Influenza is a highly contagious disease and occasionally pandemics. Current available options to counter influenza infections include both antivirals and active/passive immunization. Till now licensed therapeutic choices are limited to two classes of FDA-approved antivirals targeting the viral matrix 2 (M2) ion channel protein (amantadine or rimantadine) or the sialidase active site of the viral NA protein (oseltamivir, zanamivir, or peramivir). M2 inhibitors are only efficient against Influenza A virus (IAV), and they have shown serious side effects and low efficacy since most of the currently circulating IAV have acquired resistance to them. On the other hand, NA blockers are efficient against IAV and IBV, but the emergence of drug-resistant strains is currently increasing.1 Annual vaccination may not adequately provide protection; epidemic strains may drift from vaccine strains and the natural immune response of the high risk patient population may not be as effective as in healthy adults. Multiple strategies and technology approaches have been developed for the generation of vaccines with safety profiles and immunogenic and protective characteristics. Investigations on human monoclonal antibodies for passive immunization against influenza is also focus of intensive research.

Vaccines are the most cost-effective approach, also they are the primary prophylactic means to prevent influenza viral infections. Antibodies vaccines have been used with high success in influenza therapy.2 The effective monoclonal antibodies should be important for both pre and post viral exposure for intervention of morbidity or reduction of symptoms, especially in the immunocompromised subjects.3,4  However, new viral strains emerge continuously due to antigenic evolution, antigenic drift and antigenic shift. Antigenic drift occurs when the virus accumulates mutations at antigenic sites during replication through the actions of the inherently error-prone RNA polymerase, producing variant viruses that can escape existing immunity. Whereas antigenic shift occurs when a virus acquires an antigenically novel HA (surface exposed protein) through reassortment, a property made possible due to the segmented nature of the viral genome. Therefore, the vaccine for active immunization as well as the antibodies for passive immunization should target the highly conserved epitopes of the virus proteins. The therapeutic antibody candidates in development for severe influenza A either target a highly conserved epitope on the N-terminal ectodomain of M2 (M2e) or the stalk region of hemagglutinin (HA) common to all influenza A viruses. In contrast to the anti-HA-stalk mAbs, anti-M2e does not neutralize influenza virus; instead anti-M2e mediates the killing of infected cells.5  A research group isolated a panel of monoclonal antibodies derived from the IgG+ memory B cells of healthy, human subjects that recognize a previously unknown conformational epitope within the ectodomain of the influenza matrix 2 protein, M2e.5 This antibody binding region is highly conserved in influenza A viruses, being present in nearly all strains detected to date, including highly pathogenic viruses that infect primarily birds and swine, and the current 2009 swine-origin H1N1 pandemic strain (S-OIV). Also they observed that these human anti-M2e monoclonal antibodies protected mice from lethal challenges with either H5N1 or H1N1 influenza viruses. These results suggest that viral M2e can elicit broadly cross-reactive and protective antibodies in humans. Accordingly, recombinant forms of these human antibodies may provide useful therapeutic agents to protect against infection from a broad spectrum of influenza A strains.

Figure. Schematic representation to produce inactivated (A) or live-attenuated (B) influenza vaccines by genetic reassortment in embryonated eggs: The traditional method for generating reassortant virus is based on the coinfection of two influenza viruses in eggs. Both the WHO candidate virus and the high-growth virus for influenza inactivated vaccine (IIV) (A) or the master donor virus (MDV) for live-attenuated influenza vaccine (LAIV) (B) are inoculated in eggs followed by the selection of appropriate seed viruses by amplification in the presence of antibodies against the HA and NA of the high-growth virus (A) or the MDV (B). The resulting viruses containing the HA and NA segments from the WHO-recommended strain and the six internal vRNAs of the high-growth virus (A) or the MDV (B) are used for vaccine production. PR8, Puerto Rico/8

There has been concern that intact antibody molecule might mediate antibody-dependent enhancement (ADE) potentially leading to disease exacerbation and representing a significant safety risk. The protective antibodies against HA-stalk present a unique mechanism of ADE of influenza infection. Current anti-HA-stalk clinical candidates target a common epitope region closely located to residues on the HA2 subunit that functionally mediates fusion of the viral and host cell membranes prior to transfer of viral genome into the host cell cytoplasm. In vitro analysis of these mAbs shows that their mechanism of action to neutralize influenza infection is based on blocking viral entry into cells. Due to the highly conserved nature of this epitope region, vaccines have been designed to specifically elicit antibodies against this epitope region. In mice and ferrets, such vaccines have demonstrated prophylactic efficacy against challenge by heterologous viruses.5-7 However, piglets immunized by UV irradiation inactivated H1N2 influenza developed enhanced respiratory disease upon challenge with a pandemic H1N1 virus. The finding of this particular study has raised concern regarding universal influenza vaccine development. Interestingly, despite the fact that both piglets and ferrets share various influenza disease characteristics with humans, a similar enhancement of disease has not been described for ferrets treated with HA-stalk based vaccines.

