**2. Recombinant Influenza Virus-Like Particles (VLPs) as Vaccines**

Recently, several new platform technologies including recombinant VLPs have been developed in order to facilitate the scale-up of vaccine production including overcoming the drawbacks of the egg-based vaccine production method. The first recombinant influenza vaccine FluBlok based on HA antigen has received FDA approval [22]. The surface envelope glycoproteins such as influenza HA and NA generally are viewed as the primary targets for vaccine development. HA currently represents the major target for vaccine development including approved and experimental vaccines [22]. Recombinant HA-based vaccines have been shown to be efficacious against influenza including H7N9 [17,23–31]. Experimental NA-based vaccines have been reported [32,33]. The induction of immune responses against the surface envelope proteins has advantages, because virus-neutralizing antibody response to the viral envelope proteins can prevent early steps of viral infection. Human monoclonal antibodies targeting the HA glycoprotein can neutralize H7N9 influenza virus [34]. Importantly, the neutralizing antibodies protected against A/Shanghai/2/2013 (H7N9) virus challenge [35].

The novel recombinant VLP vaccine platforms included VLPs prepared in mammalian, insect, and plant expression systems. Recombinant VLPs are morphologically and biochemically similar to the wild type influenza virus (Figure 2); however, they lack viral genetic material and are unable to replicate and cause infection. Table 2 shows examples of various VLPs prepared from H7N9 virus antigens. Cell culture-based production of VLPs can potentially overcome limitations of classic influenza vaccines [36].

**Figure 2.** Electron microscopy images of influenza VLPs prepared in Sf9 cells using baculovirus expression system. Recombinant VLPs contain three subtypes (H5, H7, H9), morphologically and biochemically are similar to the wild type influenza virus, and can be found as individual particles (left) or groups of particles (right). Characterization of triple-subtype VLPs was done by negative staining transmission electron microscopy. (Adapted from [50], Copyright 2013, with permission from Elsevier, license 4798470004544).



mLD50, mouse median lethal dose.

As VLPs closely resemble viruses, they contain immunological epitopes in the natural conformation, are highly immunogenic, but they are non-infectious because they are prepared in the absence of viral genomic RNA segments. Each VLP represents a repetitive and highly organized molecular array of antigens displayed on the VLP surface. VLPs have been shown to be better immunogens than subunit vaccines because of their self-adjuvant properties [51]. The antigens present in VLP bind pattern recognition receptors on innate immune effector cells and trigger the innate immunity of host cells [52,53]. The molecular arrays displayed on VLP's surface are also potent inducers of Type 2 T-independent B-cell response [54]. Cryo-electron microscopy of VLPs from 1918 pandemic influenza virus demonstrated a uniform distribution of HA molecules on the surface of VLPs along with prefusion state confirmation [55]. Two types of spike on the surface of virus are formed by trimers of HA and tetramers of NA. It has been estimated that a spherical influenza virion of average diameter 120 nm has ~375 spikes [56].
