-Romanko Ekaterina E Morokutti Alexander A Shurygina Anna-Polina AP Nakowitsch Sabine S Ruthsatz Tanja T Kiefmann Bettina B König Ulrich U Bergmann Michael M Sachet Monika M Balasingam Shobana S Mann Alexander A Oxford John J Slais Martin M Kiselev Oleg O Muster Thomas T Egorov Andrej A

PloS one 20090619 6

<h4>Background</h4>We developed a novel intranasal influenza vaccine approach that is based on the construction of replication-deficient vaccine viruses that lack the entire NS1 gene (DeltaNS1 virus). We previously showed that these viruses undergo abortive replication in the respiratory tract of animals. The local release of type I interferons and other cytokines and chemokines in the upper respiratory tract may have a "self-adjuvant effect", in turn increasing vaccine immunogenicity. As a resu ...[more]

PMID: 19543385

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Project description:Abstract Background Influenza vaccines are needed with greater effectiveness and breadth of coverage. FluGen is developing M2SR (M2 deficient Single Replication), an investigational, live virus vaccine. M2SR contains the internal proteins of donor A/Puerto Rico/8/34 and hemagglutinin (HA) and neuraminidase (NA) selected from targeted Type A influenza strains. M2SR undergoes only a single round of infection in the respiratory epithelium but evokes an immune response profile similar to wild-type influenza viruses. In influenza naïve and pre-immune ferrets, M2SR protects against multiple influenza A subtypes. Methods A Phase 1, first-in-human, randomized, placebo-control study (FluGen-H3N2-V001; ClinicalTrials.gov identifier NCT02822105) was conducted at a single USA site, with 96 adults, ages 18–49 years. Study vaccine contained HA and NA from A/Brisbane/10/2007 (H3N2). Study volunteers received a single intranasal (IN) inoculation with either M2SR at dose levels of 106, 107 or 108 TCID50 or saline placebo (N = 24/cohort). Study subjects were evaluated for virus replication and solicited local and systemic reactions for 7 days, all adverse events (AE) for 28 days and serious AE (SAE) for 180 days. Results No infectious virus was detected in nasal swabs from any vaccinated subject. The most commonly reported AE was mild nasal rhinorrhea/congestion during the first 7 days after vaccination (Figure 1). No subject had fever or a severe reaction to the vaccine. No SAEs were reported. At least one AE was reported among 29%, 58%, and 83% of M2SR subjects administered 106, 107, or 108 TCID50, respectively, and 46% among placebo subjects. There were no notable imbalances among study groups for other events. T- and B-cell responses, including influenza-specific serum and mucosal antibody responses were detected at a significantly higher frequency among vaccine than placebo subjects (Figure 2).Figure 1. Common adverse events first 7 days post-vaccinationFigure 2. Influenza-specific immune responses Conclusion M2SR vaccine was safe and well tolerated at all dose levels, generated a dose-response effect for humoral (HA antibody) and mucosal antibodies against both homologous and heterologous influenza variants, and elicited robust T-cell responses. No infectious virus was detected in nasal swabs from any vaccinated subject. Disclosures J. Eiden, FluGen, Inc.: Consultant, Consulting fee and Stock Options. G. Gordon, FluGen: Consultant, Consulting fee. C. Fierro, FluGen: Investigator, Fee for service. R. Belshe, FluGen: Consultant, Consulting fee and Stock Options. H. Greenberg, FluGen: Consultant, Consulting fee and Stock OpTions. D. Hoft, FluGen: Research Contractor, Fee for service. Y. Hatta, FluGen: Employee, Salary and Stock Options. M. Imboden, FluGen: Employee, Salary and Stock Options. M. Moser, FluGen: Employee, Salary and Stock Options. R. Herber, FluGen: Employee, Salary and Stock Options. D. Boltz, FluGen: Research Contractor, fee for service. M. Tary-Lehmann, FluGen: Research Contractor, Fee for service. CTL: Founder and Owner, Salary. Y. Kawaoka, FluGen: Founder, Stock Options. G. Neumann, FluGen: Founder, Stock Options. P. Radspinner, FluGen: Board Member and Founder, Salary and Stock Options. R. Aitchison, FluGen: Consultant, Consulting fee. P. Bilsel, FluGen: Employee, Salary and Stock Options.