Another strategy for passive immunization against influenza is the use of fully human single-chain antibodies (HuscFvs) that target conserved regions of pivotal proteins of the influenza viruses including surface-exposed, secreted, and internal proteins. The fully human single-chain antibodies should be safe as they are devoid of the Fc portion; thus, they cannot cause ADE (antibody-dependent enhancement), which is a concern when utilizing mAbs as therapeutic intervention. Human scFv phage display library6 has been used as a biological tool for providing HuscFv display phage clones that bound to the desired influenza virus targets. Recombinant influenza virus proteins with the inherent functional activities or intact virus adsorbed to cell surface were used as antigens in the phage biopanning process.8-10 The antigen bound phages were then put in non-suppressor E. coli that could not produce tRNA for the stop codon located between the antibody coding gene (huscfv) and the phage p3 gene. These phage-transformed E. coli were grown in appropriate medium to express soluble HuscFvs. The HuscFvs produced by individual phage transformed E. coli clones were tested for specific binding to the targets by appropriate immunoassays. Therapeutic efficacies of the HuscFvs were tested in a mouse model of influenza. HuscFvs from one of the E. coli clones readily rescued C57BL/6 mice from lethal challenge with heterologous H5N1. 11

The prophylactic options include inactivated vaccines and live attenuated influenza vaccine (LAIV). 12  LAIV are more efficacious than inactivated vaccines because of their ability to mount efficient innate and adaptive humoral and cellular immune responses. Its administration mimics the usual route of influenza virus infection that provides more efficient cross-reactive cellular-mediated protection against heterologous influenza viruses. However, current LAIV remain restricted for use in healthy children and non-pregnant adults. LAIVs are generated either by classical reassortment in eggs (Figure 1) or by reverse genetics, an approach that utilizes molecular techniques to generate a specific virus phenotype.

Several mutations in the PB2 and NS1 genes, for example, could also attenuate the virus and confer the temperature sensitivity phenotype to the virus. 12-14 A commercial LAIV produced by MedImmune was approved in 2003 by the FDA under the trade name FluMist.  Attenuated human LAIVs were developed in the 1960s by serial passage of the virus in eggs using suboptimal conditions of temperature. The resulting attenuated viruses displayed a temperature-sensitive (ts) cold-adapted (ca) attenuated (att) phenotype that grew at 25 ◦C, but not at temperatures found in the lower respiratory tract (>35 ◦C). 15-17 Because this ts, ca, att phenotype restricts virus replication to the upper respiratory track, these viruses could induce local protective immunological responses. To date, reverse genetics has remained the only working method to produce safe, matching H5 vaccine seed strains. One limitation to the plasmid-based reverse genetic systems, however, is the host cell specificity of the RNA Pol I promoter, which is used to produce the negative-sense viral RNA in transfected cells. There are two areas where the reverse genetics technologies have proved invaluable. First is the development of reassortant strains for some of the LAIVs. In this case, as the seed viruses are required to contain 6 gene segments from the master strain, which encode the attenuating mutations, and the HA and NA from the target virus, reverse genetics has streamlined the process of seed virus development. The second area of use is where specific mutations have had to be introduced into the virus e.g. an attenuated H5N1 vaccine strain that lacks the HA cleavage sequence associated with high-level virulence in avian and mammalian hosts.18 The current LAIVs consist of the internal viral segments (PB2, PB1, PA, NP, M and NS) of an attenuated master donor virus (MDV) and the HA and NA viral segments from the selected seasonal virus strain.

The frequent mutations of the surface-exposed proteins, particularly receptor binding domain of influenza virus hemagglutinin, lead to reduction or abrogation of affinity and efficacy of anti-hemagglutinin antibody which is the principal protective antibody against influenza. Therefore, the vaccine for active immunization as well as the antibodies for passive immunization should target the highly conserved epitopes of the virus proteins. Techniques like cell culture, reverse genetics allow for safe and scalable production, while adjuvants, dose variation, and alternate routes of delivery aim to improve vaccine immunogenicity. The potential to manipulate the influenza viral RNA-dependent RNA polymerase (RdRp) complex to generate attenuated forms of the virus that can be used as LAIV for the treatment of influenza viral infections, one of the current and most effective prophylactic options for the control of influenza in humans. In addition, the spread of new pandemic strains is difficult to contain because of the time required to engineer and manufacture effective vaccines. The strain composition of influenza vaccines must be determined before the influenza season on an annual basis, and predicting in advance which strains will become dominant is challenging. Fundamentally different approaches that are currently under development hope to signal new generations of influenza vaccines.

