**3. Prevention and treatment**

Early anti-influenza drugs were synthesized by large scale screening methods without the knowledge of their chemical structures and mechanisms of action [29], whereas, the recent antivirals have been discovered based on the structure of influenza virus protein as drug targets using X-ray crystallography method. This is structure based, involving the use of organic compounds that are able to bind to viral target protein receptors [30]. These structures have high binding affinity to the viral target following chemical synthesis and effective antiviral screening using standard in vitro assays such as cell based antiviral screening [31] and biochemical evaluation [32]. Some cell based antiviral screening includes plaque assays for studying replication in virus, yield-reduction assays for quantifying specific viral antigens and dye uptake for measuring cytopathic effect. The application of bioinformatics, robotics, miniaturization strategies have led to an advanced and high-throughput drug screening of large drug libraries with unique chemical structures [33] and computational screening [34]. In vivo drug screening using various animal models such as chicken, mouse, ferret have been used to evaluate new drugs [35] this is followed by clinical trials to study its bio-safety, kinetics and tolerance in human [36].

Advances has since then seen the treatment of influenza virus infection basically through vaccines, monoclonal antibodies and antivirals drugs. Antiviral influenza drugs are mostly NA inhibitors; however, they generally have short therapeutic window and current show emerging drug resistance [37]. Till date, four (4) antiviral drugs have been approved for the treatment of influenza: the NA inhibitors oseltamivir (Tamifu), peramivir (Rapivab), zanamivir (Relenza), and the cap-dependent endonuclease inhibitor baloxavir (Xofuza) [23, 37]. Oseltamivir is the preferred treatment for patients with severe influenza. Intravenous peramivir is an option for these patients if there are contraindications to or concerns about reduced bioavailability of oral oseltamivir [24]. Baloxavir is preferred for the treatment of uncomplicated influenza in patients of age 12 years and older. A study was conducted to compare baloxavir with oseltamivir and placebo in 1436 healthy people between 12 to 65 years of age who had influenza, baloxavir and oseltamivir reduced symptom duration by approximately one day compared with placebo. Adamantanes (amantadine and rimantadine [Flumadine]) are also approved for influenza treatment but are not currently recommended because these medications are not active against influenza B, and most influenza A strains have shown resistance to adamantane for the past 10 years [24].

Vaccines remain extremely essential to the prevention of the infection. Vaccination is the most preferred method for prevention, and routine chemoprophylaxis within the community is not recommended. The first influenza vaccine was developed in 1945, and since seen several others produced. Multiple formulations of the influenza vaccine are available, including inactivated influenza vaccines (IIV); a recombinant inactivated vaccine (RIV); and a live, attenuated influenza vaccine (LAIV). LAIV shows one of the best efficacies at around 70% and tends to be more effective in children. It delivers more NA and M2 antigens, triggers mucosal responses including IgA, and has the potential for inducing CD8 T cell responses [38].

As a primary prophylactic countermeasure, annual influenza vaccination is engaged globally with the aim of limiting influenza burden. However, the effectiveness of the current available influenza vaccines is limited because they only confer protective immunity when there is antigenic similarity between the selected vaccine strains and circulating influenza isolates. The consequences of antigenic drift or shift, results in an antigenic mismatch between the current vaccines and circulating influenza isolates. Accumulation of mutations, especially at key antigenic sites in the HA globular head, due to the absence of the proofreading activity of the viral RNA polymerase and then to the selective pressure exerted by the host immune system is often responsible for the escape of influenza virus from pre-existing immunity in the case of antigenic drift [39]. There is therefore a crucial need to develop a more effective broadly-reactive (universal) influenza vaccine with the capability to confer protection against both seasonal and newly emerging pre-pandemic strains.

### **3.1 The journey to a universal flu vaccine**

In influenza virus vaccine design, the major targets of the antibody response against the virus are the surface glycoprotein antigens hemagglutinin (HA) and neuraminidase (NA). As earlier stated, Hemagglutinin (HA) and neuraminidase (NA), are the main surface glycoproteins on influenza viral particles. NA is however less abundantly expressed on the virion in comparison HA expression, with HA to NA ratio often ranging from 4:1 to 5:1 [38]. The influenza HA is responsible for binding to sialic acid, the receptor on target host cells, and there are approximately 500 molecules of HA per virion [40]. The mature form of the HA glycoprotein exists as a homotrimer containing three HA monomers that are composed of a globular head and a stem/stalk region. The receptor binding site (RBS) is present in the globular head, which is however a hypervariable region of the protein, while the stem region is majorly involved in the pH-induced fusion event triggered by endosome acidification following viral adsorption. The stem/stalk region of the HA is more conserved among and across HA subtypes belonging to the same group [38]. Antibody response elicited against this stem/stalk region forms one of the major approaches towards developing a more responsive vaccine to both current and future strains of influenza viruses (**Figure 2**).

#### *3.1.1 Stem-based universal vaccine approaches*

Influenza virus infection can elicit neutralizing antibodies against both the globular head and the stem structures of the HA viral protein. Currently, ongoing strategies for more efficacious vaccine development is aimed at eliciting antibodies that target the conserved stem region of HA since previously existing influenza vaccines only show minimal induction of stem-directed humoral immunity [3]. Several studies describe ongoing approaches to elicit stem-directed antibodies including sequential immunization with heterologous influenza strains, immunization with modified proteins by removing or glycan-masking the globular head, referred to as headless HA, through minimizing epitopes of the stem region, hyperglycosylated HA head domain, Chimeric HA, and Mosaic HA [3, 38]. Self-reactivity of this antibodies may occur due to their polyreactive profile and the proximity of the HA stem region to the cell membrane which is a crucial limitation described by scientists to this approach.

Nachbagauer et al. [40] recently presented a unique concept in the stem-based approach using the context of a LAIV with a H8 head domain and an H1 stem domain *Influenza Viruses: Targetting Conserved Viral Ha-Stem, Matrix and Nucleo-Proteins to Disarm… DOI: http://dx.doi.org/10.5772/intechopen.104770*

#### **Figure 2.**

*Showing the phylogenetic trees of (a) hemagglutinin (HA) and (b) neuraminidase (NA). The primary surface glycoproteins of influenza viruses, HA and NA, are divided into several categories and subtypes. During the last century, only viruses producing H1, H2, or H3 HAs and N1 or N2 NAs (such as H1N1, H2N2, or H3N2; circled in purple) have spread widely in the human population. The scale bars represent a 6% change in amino acid levels (source: [41]).*

(cH8/1) and a split-inactivated vaccine with an H5 head domain and an H1 stem domain (cH5/1) [40]. Using preclinical ferret investigations, the scientists assessed protection against pandemic H1N1 virus challenge using several sequential primeboost combinations and vaccination regimens. These studies show that a sequential live-attenuated followed by split-inactivated viral vaccination strategy provides superior protection against pandemic H1N1 infection. Scientists have characterized this notion as a sequential immunization and chimeric HA proteins approach to stembased universal vaccine design.

Furthermore, in a stem-based immunogens approach to the universal influenza vaccine design, based on the H1 subtype, Impagliazzo et al. created stable mini-HA stem antigens, where the best candidate demonstrated structural and binding characteristics with widely neutralizing antibodies equivalent to full-length HA, indicating correct folding. This immunogen totally protected mice in lethal heterologous and heterosubtypic challenge scenarios and lowered fever in cynomolgus monkeys following a sublethal challenge [42]. However, determining the effectiveness of antibodies targeting conserved epitopes in the HA stem region to offer protection remains a critical challenge [43].
