Generation and characterization of a cold-adapted influenza A H9N2 reassortant as a live pandemic influenza virus vaccine candidate

H9N2 subtype influenza A viruses have been identified in avian species worldwide and were isolated from humans in 1999, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this subtype of influenza A virus. Reassortant H1N1 and H3N2 human influenza A viruses with the internal genes of the influenza A/Ann Arbor/6/60 (H2N2) (AA) cold-adapted (ca) virus have proven to be attenuated and safe as live virus vaccines in humans. Using classical genetic reassortment, we generated a reassortant virus (G9/AA ca) that contains the hemagglutinin and neuraminidase genes from influenza A/chicken/Hong Kong/G9/97 (H9N2) (G9) and six internal gene segments from the AA ca virus. When administered intranasally, the reassortant virus was immunogenic and protected mice from subsequent challenge with wild-type H9N2 viruses, although it was restricted in replication in the respiratory tract of mice. The G9/AA ca virus bears properties that are desirable in a vaccine for humans and is available for clinical evaluation and use, should the need arise.     

Evaluation of replication, immunogenicity and protective efficacy of a live attenuated cold-adapted pandemic H1N1 influenza virus vaccine in non-human primates

We studied the replication of influenza A/California/07/09 (H1N1) wild type (CA09wt) virus in two non-human primate species and used one of these models to evaluate the immunogenicity and protective efficacy of a live attenuated cold-adapted vaccine, which contains the hemagglutinin and neuraminidase from the H1N1 wild type (wt) virus and six internal protein gene segments of the A/Ann Arbor/6/60 cold-adapted (ca) master donor virus. We infected African green monkeys (AGMs) and rhesus macaques with 2×10(6) TCID(50) of CA09wt and CA09ca influenza viruses. The virus CA09wt replicated in the upper respiratory tract of all animals but the titers in upper respiratory tract tissues of rhesus macaques were significant higher than in AGMs (mean peak titers 10(4.5) TCID(50)/g and 10(2.0) TCID(50)/g on days 4 and 2 post-infection, respectively; p<0.01). Virus replication was observed in the lungs of all rhesus macaques (10(2.0)-10(5.4) TCID(50)/g) whereas only 2 out of 4 AGMs had virus recovered from the lungs (10(2.5)-10(3.5) TCID(50)/g). The CA09ca vaccine virus was attenuated and highly restricted in replication in both AGMs and rhesus macaques. We evaluated the immunogenicity and protective efficacy of the CA09ca vaccine in rhesus macaques because CA09wt virus replicated more efficiently in this species. One or two doses of vaccine were administered intranasally and intratracheally to rhesus macaques. For the two-dose group, the vaccine was administered 4-weeks apart. Immunogenicity was assessed by measuring hemagglutination-inhibiting (HAI) antibodies in the serum and specific IgA antibodies to CA09wt virus in the nasal wash. One or two doses of the vaccine elicited a significant rise in HAI titers (range 40-320). Two doses of CA09ca elicited higher pH1N1-specific IgA titers than in the mock-immunized group (p<0.01). Vaccine efficacy was assessed by comparing titers of CA09wt challenge virus in the respiratory tract of mock-immunized and CA09ca vaccinated monkeys. Significantly lower virus titers were observed in the lungs of vaccinated animals than mock-immunized animals (p≤0.01). Our results demonstrate that AGMs and rhesus macaques support the replication of pandemic H1N1 influenza virus to different degrees and a cold-adapted pH1N1 vaccine elicits protective immunity against pH1N1 virus infection in rhesus macaques.     