Project description:Abstract Background A single intranasal (IN) dose of 108 TCID50 M2SR protected a responder subset of adults against infection and disease in a prior human influenza challenge study (EudraCT number: 2017-004971-30). Higher dose levels of M2SR were assessed in this phase 1b study to enhance immune responses and further increase protection levels in adults. Methods A double-blind, randomized, placebo-controlled dose escalation study (NCT03999554) was conducted at 4 US study sites with two different H3N2 M2SR vaccines that contained HA & NA from either A/Brisbane/10/2007 or A/Singapore/INFIMH-16–0019/2016. Serosusceptible 18–49 year old subjects (n = 206) received 2 IN doses of either saline or 1 of 3 different dose levels of vaccine (108 – 109 TCID50), administered 28 days apart. Results Study vaccination was well-tolerated at all dose levels. A single 109 dose of A/Singapore/2016 M2SR generated significantly increased serum HAI responses compared to the 108 dose of A/Brisbane/10/2007 M2SR that had provided protection against infection & illness in an earlier human influenza challenge study (Fig. 1). HAI titers ≥40 were achieved in 0%, 23% & 58% of subjects after the first dose of placebo, 108 or 109 M2SR, respectively (p< 0.003). Increases also were stimulated in serum microneutralization titers (MNT) to drifted strains of H3N2 (Fig 2) & in serum NAI (Fig 3) and mucosal sIgA (Fig 4) titers. Further increases in serum and mucosal immune response were noted after a 2nd IN vaccination. Proportion of subjects with seroprotective HAI titers after vaccination Proportion of subjects with increased microneutralization titers against drifted H3N2 viruses after vaccination Geometric mean fold rise in serum neuraminidase inhibition antibody titers after vaccination Conclusion An earlier clinical trial with a 108 dose of M2SR provided protection against infection and illness upon challenge with a highly drifted strain of H3N2. Protection correlated with vaccine induced serum MNT responses. In the current study, a single, 109 dose of M2SR significantly increased serum MNT, HAI & NAI titers as well as mucosal immune responses among greater proportions of study subjects. Since HAI, alone, is a well-accepted surrogate marker for vaccine protection against influenza, these broader enhancements indicate the potential for M2SR to protect against both matched and drifted strains of influenza in a high proportion of adults and strongly support clinical assessment in younger and older age groups as well as development of multivalent M2SR. Geometric mean fold rise in nasal mucosal secretory IgA antibody titers after vaccination Disclosures Joseph Eiden, MD, PhD, FluGen (Consultant) Ruth Ellis, MD, FluGen (Consultant) Roger Aitchison, ScM, FluGen (Consultant) Renee Herber, BS, FluGen (Employee) Pamuk Bilsel, PhD, FluGen (Employee)

Project description:UNLABELLED:The recent swine H1N1 influenza outbreak demonstrated that egg-based vaccine manufacturing has an Achille's heel: its inability to provide a large number of doses quickly. Using a novel manufacturing platform based on transient expression of influenza surface glycoproteins in Nicotiana benthamiana, we have recently demonstrated that a candidate Virus-Like Particle (VLP) vaccine can be generated within 3 weeks of release of sequence information. Herein we report that alum-adjuvanted plant-made VLPs containing the hemagglutinin (HA) protein of H5N1 influenza (A/Indonesia/5/05) can induce cross-reactive antibodies in ferrets. Even low doses of this vaccine prevented pathology and reduced viral loads following heterotypic lethal challenge. We further report on safety and immunogenicity from a Phase I clinical study of the plant-made H5 VLP vaccine in healthy adults 18-60 years of age who received 2 doses 21 days apart of 5, 10 or 20 µg of alum-adjuvanted H5 VLP vaccine or placebo (alum). The vaccine was well tolerated at all doses. Adverse events (AE) were mild-to-moderate and self-limited. Pain at the injection site was the most frequent AE, reported in 70% of vaccinated subjects versus 50% of the placebo recipients. No allergic reactions were reported and the plant-made vaccine did not significantly increase the level of naturally occurring serum antibodies to plant-specific sugar moieties. The immunogenicity of the H5 VLP vaccine was evaluated by Hemagglutination-Inhibition (HI), Single Radial Hemolysis (SRH) and MicroNeutralisation (MN). Results from these three assays were highly correlated and showed similar trends across doses. There was a clear dose-response in all measures of immunogenicity and almost 96% of those in the higher dose groups (2 × 10 or 20 µg) mounted detectable MN responses. Evidence of striking cross-protection in ferrets combined with a good safety profile and promising immunogenicity in humans suggest that plant-based VLP vaccines should be further evaluated for use in pre-pandemic or pandemic situations. TRIAL REGISTRATION:ClinicalTrials.gov NCT00984945.

Project description:Abstract Background Demonstration of protection by a M2SR (M2 deficient Single Replication) monovalent H3N2 vaccine was assessed in a phase 2a clinical trial in which the challenge virus was substantially drifted from the vaccine. M2SR is an investigational, live virus vaccine containing hemagglutinin (HA) and neuraminidase (NA) selected from targeted Type A influenza strains. M2SR undergoes only a single round of infection in the respiratory epithelium but evokes an immune response profile similar to wild-type influenza virus and protects ferrets against both homologous and heterologous influenza variants. Methods A blinded, randomized, placebo-controlled human challenge study (EudraCT #: 2017-004971-30) was conducted with M2SR containing HA and NA from A/Brisbane/10/2007 (H3N2). 18–55-year-old subjects received 1 IN dose of saline or 108 TCID50 of vaccine. 4 weeks later, 99 subjects were challenged IN with 106 TCID50 H3N2 A/Belgium/4217/2015 (Figures 1 and 2). Results Adverse events (AE) were similar between placebo (N = 51) and M2SR recipients (N = 48) during the 28 days after immunization. After challenge with A/Belgium/4217/2015, 35% of M2SR recipients experienced influenza infection and illness, compared with 49% of placebo subjects (Figure 3). An 18% reduction in viral load was noted after challenge for M2SR subjects. Serum microneutralization response to vaccine was detected in 54% of M2SR subjects (vs. 0/51 placebo subjects), and among these subjects a 34% reduction in viral load and 51% reduction in symptom scores was noted after challenge vs placebo. Among the 29% of subjects with post-vaccine response to both vaccine and challenge strains, a 62% reduction in viral load and 56% reduction in symptom scores was noted after challenge with highly drifted H3N2 (Figure 4). Conclusion One dose of M2SR protected healthy adults against influenza infection and illness with a highly drifted challenge strain. This is believed to be the first study to demonstrate protection against challenge with an influenza strain substantially different from the vaccine and indicates potential for improved breadth of protection by M2SR compared with current vaccines. The mild vaccine AE profile supports clinical trials of additional dose levels and regimens to enhance drifted strain protection by M2SR. Disclosures All authors: No reported disclosures.

