An alternative method for preparation of pandemic influenza strain-specific antibody for vaccine potency determination

Division of Viral Products, Center for Biologics Evaluations and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892, United States.
Vaccine (Impact Factor: 3.62). 03/2010; 28(12):2442-9. DOI: 10.1016/j.vaccine.2009.12.079
Source: PubMed


The traditional assay used to measure potency of inactivated influenza vaccines is a single-radial immunodiffusion (SRID) assay that utilizes an influenza strain-specific antibody to measure the content of virus hemagglutinin (HA) in the vaccine in comparison to a homologous HA reference antigen. Since timely preparation of potency reagents by regulatory authorities is challenging and always a potential bottleneck in influenza vaccine production, it is extremely important that additional approaches for reagent development be available, particularly in the event of an emerging pandemic influenza virus. An alternative method for preparation of strain-specific antibody that can be used for SRID potency assay is described. The approach does not require the presence or purification of influenza virus, and furthermore, is not limited by the success of the traditional technique of bromelain digestion and purification of virus HA. Multiple mammalian expression vectors, including plasmid and modified vaccinia virus Ankara (MVA) vectors expressing the HAs of two H5N1 influenza viruses and the HA of the recently emerging pandemic H1N1 (2009) virus, were developed. An immunization scheme was designed for the sequential immunization of animals by direct vector injection followed by protein booster immunization using influenza HA produced in vitro from MVA vector infection of cells in culture. Each HA antibody was highly specific as shown by hemagglutination inhibition assay and the ability to serve as a capture antibody in ELISA. Importantly, each H5N1 antibody and the pandemic H1N1 (2009) antibody preparation were suitable for use in SRID assays for determining the potency of pandemic influenza virus vaccines. The results demonstrate a feasible approach for addressing one of the potential bottlenecks in inactivated pandemic influenza vaccine production and are particularly important in light of the difficulties in preparation of potency reagent antibody for pandemic H1N1 (2009) virus vaccines.

  • Source
    • "Although such assays might represent a major improvement over SRID in many regards, the experimental denaturation of HA during sample preparation for the assays or during the assays themselves makes the assays incapable of providing sufficient information on HA conformational integrity. Monoclonal antibody based assays (such as enzyme-linked immunosorbent assay [ELISA] [26] [27] and antibody-dependent surface plasmon resonance [SPR] [28]) can potentially measure conformational integrity. However these assays rely on a strain-specific monoclonal antibody panel that would need to be updated and calibrated along with a reference antigen that likely would be quantified with a conformationally insensitive biophysical assay. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Influenza vaccines are the primary intervention for reducing the substantial health burden from pandemic and seasonal influenza. Hemagglutinin (HA) is the most important influenza vaccine antigen. Subunit and split influenza vaccines are formulated, released for clinical use, and tested for stability based on an in vitro potency assay, single-radial immunodiffusion (SRID), which selectively detects HA that is immunologically active (capable of eliciting neutralizing or hemagglutination inhibiting antibodies in an immunized subject). The time consuming generation of strain-specific sheep antisera and calibrated antigen standards for SRID can delay vaccine release. The limitation in generating SRID reagents was evident during the early days of the 2009 pandemic, prompting efforts to develop more practical, alternative, quantitative assays for immunologically active HA. Here we demonstrate that, under native conditions, trypsin selectively digests HA produced from egg or mammalian cell in monovalent vaccines that is altered by stress conditions such as low pH, elevated temperature, or deamidation, leaving native, pre-fusion HA, intact. Subsequent reverse-phase high pressure liquid chromatography (RP-HPLC) can separate trypsin-resistant HA from the digested HA. Integration of the resulting RP-HPLC peak yields HA quantities that match well the values obtained by SRID. Therefore, trypsin digestion, to pre-select immunologically active HA, followed by quantification by RP-HPLC is a promising alternative in vitro potency assay for influenza vaccines.
    Full-text · Article · Sep 2015 · Vaccine
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Pandemic flu had at least two waves in Iran. Knowing how many of the general population were already exposed to this infection has a major impact on national preventive measures. As of December 30, 2009, a total of 3672 confirmed cases of human infection with a novel Influenza A (2009 H1N1) virus had been reported in Iran with 140 deaths. In this study we aim to measure, as a pilot study, the seroprevalence of positive antibody titer (humoral immunity) against 2009 H1N1 virus in Iranian population in Shiraz, Southern Iran. Through cluster random sampling of families residing in Shiraz, 2553 subjects were selected and after a medical interview blood samples were taken and checked for polyclonal antibody against 2009 H1N1 antigen using hemagglutination inhibition assay. An antibody titer of more than 1:40 dilution was considered positive. Data were analyzed considering the demographic characteristics of the population and were compared among different age groups. 1504 (58.91%) samples were tested positive for the presence of polyclonal antibody against 2009 H1N1 virus. The prevalence of positive titers were significantly higher in 60 to 64 years old group and significantly lower in 20 to 24 years old group (p<0.05). Data did not differ based on other demographic characteristics or the history of flu like illnesses in the past 6 months. High seroprevalence of antibody against 2009 H1N1 in the sera of our subjects describes either a high level of pre-existing immunity against H1N1 in Iranian population or a high rate of asymptomatic infection in our area compared to other countries.
    Full-text · Article · Mar 2010 · Iranian journal of immunology: IJI
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The zoonotic transmissions of highly pathogenic avian influenza viruses of the H5N1 subtype that have occurred since 1997 have sparked the development of novel influenza vaccines. The advent of reverse genetics technology, cell-culture production techniques and novel adjuvants has improved the vaccine strain preparation, production process and immunogenicity of the vaccines, respectively, and has accelerated the availability of pandemic influenza vaccines. However, there is still room for improvement, and alternative vaccine preparations can be explored, such as viral vectors. Modified vaccinia virus Ankara (MVA), originally developed as a safe smallpox vaccine, can be exploited as a viral vector and has many favourable properties. Recently, we have demonstrated that an MVA-based vaccine could protect mice and macaques against infection with highly pathogenic influenza viruses of the H5N1 subtype. In the present study, recombinant MVA expressing the haemagglutinin (HA) gene of pandemic influenza A/H1N1 virus was evaluated in the ferret model. A single immunization induced modest antibody responses and afforded only modest protection against the development of severe disease upon infection with a 2009(H1N1) strain. In contrast, two immunizations induced robust antibody responses and protected ferrets from developing severe disease, confirming that MVA is an attractive influenza vaccine production platform.
    Full-text · Article · Nov 2010 · Journal of General Virology
Show more