Characterization of an influenza A H5N2 reassortant as a candidate for live-attenuated and inactivated vaccines against highly pathogenic H5N1 viruses with pandemic potential

We generated a high-growth 7:1 reassortant (Len17/H5) that contained the hemagglutinin (HA) gene from non-pathogenic A/Duck/Potsdam/1402-6/86 (H5N2) virus and other genes from the cold-adapted (ca) attenuated A/Leningrad/134/17/57 (H2H2) strain. Len17/H5 demonstrated an attenuated phenotype in mice and did not infect chickens. Mice administered Len17/H5 either as a live-attenuated intranasal vaccine or as an inactivated intramuscular vaccine were substantially protected from lethal challenge with highly pathogenic A/Hong Kong/483/97 (H5N1) virus and were protected from pulmonary infection with antigenically distinct A/Hong Kong/213/2003 (H5N1) virus. The cross-protective effect correlated with the levels of virus-specific mucosal IgA and/or serum IgG antibodies. Our results suggest a new strategy of using classical genetic reassortment between a high-growth ca H2N2 strain and antigenically related non-pathogenic avian viruses to prepare live-attenuated and inactivated vaccines for influenza pandemic.     

Efficacy of seasonal live attenuated influenza vaccine against virus replication and transmission of a pandemic 2009 H1N1 virus in ferrets

In March 2009, a swine origin influenza A (2009 H1N1) virus was introduced into the human population and quickly spread from North America to multiple continents. Human serologic studies suggest that seasonal influenza virus vaccination or infection would provide little cross-reactive serologic immunity to the pandemic 2009 H1N1 virus. However, the efficacy of seasonal influenza infection or vaccination against 2009 H1N1 virus replication and transmission has not been adequately evaluated in vivo. Here, ferrets received one or two doses of the US licensed 2008-2009 live attenuated influenza vaccine (LAIV) intranasally. An additional group of ferrets were inoculated with the A/Brisbane/59/07 (H1N1) virus to model immunity induced by seasonal influenza virus infection. All vaccinated and infected animals possessed high titer homologous hemagglutination-inhibition (HI) and neutralizing antibodies, with no demonstrable cross-reactive antibodies against 2009 H1N1 virus. However, in comparison to non-immune controls, immunized ferrets challenged with pandemic A/Mexico/4482/09 virus displayed a significant reduction in body temperature and virus shedding. The impact of single-dose LAIV inoculation on 2009 H1N1 disease and virus transmission was also measured in vaccinated ferrets that were challenged with pandemic A/Netherlands/1132/09 virus. Although a single dose of LAIV reduced virus shedding and the frequency of transmission following homologous seasonal virus challenge, it failed to reduce respiratory droplet transmission of 2009 H1N1 virus. The results demonstrate that prior immunization with seasonal LAIV or H1N1 virus infection provides some cross-protection against the 2009 H1N1 virus, but had no significant effect on the transmission efficiency of the 2009 H1N1 virus.     

Evaluation of the attenuation,immunogenicity, and efficacy of a live virus vaccine generated by codon-pair bias de-optimization of the 2009 pandemic H1N1 influenza virus,in ferrets

Codon-pair bias de-optimization (CPBD) of viruses involves re-writing viral genes using statistically underrepresented codon pairs, without any changes to the amino acid sequence or codon usage. Previously, this technology has been used to attenuate the influenza A/Puerto Rico/8/34 (H1N1) virus. The de-optimized virus was immunogenic and protected inbred mice from challenge. In order to assess whether CPBD could be used to produce a live vaccine against a clinically relevant influenza virus, we generated an influenza A/California/07/2009 pandemic H1N1 (2009 pH1N1) virus with de-optimized HA and NA gene segments (2009 pH1N1-(HA + NA)^Min), and evaluated viral replication and protein expression in MDCK cells, and attenuation, immunogenicity, and efficacy in outbred ferrets. The 2009 pH1N1-(HA + NA)^Min virus grew to a similar titer as the 2009 pH1N1 wild type (wt) virus in MDCK cells (∼10⁶ TCID₅₀/ml), despite reduced HA and NA protein expression on western blot. In ferrets, intranasal inoculation of 2009 pH1N1-(HA + NA)^Min virus at doses ranging from 10³ to 10⁵ TCID₅₀ led to seroconversion in all animals and protection from challenge with the 2009 pH1N1 wt virus 28 days later. The 2009 pH1N1-(HA + NA)^Min virus did not cause clinical illness in ferrets, but replicated to a similar titer as the wt virus in the upper and lower respiratory tract, suggesting that de-optimization of additional gene segments may be warranted for improved attenuation. Taken together, our data demonstrate the potential of using CPBD technology for the development of a live influenza virus vaccine if the level of attenuation is optimized.     

