**2.2. Virus-like particles as a VADS**

Virus-like particles (VLPs) consist mainly of viral proteins devoid of viral genomes to mimic the natural structure of virions and have been engineered to carry agents for various applications, including, particularly, for constructing a VADS for delivering subunit vaccines based on their viral envelop structures suitable for presenting functional spikes on NP surfaces to maintain the intrinsic immunogenicity apt to trigger immunoresponses [16]. VLPs are usually manufactured using protein expression systems based on bioengineered bacteria, yeast, insect, avian, mammalian or plant cells, or using cell-free protein synthesis system (CFPS), which provide an alternative to construct effective VADSs with beneficial characteristics, such as having defined structure formed through self-assembling, large cargo loading capacity, easy functionalization with ligands, and high stability and low toxicity [17]. As a VADS for producing vaccine candidates, VLPs are often designed with characteristics identical to native virus especially in the aspects of the immunochemical properties, 3-D (3-dimensional) architectures and morphological conformations, through engineering on their particulate structures, which, like the native virus, include the nonenveloped and enveloped types. The nonenveloped VLPs mainly consist of one or more pathogenic components, but do not contain any components of the expression hosts, while the enveloped VLPs generally consist of matrix proteins which are enveloped in a lipid membrane derived from the expression hosts, possibly, with glycoproteins embedded into the bilayered membranes.

VLPs are widely used as a VADS because they possess several clear advantages, including induction of immunity with a broad cross-protection rendering onetime immunization to protect against different virus genotypes, high potency to trigger immune responses providing an option to conquering intractable pathogens such as HIV and HCV (hepatitis C virus), high thermostability possibly requiring no integral cold chain to keep viability favoring global vaccination, and high manufacturing efficiency in large scale while at low cost offering a strategy to handle the emergency arising from infectious diseases, such as Ebola outbreak and epidemic. For example, HCV infection is still a significant public health problem, though it has been partially addressed with the advent of directly acting antiviral agents (DAAs), which represent a major advance toward controlling HCV but confer little protection against reinfection [18]. Presently, around 71 million people in the world are living with chronic HCV infection, and each year nearly half a million of them will die of HCV infection or its complications, rendering it urgent to develop an effective vaccine capable of eradicating HCV, which may well be produced using the VLP-based VADS, in reference to HBV vaccines. Recently, a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine was successfully engineered by researchers using scale-up production methods of Huh7 cell factories containing a recombinant adenoviral expression system representing each HCV genotype, followed by cell lysing and purification with iodixanol ultracentrifugation and stirred cell ultrafiltration [19]. When given subcutaneously to mice, whether in the presence of an adjuvant (system) or not, the quadrivalent vaccine consistently induced production of Ab and nAb (neutralizing Ab) together with robust T and B cell responses for eliciting broad humoral and cellular immunity, indicating the VLP-based VADS a useful tool which may be employed for the production of an effective HCV vaccine [20].

that among different particles with a size ranging from 20 to 2000 nm, 40 nm NPs induced the strongest cellular and humoral immunity [12]. Further investigations demonstrated that covalent linkage of peptide to NPs is a requirement for eliciting immunization efficacy and also proved that 40-nm-sized NPs serve as a VADS owing to their preferential uptake by APCs and their ability to traffic to lymph nodes to induce strong immune responses compared to their

Notably, Schöttler et al*.* recently reported the counterintuitive research results on cellular uptake of NPs with PEGylation (modification with polyethylene glycol), which is gold standard in removal of immune clearance of in vivo NPs through the mechanism of reducing nonspecific cellular uptake of nanocarriers [14]. The researchers documented that polystyrene NPs, which had been modified with PEG or poly(ethyl ethylene phosphate) (PEEP), only had been exposed to plasma proteins, could exhibited a lowered cellular uptake by macrophages (RAW264.7 cells), whereas those not exposed to plasma proteins showed high nonspecific uptake. Further mass spectrometric analysis revealed that the plasma-exposed nanocarriers formed a protein corona which was identified to contain just an abundance of clusterin proteins (known as apolipoprotein J) and to be the decisive factor controlling lowered nonspecific cellular uptake of the PEGylated or PEEPylated polystyrene NPs, and to contrast, the classic conception that PEGylated NPs free of immune clearance is resulted from avoidance of protein adsorption. These outcomes indicated that PEG as well as PEEP can affect the composition of protein adsorption by polystyrene NPs, and that the presence of certain type proteins may be just a prerequisite in preventing nonspecific cellular uptake of NPs, defying the conventional belief that PEGylation reduces protein adsorption thereby conferring a stealth effect [15].

