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  1. Biologics Research Projects

Influenza neuraminidase antigenicity and efficacy in vaccines

Robert Daniels, PhD

Office of Vaccines Research and Review
Division of Viral Products
Laboratory of Respiratory and Special Pathogens



Dr. Robert Daniels received his PhD in molecular and cellular biology from the University of Massachusetts, Amherst in 2007 and completed post-doctoral training at the Karolinska Institute in Sweden. In 2010 he became an assistant professor in the Department of Biochemistry and Biophysics at Stockholm University where he used his expertise in biochemistry and secretory protein folding to establish a research group that examined the maturation and evolution of the influenza virus surface antigen, neuraminidase (NA). He also received the 2018 Stockholm University teacher of the year award. In 2019, Dr. Daniels joined the Laboratory of Pediatric and Respiratory Viral Diseases (LPRVD) in the Division of Viral Products (DVP), in the Office of Vaccine Research and Review (OVRR) at CBER. His group focuses on increasing influenza vaccine efficacy and cross protection by developing methodologies for improving the response against the NA antigen in vaccines and for assessing NA in circulating strains and vaccine preparations.

General Overview

Influenza viruses are estimated to cause symptomatic infections in 3-11% of the U.S. population annually and severe disease in about 1.5% of those infected. Although several drugs now available can limit the severity of an influenza infection, yearly vaccination remains the most effective approach to reduce the disease burden caused by influenza viruses.

Current influenza vaccines include split inactivated influenza viruses, live attenuated influenza viruses, and recombinant hemagglutinin (HA) antigens. Each vaccine type has advantages and all of them protect against the two influenza A subtypes (H1N1 and H3N2) and at least one of the influenza B lineages (Yamagata and Victoria) that are responsible for seasonal infections in humans.

Manufacturing split inactivated influenza vaccines generally involves propagating candidate vaccine viruses (CVVs) in eggs or mammalian cells, whereas recombinant HA vaccines are produced using insect cells. Despite these differences, both products are standardized based on the HA antigen content, as responses against HA correlate well with protection.

Each season, several inter-connected challenges can affect the influenza vaccine efficacy: 1) Influenza viruses are constantly evolving, which  can cause antigenic drift and occasional antigenic shift in type A viruses; 2) vaccine strains must be selected months in advance to meet manufacturing deadlines; 3) viral propagation in eggs or cells can lead to unexpected adaptations that can alter important antigens in the vaccine.

Although influenza vaccines have mainly been developed to generate an optimal immune response against HA, influenza viruses do possess a second, less abundant surface antigen, neuraminidase (NA). Like HA, antibodies that recognize NA can provide both matched and cross-protection against influenza virus strains. NA also evolves and drifts independently of HA. These properties imply that by improving the NA response, it might be possible to increase the breadth of the vaccine coverage and mitigate many of the yearly challenges that influenza vaccines face.

In the split inactivated and the live attenuated influenza virus vaccines, NA is present. However, many technical issues must first be solved before the NA component of the annual vaccines can be regulated. Our laboratory is systematically addressing several of these issues to establish a framework for improving the ability of NA to increase the breadth and efficacy of the annual vaccine.

Scientific Overview

Influenza viruses contain two surface antigens, the receptor-binding protein, hemagglutinin (HA), and the receptor-destroying enzyme, neuraminidase (NA). However, influenza vaccines have primarily been developed using methodologies centered on the more abundant HA antigen. Our long-term objectives are to increase the breadth and efficacy of the annual influenza vaccine by establishing a similar framework for assessing the NA antigen.

To reach this goal, we are working to create methods capable of rapidly monitoring changes in NA antigenicity and to define the NA parameters that correlate with protection. We will use these parameters to assess the immunogenic NA content during the vaccine manufacturing process and to determine if the quantity is sufficient. In parallel, we are developing approaches to increase the NA content in CVVs, retain its immunogenicity throughout the different manufacturing processes, and to rationally design NAs for improved immunogenicity in recombinant-based vaccines.