Engineering temperature sensitive live attenuated influenza vaccines from emerging viruses

The licensed live attenuated influenza A vaccine (LAIV) in the United States is created by making a reassortant containing six internal genes from a cold-adapted master donor strain (ca A/AA/6/60) and two surface glycoprotein genes from a circulating/emerging strain (e.g., A/CA/7/09 for the 2009/2010 H1N1 pandemic). Technologies to rapidly create recombinant viruses directly from patient specimens were used to engineer alternative LAIV candidates that have genomes composed entirely of vRNAs from pandemic or seasonal strains. Multiple mutations involved in the temperature-sensitive (ts) phenotype of the ca A/AA/6/60 master donor strain were introduced into a 2009 H1N1 pandemic strain rA/New York/1682/2009 (rNY1682-WT) to create rNY1682-TS1, and additional mutations identified in other ts viruses were added to rNY1682-TS1 to create rNY1682-TS2. Both rNY1682-TS1 and rNY1682-TS2 replicated efficiently at 30 °C and 33 °C. However, rNY1682-TS1 was partially restricted, and rNY1682-TS2 was completely restricted at 39 °C. Additionally, engineering the TS1 or TS2 mutations into a distantly related human seasonal H1N1 influenza A virus also resulted pronounced restriction of replication in vitro. Clinical symptoms and virus replication in the lungs of mice showed that although rNY1682-TS2 and the licensed FluMist^®-H1N1pdm LAIV that was used to combat the 2009/2010 pandemic were similarly attenuated, the rNY1682-TS2 was more protective upon challenge with a virulent mutant of pandemic H1N1 virus or a heterologous H1N1 (A/PR/8/1934) virus. This study demonstrates that engineering key temperature sensitive mutations (PB1-K391E, D581G, A661T; PB2-P112S, N265S, N556D, Y658H) into the genomes of influenza A viruses attenuates divergent human virus lineages and provides an alternative strategy for the generation of LAIVs.     

Cold-adapted X-31 live attenuated 2009 pandemic H1N1 influenza vaccine elicits protective immune responses in mice and ferrets

The 2009 pandemic influenza H1N1 (pdmH1N1) is characterized by rapid transmission among humans and disproportionate infection to children and young adults. Although the pdmH1N1 demonstrated less lethality than initially expected and has now moved into its post-pandemic period, it remains highly possible that through antigenic shift or antigenic drift the pdmH1N1 might re-emerge in the future as a more virulent strain than before, underscoring the need for vaccination prior to an outbreak. Using X-31 ca as a backbone strain, we generated a live attenuated pdmH1N1 vaccine and evaluated its potential as a safe and effective vaccine using mouse and ferret models. Despite an acceptable level of attenuation phenotypes, single dose of immunization with the vaccine efficiently stimulated both systemic and mucosal antibody responses and provided complete protection against lethal challenge with wild type pdmH1N1 virus, even at the lowest immunization dose of 10³ PFU. The promising results of safety, immunogenicity, and protective efficacy of the vaccine not only contribute to expanding the repertoire of live vaccines as a judicious choice for pandemic H1N1 preparedness, but also suggest the great potential of X-31 ca donor strain to serve as reliable platform for generating diverse live vaccine constructs against seasonal influenza viruses and other pandemic strains.     

Comparison of egg and high yielding MDCK cell-derived live attenuated influenza virus for commercial production of trivalent influenza vaccine: In vitro cell susceptibility and influenza virus replication kinetics in permissive and semi-permissive cells