Virus-like particles (VLPs) consist mainly of viral proteins devoid of viral genomes to mimic the natural structure of virions and have been engineered to carry agents for various applications, including, particularly, for constructing a VADS for delivering subunit vaccines based on their viral envelop structures suitable for presenting functional spikes on NP surfaces to maintain the intrinsic immunogenicity apt to trigger immunoresponses [16]. VLPs are usually manufactured using protein expression systems based on bioengineered bacteria, yeast, insect, avian, mammalian or plant cells, or using cell-free protein synthesis system (CFPS), which provide an alternative to construct effective VADSs with beneficial characteristics, such as having defined structure formed through self-assembling, large cargo loading capacity, easy functionalization with ligands, and high stability and low toxicity [17]. As a VADS for producing vaccine candidates, VLPs are often designed with characteristics identical to native virus especially in the aspects of the immunochemical properties, 3-D (3-dimensional) architectures and morphological conformations, through engineering on their particulate structures, which, like the native virus, include the nonenveloped and enveloped types. The nonenveloped VLPs mainly consist of one or more pathogenic components, but do not contain any components of the expression hosts, while the enveloped VLPs generally consist of matrix proteins which are enveloped in a lipid membrane derived from the expression hosts, possibly, with glycoproteins embedded into the bilayered membranes. VLPs are widely used as a VADS because they possess several clear advantages, including induction of immunity with a broad cross-protection rendering onetime immunization to

larger counterparts [4, 13].

30 Immunization - Vaccine Adjuvant Delivery System and Strategies

**2.2. Virus-like particles as a VADS**

Globally, HIV continues to be a major public health issue and also claims approximately 1 million lives each year, although total people living with HIV have the opportunity to receive antiretroviral drugs (ARVs), which may effectively control the virus from transmission and causing illness but are also found to be undermined in efficacy by pathogens that had evolved with drug resistance [21]. Still, a highly effective vaccine is believed to be the ultimate weapon able to erase HIV and associated disease AIDS (acquired immune deficiency syndrome), though at present, there are no such a product in markets and, moreover, many products developed in previous years bearing such expectations failed to show clinical efficacy in fighting this rapidly mutating pathogen, including especially the big trial, known as STEP, which was halted in 2007 after the vaccine was found to increase the risk, instead of prophylaxis, of HIV infection [22]. Nevertheless, scientists are getting closer than ever to developing such an effective product, as evidenced by a large-scale clinical study conducted in 2009 in Thailand (called RV144) showing that immunization with a combination of two HIV vaccines prevented about 31% of new infections through a prime-boost combination regime [23], which comprises four priming intramuscular injections of ALVAC-HIV, which is a recombinant canarypox vaccine express HIV-1 Gag, Pro and gp120-gp41, plus two boosting intramuscular injections of AIDSVAX® B/E, which is an alum-adjuvanted bivalent HIV-1 gp120 vaccine of subtypes E and B [24]. Though the low prevention rate of vaccination with ALVAC/AIDSVAX combination excludes the products from approval for clinical prophylaxis of HIV, the moderate effects displayed in human trial not only provoked scientists to make deep explorations on the causes for the failures, but also presented researchers a great encouragement to commit further efforts to developing efficacious HIV vaccines. Subsequently, based on the virion features which are more and more clearly elaborated in structure and function, scientists set out to handle the obstacles identified to the development of a preventive HIV vaccine from several aspects, such as accurately targeting the conserved antigenic proteins, seeking Ags able to induce the broadly neutralizing Abs (bnAbs), and formulating a highly efficient VADS [25]. As mentioned above, a VLP-based VADS proves a highly potent inducer for Abs and helper T cell responses and also able to elicit robust cytotoxic T cell responses necessary for preventing primary infections and erasing infected cells, thus offering researchers an alternative tool to engineer effective vaccines against HIV, which is regarded as the most challenging foe owing to its poor immunogenicity, fragile surface glycoprotein, and the ability to overpower the cell immune system [26].