Project description:Aim:The need for non-parenteral administration of inactivated avian influenza virus of H5N1 subtype (AIV-H5N1) vaccine is paramount. Here, we provide preliminary data on the immune response of chicken and mice after intranasal administration of AIV-H5N1-inactivated vaccine with ISCOMS, Inmunair (INM), and combined ISCOMS and INM as an adjuvant. Materials and Methods:The AIV isolate of A/Chicken/Denpasar/01/2004 (H5N1) was cultivated in specific pathogen-free chicken eggs and inactivated with formaldehyde. The vaccine preparation was added with those adjuvants for intranasal administration and aluminum hydroxide for subcutaneous injection. The chicken and mouse were vaccinated at the age of 3 weeks or 1 month and repeated 2 weeks thereafter. In one experiment, chicken was injected with Newcastle disease virus (NDV) at the same time with AIV vaccine. The sera were collected at one (serum 1) and 2 w (serum 2) after booster vaccination. The anti-AIV-H5 and NDV antibodies in chicken sera were detected using hemagglutination inhibition (HI) assay. Mouse IgG anti-AIV-H5N1 antibody was detected using ELISA. Results:The result shows that the geometric mean titers (GMTs) of chicken sera of intranasal vaccinated with inactivated AIV-H5N1 vaccine with mixed ISCOM- INM as adjuvant were <20.0 and 22.7 unit HI-unit (HIU) in serum 1 and serum 2, respectively. The GMTs of the positive control group were 23.7 and 25.7 HIU in serum 1 and serum 2, respectively. The result of the second experiment shows that IgG anti-AIV-H5N1 was detected in mouse sera. In the third experiment, the GMTs of anti-NDV in chicken vaccinated subsequently with inactivated NDV vaccine and AIV-H5N1 with mixed ISCOMS-INM administrated intranasally and aluminum hydroxide adjuvant administrated through subcutaneous injection as well as positive control group receiving NDV vaccine only were 28.0, 28.0, and 27.4 HIU in serum 1 while were 29.6, 29.2, and 28.2 HIU in serum 2, respectively. Conclusion:Intranasal administration of inactivated AIV-H5N1 vaccine-induced a systemic immune response in chicken and mice after adding ISCOMS and/or INM as adjuvants. The adjuvant and the intranasal administration caused no immunosuppressive effect on the chicken immune response to NDV vaccine.

Project description:BACKGROUND: H5N1 influenza vaccines, including live intranasal, appear to be relatively less immunogenic compared to seasonal analogs. The main influenza virus surface glycoprotein hemagglutinin (HA) of highly pathogenic avian influenza viruses (HPAIV) was shown to be more susceptible to acidic pH treatment than that of human or low pathogenic avian influenza viruses. The acidification machinery of the human nasal passageway in response to different irritation factors starts to release protons acidifying the mucosal surface (down to pH of 5.2). We hypothesized that the sensitivity of H5 HA to the acidic environment might be the reason for the low infectivity and immunogenicity of intranasal H5N1 vaccines for mammals. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that original human influenza viruses infect primary human nasal epithelial cells at acidic pH (down to 5.4), whereas H5N1 HPAIVs lose infectivity at pH ? 5.6. The HA of A/Vietnam/1203/04 was modified by introducing the single substitution HA2 58K?I, decreasing the pH of the HA conformational change. The H5N1 reassortants containing the indicated mutation displayed an increased resistance to acidic pH and high temperature treatment compared to those lacking modification. The mutation ensured a higher viral uptake as shown by immunohistochemistry in the respiratory tract of mice and 25 times lower mouse infectious dose??. Moreover, the reassortants keeping 58K?I mutation designed as a live attenuated vaccine candidate lacking an NS1 gene induced superior systemic and local antibody response after the intranasal immunization of mice. CONCLUSION/SIGNIFICANCE: Our finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may play a substantial role in the infectivity of HPAIVs for mammals.

Dataset Information

Preclinical evaluation of a replication-deficient intranasal DeltaNS1 H5N1 influenza vaccine.

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Preclinical evaluation of a replication-deficient intranasal DeltaNS1 H5N1 influenza vaccine.

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