Currently MedImmune manufactures cold-adapted (ca) live, attenuated influenza vaccine (LAIV) from specific-pathogen free (SPF) chicken eggs. Difficulties in production scale-up and potential exposure of chicken flocks to avian influenza viruses especially in the event of a pandemic influenza outbreak have prompted evaluation and development of alternative non-egg based influenza vaccine manufacturing technologies. As part of MedImmune's effort to develop the live attenuated influenza vaccine (LAIV) using cell culture production technologies we have investigated the use of high yielding, cloned MDCK cells as a substrate for vaccine production by assessing host range and virus replication of influenza virus produced from both SPF egg and MDCK cell production technologies. In addition to cloned MDCK cells the indicator cell lines used to evaluate the impact of producing LAIV in cells on host range and replication included two human cell lines: human lung carcinoma (A549) cells and human muco-epidermoid bronchiolar carcinoma (NCI H292) cells. The influenza viruses used to infect the indicators cell lines represented both the egg and cell culture manufacturing processes and included virus strains that composed the 2006–2007 influenza seasonal trivalent vaccine (A/New Caledonia/20/99 (H1N1), A/Wisconsin/67/05 (H3N2) and B/Malaysia/2506/04). Results from this study demonstrate remarkable similarity between influenza viruses representing the current commercial egg produced and developmental MDCK cell produced vaccine production platforms. MedImmune's high yielding cloned MDCK cells used for the cell culture based vaccine production were highly permissive to both egg and cell produced ca attenuated influenza viruses. Both the A549 and NCI H292 cells regardless of production system were less permissive to influenza A and B viruses than the MDCK cells. Irrespective of the indicator cell line used the replication properties were similar between egg and the cell produced influenza viruses. Based on these study results we conclude that the MDCK cell produced and egg produced vaccine strains are highly comparable.     

Efficacy of live attenuated vaccines against 2009 pandemic H1N1 influenza in ferrets

The advent of the H1N1 influenza pandemic (pH1N1) in 2009 triggered the rapid production of pandemic influenza vaccines, since seasonal influenza vaccines were expected and demonstrated not to provide significant cross-protection against the newly emerged pandemic virus. To increase vaccine production capacity and further evaluate the effectiveness of different candidate pandemic influenza vaccines, the World Health Organization stimulated the evaluation of different vaccination concepts including the use of live attenuated influenza vaccines (LAIVs). Therefore, we have immunized ferrets intranasally with a single dose of pH1N1-LAIV from different manufacturers. They all induced adequate serum HI antibody titers in the ferrets and protected them against intratracheal wild-type pH1N1 virus challenge: pH1N1 virus replication in the upper respiratory tract and lungs was reduced and no disease signs or severe broncho-interstitial pneumonia were observed in any of the vaccinated ferrets. These data together with the relatively efficient production process emphasize the potential of the LAIV concept for pandemic preparedness.     

An elastase-dependent attenuated heterologous swine influenza virus protects against pandemic H1N1 2009 influenza challenge in swine

Influenza virus infections continue to cause production losses in the agricultural industry in addition to being a human public health concern. The primary method to control influenza is through vaccination. However, currently used killed influenza virus vaccines must be closely matched to the challenge virus. The ability of an elastase-dependent live attenuated influenza A virus was evaluated to protect pigs against the pandemic H1N1 2009 influenza virus. Pigs vaccinated intranasally or intratracheally with the elastase-dependent swine influenza virus (SIV) vaccine had significantly reduced macroscopic and microscopic lung lesions and lower viral loads in the lung and in nasal swabs. Thus, elastase-dependent SIV mutants can be used as live-virus vaccines against swine influenza in pigs. In addition, low levels of cross-neutralizing antibodies to H1N1 2009 were elicited prior to challenge by the swine adapted H1N1 avian strain vaccine.     

Vaccination for 2009 pandemic H1N1 influenza A did not induce conserved epitope-specific memory CD8 T cell responses in HIV+ northern Thai children

The influenza virus causes severe illness in susceptible populations, including children and people living with human immunodeficiency virus (HIV). Here, we investigated cell-mediated immune responses (CMI) against influenza CD8 T cell conserved epitopes in HIV-infected (HIV+) northern Thai children following the 2009 pandemic H1N1 influenza A vaccination. Sixty HIV+ children were vaccinated with two doses of the 2009 pandemic influenza vaccine and their CD8 T cell responses were assessed. We found no significant differences in the increase of cytokines-producing and CD107a-expressing CD8+ T cells or CD8+ memory T cells in response to pooled conserved epitopes stimulation in vitro between children with different serologic responses to the vaccine at all time points of the study. Our results suggest that the 2009 pandemic H1N1 vaccine did not induce the conserved epitope-specific immune responses in HIV+ children. Vaccine design and vaccination strategy against influenza in these populations warrant further studies.     