HEK293 cell (Human embryonic kidney 293 cell) expression system, Salvo et al*.* engineered a VLP-based VADS composed of Zika prM/E (pre-membrane and envelope) glycoproteins for making vaccines to defend against Zika and demonstrated that mice injected with Zika VLP combined with adjuvant alum secreted high levels of the Ag-neutralizing Abs [31]. In particular, the vaccinated mice all survived without morbidity or weight loss after receiving the lethal challenge with the dose of 200 PFU of Zika strain H/PF/2013, proving the protective efficacy of the VLP-based Zika vaccine which may be tested in humans as a prophylactic candidate with minimal safety concerns to protect unborn babies whose mothers become

Polymeric Nanoparticles Engineered as a Vaccine Adjuvant-Delivery System

http://dx.doi.org/10.5772/intechopen.81084

33

Similarly, Espinosa and colleagues formulated a ZIKV vaccine based on virus-like particles (VLPs) which were generated in HEK293 cells transiently transfected with the prM/E genes of Zika placed downstream from a heterologous signal sequence and observed efficient induction of neutralizing antibody and a dose-sparing effect of alum in VLP-immunized mice (C57Bl/6 x Balb/c) [32]. In addition, passive transfer experiments showed that AG129 mice received the sera from immunized mice prior to Zika infection manifested significantly reduced viral replication as indicated by viral RNA levels in the blood and successfully conquered the infection to contrast control mice which succumbed to infection, underscoring the protective effect of the humoral immunity elicited by this VLP-based Zika vaccine candidate. In summary, the VLP-based VADSs are a potent inducer of Ab and cellular responses and also possesses the prerequisite features required to prepare the vaccines that are able not only to prevent the primary infections but also to clear infected cells, thus representing an alternative tool promising to engineer efficacious vaccines against the intractable pathogens, such as

Chitosan, a linear polysaccharide composed of randomly distributed β-(1,4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit), is usually made through hydrolysis of the chitin shells of shrimp and other crustaceans with an alkaline substance, such as sodium hydroxide [34]. Chitosan has a positive charge under neutral conditions due to protonation of basic amine groups, which contributes to the enhancement of solubility of the compound; however, the dissolution behavior of chitosan in aqueous media is also negatively influenced by the number and structural form of acetylated groups, allowing chitosan able to be used for agent delivery in several distinct forms, including solution, hydrogel, and especially nano/microparticle, which can be obtained via crosslinking, ionotropic gelation or precipitation-coacervation [35]. Interestingly, novel chitosan derivatives with customized biochemical properties are still continuously synthesized through facile conjugation of side chain moieties of functional molecules to solvent-accessible amine and hydroxyl groups, rapidly expanding chitosan in application range and dosage form [36].

In retrospect, in 1980s, researchers observed that chitosan of 70% deacetylated chitin could activate peritoneal macrophages [37] and induce production of various cytokines in mice [38], which was subsequently further explored by Illum et al*.* to demonstrate that in mouse model chitosan, via nasal immunization, could act as an efficient VADS able to remarkably enhance

infected with Zika during pregnancy.

HCV, HIV, and even parasites [33].