Within the lab, these objectives are separated into the following research areas: 1) NA assay development for characterizing circulating strains; 2) engineering CVVs to increase NA responses from viral-based vaccines; 3) rationally designing recombinant NAs for improved production and immunogenicity.

We address each research area using a similar systematic approach that generally involves in vitro biochemical analysis followed by validation tests that include cell-based assays and in vivo animal models. The techniques we utilize include: enzyme kinetics, protein/viral purification, analytical assays, viral reverse genetics with propagation in cells and eggs, and viral immunization and challenge models. This broad range of approaches are carried out using the most up to date equipment and techniques so that advancements in one area of vaccine manufacturing can be rapidly assessed in another.

The results from this work will likely help to establish the frameworks that are necessary to better utilize the NA antigen in the influenza vaccine and to identify the NAs in circulating strains that will provide the greatest breadth of coverage for upcoming seasons. Together, these concepts should help to advance influenza vaccine manufacturing and to improve the efficacy of the yearly vaccine.

The main goal of the lab is to increase the vaccine efficacy and cross-protection against influenza viruses by improving the NA antigen response. The illustration depicts the analytical and experimental approaches that the lab utilizes in an effort to enhance the NA response elicited by viral and protein-based influenza vaccines.


  1. Nature Microbiology, Dec 4 (12); 2565-2577 (doi:10.1038/s41564-019-0537-z)
    Structural restrictions for influenza NA activity promote adaptation and diversification.
    Wang H, Dou D, Östbye H, Revol R, Daniels R (2019)
  2. ACS Nano, June 25; 13(6):6689-6701 (doi:10.1021/acsnano.9b01052)
    Curvature- and phase-induced protein sorting quantified in transfected cell-derived giant vesicles.
    Moreno-Pescador G, Florentsen CD, Østbye H, Sønder SL, Boye TL, Veje EL, Sonne AK, Semsey S, Nylandsted J, Daniels R, Bendix PM (2019)
  3. Frontiers in Microbiology, July 23; 10: 1511 (doi: 10.3389/fmicb.2019.01511)
    Enhancing recombinant protein yields in the E. coli periplasm by combining signal peptide and production rate screening.
    Karyolaimos A, Ampah-Korsah H, Hillenaar T, Borras AM, Dolata KM, Sievers S, Riedel K, Daniels R, de Gier JW (2019)
  4. Frontiers in Immunology, July 20; 9: 1581 (doi: 10.3389/fimmu.2018.01581)
    Influenza A virus cell entry, replication, virion assembly and movement.
    Dou D, Revol R, Östbye H, Wang H, Daniels R (2018)
  5. Proceedings of the National Academy of Sciences, April 17; 115 (16) E3808-E3816. (doi: 10.1073/pnas.1722333115)
    Multiple nuclear-replicating viruses require the stress-induced protein ZC3H11A for efficient growth.
    Younis S, Kamel W, Falkeborn T, Wang H, Yu D, Daniels R, Essand M, Hinkula J , Akusjärvi G, Andersson L (2018)
  6. Journal of Cell Biology, 2017 Aug 7; 216(8):2283-2293 (doi: 10.1083/jcb.201702102)
    Translational regulation of viral secretory proteins by the 5’ coding regions and a viral RNA-binding protein.
    Nordholm J, Petitou J, Östbye H, da Silva DV, Dou D, Wang H, Daniels R (2017)
  7. Cell Reports, Jul 5; 20(1):251-263 (doi: 10.1016/j.celrep.2017.06.021)
    Analysis of IAV replication and co-infection dynamics by a versatile RNA viral genome labeling method.
    Dou D, Hernandez-Neuta I, Wang H, Östbye H, Qian X, Thiele S, Resa-Infante P, Kouassi N, Sender V, Hentrich K, Mellroth P, Henriques-Normark B, Gabriel G, Nilsson M, Daniels R (2017).


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