Pandemic (H1N1) 2009 and Hajj Pilgrims Who Received Predeparture Vaccination, Egypt

In Egypt, vaccination against pandemic (H1N1) 2009 virus was required of pilgrims departing for the 2009 Hajj. A survey of 551 pilgrims as they returned to Egypt found 542 (98.1% [weighted]) reported receiving the vaccine; 6 (1.0% [weighted]) were infected with influenza virus A (H3N2) but none with pandemic (H1N1) 2009 virus.     

Recombinant H1N1 virus-like particle vaccine elicits protective immunity in ferrets against the 2009 pandemic H1N1 influenza virus

The pandemic virus of 2009 (2009 H1N1) continues to cause illness worldwide, especially in younger age groups. The widespread H1N1 virus infection further emphasizes the need for vaccine strategies that are effective against emerging pandemic viruses and are not dependent on the limitations of traditional egg-based technology. This report describes a recombinant influenza virus-like particle (VLP) vaccine consisting of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins of influenza A/California/04/2009 (H1N1) virus. Influenza H1N1 VLPs with a diameter of approximately 120 nm were released into the culture medium from Sf9 insect cells infected with recombinant baculovirus coexpressing HA, NA, and M1 proteins. Purified recombinant H1N1 VLPs morphologically resembled influenza virions and exhibited biological characteristics of influenza virus, including HA and NA activities. In the ferret challenge model, 2009 influenza H1N1 VLPs elicited high-titer serum hemagglutination inhibition (HI) antibodies specific for the 2009 H1N1 virus and inhibited replication of the influenza virus in the upper and lower respiratory tract tissues following A/Mexico/4482/09 (H1N1) virus challenge. Moreover, a single 15 μg dose of H1N1 VLPs resulted in complete virus clearance in the ferret lung. These results provide support for the use of recombinant influenza VLP vaccine as an effective strategy against pandemic H1N1 virus.     

Intranasal c-di-GMP-adjuvanted plant-derived H5 influenza vaccine induces multifunctional Th1 CD4+ cells and strong mucosal and systemic antibody responses in mice

Vaccination is the best available measure of limiting the impact of the next influenza pandemic. Ideally, a candidate pandemic influenza vaccine should be easy to administer and should elicit strong mucosal and systemic immune responses. Production of influenza subunit antigen in transient plant expression systems is an alternative to overcome the bottleneck in vaccine supply during influenza pandemic. Furthermore, a needle-free intranasal influenza vaccine is an attractive approach, which may provide immunity at the portal of virus entry.The present study investigated the detailed humoral and cellular immune responses in mice vaccinated intranasally or intramuscularly with plant-derived influenza H5N1 (A/Anhui/1/05) antigen alone or formulated with bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) as adjuvant. The use of c-di-GMP as intramuscular adjuvant did not enhance the immune response to plant-derived influenza H5 antigen. However, intranasal c-di-GMP-adjuvanted vaccine induced strong mucosal and systemic humoral immune responses. Additionally, the intranasal vaccine elicited a balanced Th1/Th2 profile and, most importantly, high frequencies of multifunctional Th1 CD4⁺ cells. Our results highlight that c-di-GMP is a promising mucosal adjuvant for pandemic influenza vaccine development.     

Characterization of live influenza vaccine donor strain derived from cold-adaptation of X-31 virus

A human influenza A virus X-31 (high-yielding strain) was cold-adapted for possible future use as live attenuated vaccine. Mutant influenza viruses were selected during successive serial passage in embryonated hens' eggs at progressively lower sub-optimal temperature (30, 27 degrees C followed by 24 degrees C). The cold-passaged mutant exhibited both temperature-sensitivity (ts) and cold-adapted (ca) phenotypes. The pathogenicity and immunogenicity of X-31 ca virus were studied in mice following intranasal inoculation. The mice did not show clinical signs even at high titer infection. Immunization of mice with X-31 ca virus elicited high titers of neutralizing antibody and provided complete protection against homologous and heterologous virus challenges. To assess the genetic stability, the X-31 ca virus was passaged at 37 degrees C in MDCK cells or inoculated into mice. Revertant virus was not found in the lungs of any of the mice and the supernatants of the MDCK culture. We conclude that the X-31 ca candidate vaccine virus exhibits the desired level of attenuation, immunogenicity, and protective efficacy required for live attenuated vaccine and merits further evaluation at clinical level.     