**2.3. Chitosan NPs**

Recently, Chapman et al*.* constructed an MVA (modified vaccinia Ankara)-mGag (an HIV-1 subtype C mosaic Gag immunogen) and a DNA-mGag vaccine, which were designed to address the tremendous diversity of HIV, and showed that mGag budded from cells infected and transfected with MVA-mGag and DNA-mGag, respectively, formed VLPs [27]. In mice, the DNA-mGag homologous prime boost vaccination elicited predominantly CD8+ T cells, and the homologous MVA-mGag vaccination induced predominantly CD4+ T cells; in contrast, a heterologous DNA-mGag prime MVA-mGag boost induced strong, more balanced Gag CD8+ and CD4+ T cell responses that were predominantly of an effector memory phenotype. Also, it was found that DNA-mGag homologous vaccination induced much higher cumulative Ag-specific IFN-γ secretion responses and generated significant higher levels of cytokine-positive CD8+ T cells than DNA-nGag (natural Gag), indicating a heterologous prime-boost regimen with DNA and MVA vaccines expressing HIV-1 subtype C mosaic Gag as an Ag is highly immunogenic and may be an effective VLP-based VADS for eliciting strong immunity to HIV. Yao's group formulated a VLP-based HIV vaccine, which was composed of HIVIIIB Gag and HIVBaL gp120/ gp41 envelope as a pseudovirion vaccine capable of presenting Ags in their native conformations and was engineered through using HEK (human embryonic kidney cell)-derived cell line expression system [28]. The researchers demonstrated that mice vaccinated by intranasal prime followed by two sub-cheek boosts with VLPs adjuvanted with liposomes entrapping TLR3 ligand dsRNA were stimulated to secrete high titers of Abs against the Ags, with predominant IgG2c over IgG and produce a significantly increased germinal center B cells and T follicular cells, suggesting that the VLP-based VADS is superior for induction of a Th1-biased immune response, while prolonging lymph node germinal centers, T follicular cells, and generating neutralizing antibodies, and thus is rather suitable for making HIV vaccines [26].

Notably, certain types of pathogens that are once known to cause only a mild and self-healing illness and therefore never listed in dangerous items and may abruptly cause the unexpected problems associated with human and population health, hinting the existence of undisclosed infection mechanisms and pathophysiological processes or the emergence of mutations relevant to severe toxicity. For example, during the 2015–2016 South American Zika epidemic, the mosquito-borne virus which used to cause mild symptoms, such as fever, skin rash, and joint or muscle pain, was eventually identified able to cause severe damage to fetal brain through infecting pregnant women and thus finally recognized as the culprit responsible for thousands of microcephaly affected new borns, raising a great social problem and concern [29]. Unfortunately, up to now, still there are no licensed vaccines for prophylaxis of Zika, though several conventional approaches have been tried on developing such as an urgently needed products, including inactivated, recombinant live-attenuated viruses, protein subunit vaccines, RNA and DNA vaccines, as well as the VLP-based VADS [30]. Recently, using HEK293 cell (Human embryonic kidney 293 cell) expression system, Salvo et al*.* engineered a VLP-based VADS composed of Zika prM/E (pre-membrane and envelope) glycoproteins for making vaccines to defend against Zika and demonstrated that mice injected with Zika VLP combined with adjuvant alum secreted high levels of the Ag-neutralizing Abs [31]. In particular, the vaccinated mice all survived without morbidity or weight loss after receiving the lethal challenge with the dose of 200 PFU of Zika strain H/PF/2013, proving the protective efficacy of the VLP-based Zika vaccine which may be tested in humans as a prophylactic candidate with minimal safety concerns to protect unborn babies whose mothers become infected with Zika during pregnancy.

Similarly, Espinosa and colleagues formulated a ZIKV vaccine based on virus-like particles (VLPs) which were generated in HEK293 cells transiently transfected with the prM/E genes of Zika placed downstream from a heterologous signal sequence and observed efficient induction of neutralizing antibody and a dose-sparing effect of alum in VLP-immunized mice (C57Bl/6 x Balb/c) [32]. In addition, passive transfer experiments showed that AG129 mice received the sera from immunized mice prior to Zika infection manifested significantly reduced viral replication as indicated by viral RNA levels in the blood and successfully conquered the infection to contrast control mice which succumbed to infection, underscoring the protective effect of the humoral immunity elicited by this VLP-based Zika vaccine candidate.