References 

  1. Fiore AE, Fry A, Shay D, Gubareva L, Bresee JS, Uyeki TM, Centers for Disease Control and Prevention (CDC). Antiviral agents for the treatment and chemoprophylaxis of influenza — recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep. 2011 Jan 21; 60(1):1-24.
  1. Shriver Z., Trevejo J. M., Sasisekharan R. Antibody-based strategies to prevent and treat influenza. Frontiers in Immunology. 2015;6, article 315 doi: 10.3389/fimmu.2015.00315.

 

  1. Sparrow E, Friede M, Sheikh M, Torvaldsen S, Newall AT. Passive immunization for influenza through antibody therapies, a review of the pipeline, challenges and potential applications. 2016 Oct 26; 34(45):5442-5448.
  1. Grandea AG 3rd1, Olsen OACox TCRenshaw MHammond PWChan-Hui PYMitcham JLCieplak WStewart SMGrantham MLPekosz AKiso MShinya KHatta MKawaoka YMoyle M. Human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza A viruses. Proc Natl Acad Sci U S A.2010;107(28):12658-63
  1. Martínez-Sobrido L1, Peersen O2, Nogales A3. 1. Temperature Sensitive Mutations in Influenza A Viral Ribonucleoprotein Complex Responsible for the Attenuation of the Live Attenuated Influenza Vaccine. Viruses. 2018;15;10(10)
  1. Krammer F, Margine I, Hai R, Flood A, Hirsh A, Tsvetnitsky V, Chen D, Palese P.. H3 stalk-based chimeric hemagglutinin influenza virus constructs protect mice from H7N9 challenge. J Virol. 2014; 88:2340-3.
  1. Krammer F, Hai R, Yondola M, Tan GS, Leyva-Grado VH, Ryder AB, Miller MS, Rose JK, Palese P, García-Sastre A, et al. Assessment of influenza virus hemagglutinin stalk-based immunity in ferrets. J Virol. 2014; 88:3432-42.
  1. Dong-Din-On F., Songserm T., Pissawong T., et al. Cell penetrable human scFv specific to middle domain of matrix protein-1 protects mice from lethal influenza. Viruses. 2015;7(1):154–179.
  1. Pissawong T., Maneewatch S., Thueng-in K., et al. Human monoclonal ScFv that bind to different functional domains of M2 and inhibit H5N1 influenza virus replication. Virology Journal. 2013;10, article 148.
  1. Maneewatch S., Thanongsaksrikul J., Songserm T., et al. Human single-chain antibodies that neutralize homologous and heterologous strains and clades of influenza A virus subtype H5N1. Antiviral Therapy. 2009;14(2):221–230.
  1. Mallajosyula VV, Citron M, Ferrara F, Lu X, Callahan C, Heidecker GJ, Sarma SP, Flynn JA, Temperton NJ, Liang X, et al. Influenza hemagglutinin stem-fragment immunogen elicits broadly neutralizing antibodies and confers heterologous protection. Proc Natl Acad Sci U S A. 2014; 111:E2514-23.
  1. Wright PF, Neuman G, Kawaoka Y. 2007. Orthomyxoviruses, p 1691–1740. Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE (ed), Fields virology, 5th ed, vol 2. Lippincott Williams & Wilkins, Philadelphia, PA
  1. Maassab HF, Heilman CA, Herlocher ML. 1990. Cold-adapted influenza viruses for use as live vaccines for man. Adv. Biotechnol. Processes 14:203–242
  1. Murphy BR, Park EJ, Gottlieb P, Subbarao K. 1997. An influenza A live attenuated reassortant virus possessing three temperature-sensitive mutations in the PB2 polymerase gene rapidly loses temperature sensitivity following replication in hamsters. Vaccine 15:1372–1378
  1. Parkin NT, Chiu P, Coelingh K. 1997. Genetically engineered live attenuated influenza A virus vaccine candidates. J. Virol. 71:2772–2778
  1. Talon J, Salvatore M, O’Neill RE, Nakaya Y, Zheng H, Muster T, Garcia-Sastre A, Palese P. 2000. Influenza A and B viruses expressing altered NS1 proteins: a vaccine approach. Proc. Natl. Acad. Sci. U. S. A. 97:4309–4314
  1. Maassab HF . 1967. Adaptation and growth characteristics of influenza virus at 25 degrees C. Nature213:612–614.
  1. Webby RJ, Perez DR, Coleman JS, Guan Y, Knight JH, Govorkova EA, McClain-Moss LR, Peiris JS,  Rehg JE,  Tuomanen EI,  Webster RG. 2004. Responsiveness to a pandemic alert: use of reverse genetics for rapid development of influenza vaccines. Lancet 363:1099–1103