Live attenuated pandemic influenza vaccine: clinical studies on A/17/California/2009/38 (H1N1) and licensing of the Russian-developed technology to WHO for pandemic influenza preparedness in developing countries

In February 2009, Nobilon granted the World Health Organization (WHO) a non-exclusive licence to develop, register, manufacture, use and sell seasonal a pandemic live attenuated influenza vaccine (LAIV) produced on embryonated chicken eggs. WHO was permitted to grant sub-licences to vaccine manufacturers in developing countries within the framework of its influenza vaccine technology transfer initiative. In parallel, the Institute of Experimental Medicine (IEM), Russia, concluded an agreement with WHO for the supply of Russian LAIV reassortants for use by these manufacturers. Also in 2009, IEM carried out a study on a novel A/17/California/2009/38 (H1N1) pandemic LAIV candidate derived from the pandemic-related A/California/07/2009 (H1N1) influenza virus and the attenuated A/Leningrad/134/17/57 (H2N2) master donor virus, using routine reassortant technique in embryonated chicken eggs. Following successful preclinical studies in eggs and in ferrets, a double-blind, controlled, randomized clinical trial was carried out in immunologically na?ve study participants between 12-18 and 18-60 years old. Collectively, the immunogenicity data (haemagglutinin inhibition test, ELISA and cytokine tests for the detection of memory T cells) support the use of a single dose of the pandemic H1N1 LAIV in 12-60 year olds. The outcome of the studies showed no significant adverse reactions attributable to the vaccine, and suggests that the vaccine is as safe and immunogenic as seasonal influenza vaccines. Importantly, it was clearly demonstrated that reliance on the HAI assay alone is not recommended for testing LAIV. To date, via the licence agreement with WHO, the H1N1 LAIV has been transferred to the Government Pharmaceutical Organization in Thailand, the Serum Institute of India, and the Zhejiang Tianyuan Bio-Pharmaceutical Co., Ltd. in China.     

Protective efficacy in mice of monovalent and trivalent live attenuated influenza vaccines in the background of cold-adapted A/X-31 and B/Lee/40 donor strains

Influenza virus continues to take a heavy toll on human health and vaccination remains the mainstay of efforts to reduce the clinical impact imposed by viral infections. Proven successful for establishing live attenuated vaccine donor strains, cold-adapted live attenuated influenza vaccines (CAIVs) have become an attractive modality for controlling the virus infection. Previously, we developed the cold-adapted strains A/X-31 and B/Lee/40 as novel donor strains of CAIVs against influenza A and B viruses. In this study, we investigated the protective immune responses of both mono- and trivalent vaccine formulations in the mouse model. Two type A vaccines and one type B vaccine against A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), and B/Shangdong/7/97 in the background of the A/X-31 ca or B/Lee/40 ca were generated by a reassortment procedure and evaluated for their immunogenicity and protective efficacy. Each monovalent vaccine elicited high levels of serum antibodies and conferred complete protection against homologous wild type virus infection. As compared to the monovalent vaccines, trivalent formulation induced higher levels of type A-specific serum antibodies and slightly lower levels of type B-specific antibodies, suggesting an immunological synergism within type A viruses and an interference in the replication of type B virus. Relatively lower type B-specific immunogenicity in trivalent vaccine formulation could be effectively implemented by increasing the vaccine dose of influenza B virus. These results of immunogenicity, protection efficacy, and immunological synergism between type A vaccines provide an experimental basis for optimal composition of trivalent vaccines for subsequent developments of multivalent CAIVs against seasonal and pandemic influenza viruses.