In summary, the VLP-based VADSs are a potent inducer of Ab and cellular responses and also possesses the prerequisite features required to prepare the vaccines that are able not only to prevent the primary infections but also to clear infected cells, thus representing an alternative tool promising to engineer efficacious vaccines against the intractable pathogens, such as HCV, HIV, and even parasites [33].


*3.2. Electrochemical Methods*

3.2.1. Electrochemical Impedance Spectroscopy (EIS)

The coating efficiencies of pure PS and all PS nanocomposite coatings on C-steel rods were investigated using electrochemical impedance spectroscopy (EIS), which is a vital tool in electrochemistry research. It is a strong nondestructive tool for examining and evaluating various electrical characteristics of materials [2–4]. Figures 12 and 13 show Nyquist plots of pure PS and all samples of commercial and local clay PCNs, as well as similar circuits. Table 1 shows the EIS parameters as corrosion resistance (RCorr or Rct), electrical double-layer capacitance (Cdl or CCorr), coating resistance (Rpo) (pore resistance), and coating capacitance (CC). As illustrated in Figures 12 and 13, these parameters were derived by fitting the Nyquist plots of all the coated C-steel samples that had undergone testing to an analogous circuit using Gamry software.

**Figure 11.** TEM micrographs of pure OC and 1–5 wt.% PCN for RCKh at high magnifications.

**Table 1.** EIS data of bare C-steel, pure PS, and 1–5 wt.% PCN coating from commercial Indian and local Khulays clay in 3.5% NaCl solution at 30 ◦C.

**Figure 13.** EIS plots of coated C-steel by pure PS and all prepared (1–5% PCNs for RCKh in 3.5% NaCl solution at 30 ◦C.

The fitted EIS data of bare C-steel and PS and the 1–5% PCN data for both types of clay are presented in Table 1. It is noticed that the coating of C-steel with PS greatly increased the RCorr values and decreased the CCorr values relative to bare C-steel. As reported in the literature for the coated substrates, the first semicircle in the high-frequency region was related to the resistance and capacitance of the protective coating and its properties [13–15,22]. The second semicircle in the low-frequency region was attributed to the electrochemical reactions on the C-steel surface. The findings demonstrated that, in comparison with pure PS coating, the introduction of a tiny amount of OC to the PS polymer enhanced the corrosion resistance (RCorr) and pore resistance (Rpo) of the prepared PCNs. It was realized that, as the diameter of the second semicircle increased, so did the values of the RCorr and Rpo. By adding the small percentage of OC, the values of the corrosion and coating capacitance (CCorr, CC) decreased. These observations for the RCorr, Rpo, CCorr, and CC values indicate that the PCN coatings have higher corrosion protection

than pure PS coating. Generally, this indicates that the protective properties of pure PS coating were improved by adding OC content. This behavior was observed for all the prepared PCNs from commercial Indian clay and Khulays clay. In the present study, our impedance results are in agreement with many previous studies [2,3,19,23,24]. Moreover, adding the organic form of the commercial Indian clay at 1% PCN increased the corrosion resistance value (RCorr) of the coating by about three times the value of the local Khulays clay at 1% PCN. The value of the RCorr in the case of the commercial Indian clay was 6.26 M Ω·cm2, while in the case of the local Khulays clay, the value was 2.46 M Ω·cm2 This was probably due to the high montmorillonite content in the CCIn, which could be easily exfoliated and allows for maximum protection.

## 3.2.2. EFM Method

The electrochemical frequency modulation (EFM) method is considered a new nondestructive way of electrochemical corrosion monitoring. Consequently, it is a good choice for many metals and metal alloys in different aqueous corrosion systems. The corrosion parameters of the EFM test are good for comparison with the results from the Tafel method and linear polarization resistance [40,41]. The results from the EFM are included in Table 2, with the potentiodynamic polarization results prepared from 1–5 wt.% PCNs for commercial Indian (CCIn) and local Khulays (RCKh) clay in 3.5% NaCl solution at 30 ◦C. The percentage of coating, or the protection efficiency, was calculated from the following equation:

$$\% \text{PE} = \frac{(\text{Icorr}(\text{PS}) - \text{Icorr}(\text{PCN}))}{\text{Icorr}(\text{PS})} \tag{2}$$

**Table 2.** EFM data of bare C-steel, pure PS, and 1–5% PCN coating from commercial Indian and local Khulays clay in 3.5% NaCl solution at 30 ◦C, and relative protection efficiencies (%PE) calculated using ICorr.

The EFM parameters (the corrosion current (ICorr), corrosion potential (ECorr), corrosion rate (CR), and calculated relative coating efficiency from the ICorr values) are summarized in Tables 2 and 3. It can be noticed that the coating of C-steel with PS greatly decreased the CR and ICorr values relative to the bare C-steel. Moreover, the PCN coating further decreased the corrosion rate and increased the coating efficiency for both clay types. For the EFM in Table 2, the parameters are the current density in μA/cm2 and the corrosion rate in MPY (milli-inch per year). The goodness of fit for the EFM method is presented in causality factors 2 and 3. Almost all of our results fit this criterion. It is shown in Table 2 that for the 1% PCN of CCIn, the CR is 5.920 × 10−4 mpy, which is the lowest CR of all the formulations.

#### 3.2.3. Potentiodynamic Polarization (Tafel Plots)

Tafel plots are represented in Figures 14 and 15 for the coated C-steel samples prepared from PCNs prepared from CCIn and RCKh, respectively, in 3.5 wt.% NaCl solution at 30 ◦C. The cathodic and anodic curves were analyzed directly from a Gamry potentiostat/galvanostat to determine the Tafel corrosion parameters.

**Figure 14.** Tafel plots of coated C-steel by pure PS and from 1–5 wt.% PCN prepared from commercial Indian clay (CCIn) in 3.5% NaCl solution at 30 ◦C.

**Figure 15.** Tafel plots of coated C-steel by pure PS and 1–5 wt.% PCN prepared from local Khulays clay (RCKh) in 3.5% NaCl solution at 30 ◦C.

**Table 3.** Tafel data of bare C-steel, pure PS, and 1–5% PCN coating from CCIn and RCKh in 3.5% NaCl solution at 30 ◦C, and the relative protection efficiencies (PE%) calculated using ICorr.

In Table 3 for the Tafel plots, the parameters are the current density in μA/cm2 and the corrosion rate in mpy. For Tafel, the values of the chi-square values are included, which reflect the goodness of fit of the present data, which lies in the accepted range. As in the EFM, the best coating efficiency is shown for the 1% PCN of CCIn.

The Tafel plot results showed that the PCN-coated C-steel samples had smaller ICorr and CR values than the PS-coated sample. This indicates that an incorporation of OC in the PS matrix enhances the protective properties of this polymer as a coating for C-steel. This behavior is consistent with previous studies that evaluated the effect of adding small amounts of clay to different types of polymers, such as polyurethane [13], polyaniline [16,23], polypyrrole [22], polystyrene [17,18], and epoxy coating [9,24]. Moreover, the trend in the Tafel results was in agreement with the EFM investigation. According to the EIS, EFM, and Tafel results, the PCN-coated C-steel samples exhibited better corrosion resistance and coating efficiency than the pure PS coating. The best electrochemical protection efficacy was observed at 1% PCN for both types of clay. This is most likely due to the good dispersion of the 1 wt.% organoclay layers in the PS chains, as demonstrated by the XRD and TEM results. Other research groups [13,17] have obtained greater protection efficiencies at 3 wt.% organoclay with polymers. In the case of 1% PCN, however, the fully exfoliated structure was obtained by comparing the electrochemical results with the characterization method results (XRD, TEM). However, the corrosion protection performances of the 3% and 5% PCNs were worse than that of the 1% PCN. This suggests that the results of the characterization methods are consistent with the electrochemical investigations (EIS, EFM, and Tafel). The order of the coating efficiency for both clay types from Tables 2 and 3 is as follows:

$$1\% \text{ PCN} > 3\% \text{ PCN} > 5\% \text{ PCN}$$