**5.7 Toxicity studies**

*Current and Future Aspects of Nanomedicine*

low molecular weights [37–39].

**5.3 Nanoprecipitation**

of acetone and ethanol [41].

**5.4 Encapsulation of peptide**

• peptide release controlling,

• decrease toxicity, and

**5.5 Peptide characterization**

• design of new dosage forms [42].

peptide encapsulation is important because of

• modeling of targeted delivery systems,

• insurance of bioconjugate molecules stability,

energy methods [36]. Peptides conjugates biopolymers can be synthesized in organic media using microwave energy. Also, there are another methods, including complex formation of biopolymers and peptides and electrostatic complex formation and metal coordination via ion coordination [35]. Specific antibody titers were observed in mouse experiments against peptides containing polymeric conjugates and complexes. The molecular weights of these conjugates are also very important. Biopolymer conjugation is crucial to obtain a high immune response to antigens at

Nanoprecipitation is the most strategic method for the preparing of vaccine prototypes. Reducing the pH is very important to stabilization of system. Also, salt concentration under the solubility conditions is another important thing for the encapsulation method [70]. If the experiments cannot move on then adding a non-solvent phase in the quality of the solvent technique in which the parent compound of the NPs is dissolved can help [40]. Nanoprecipitation is frequently used in encapsulation of peptides. A pH-controlled precipitation rather than a non-solvent precipitation is a more preferred approach for passing the polymer to a non-dissolved phase with a simple pH change in the medium. For NPs or biopolymers prepared by nanoprecipitation, these solvents are known as the organic phase

Encapsulation is carried out simultaneously by synthesizing NPs and biopolymers in all of the methods mentioned the encapsulation method for peptides should be selected based on the hydrophobic or hydrophilic facilities of peptide. Using of

• mask unfavorable organoleptic properties (taste, odor, color),

• protection of peptide from immune attacks and enzyme degradation,

After purification of the peptides, they are commonly characterized by liquid chromatography-electrospray ionization-mass spectrometry (LS-ESI-MS), fluorescence spectroscopy and possible three-dimensional structures of the synthetic peptide (PEP-FOLD) server. It has validation since the chromatographic method has positive properties in terms of linearity, accuracy, precision and repeatability. Synthetic peptide vaccines are immunogens that can be used when creating vaccine

**32**

Peptide-based vaccine prototypes need to be tested in a cell culture medium to be feasible because they may have physiological, biological and chemical effects, causing cytotoxicity. The method used to investigate the cytotoxic profiles of peptide-based vaccines is also called *in vitro* cytotoxicity assays or cell culture-based measurement methods [46, 47]. Tetrazolium salts are compounds used in cell lines to measure the metabolic pathways of cells of microbial origin. Tetrazolium salts are the heterocyclic organic structure of these compounds and their reduction to colorless or weak colored aqueous solutions known as formazans has been the basis of their use as vital dyes in redox chemistry, biological and chemical applications [46, 47]. The tetrazolium ring can only be broken by active mitochondria, so viable cells and dead cells can be distinguished by discoloration. The fact that this change can be made only by living cells in vitro has made tetrazolium compounds a highly biologically important to measure toxicity of peptide-based vaccine formulas. The mechanism of toxicity assays, such as 3-(4,5-dimethylthiazolyl)-2,5 diphenyltetrazolium bromide (MTT) [19], 3-(4,5-dimethylthiazol-2yl)-5-(3 carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), 2,3-bis(2 methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT), sodium 5-(2,4-disulfophenyl)-2-(4-iodophenyl)-3-(4 nitrophenyl)-2Htetrazolium inner salt (WST), 5-methyl-phenazinium methyl sulfate(PMS), 5-[3-(carboxymethoxy)phenyl]-3-(4,5-dimethyl-2-thiazolyl)-2-(4- sulfophenyl)-2H-tetrazolium inner salt; (MTS) are used [49, 50] and in our studies, we generally use MTT analysis. For example, our technological vaccine prototype example is Zika peptide loaded PLGA nanoparticles which were determined on ECV304 human epithelial cells via MTT assay, which is the cytotoxicity test, was performed to determine the cytotoxic effects of the peptide, peptide loaded NPs [45]. The importance of toxicity studies is to determine the non-toxic vaccine prototype and to switch to in vivo animal studies.

## **5.8 Contemporary advancements in peptide based vaccine**

### *5.8.1 Liposome based subunit vaccine*

Live attenuated vaccine is highly immunogenic and considered as well-tolerant for healthy individuals. However, live attenuated vaccine should not be administered to immunocompromised individual as it would cause systemic infection. An alternative vaccine technology, subunit vaccine, is safer and more suitable for immunocompromised individual. It uses fragment of a pathogen (antigen) to trigger an immune response and stimulate immunity against the pathogen. However,

**Figure 4.** *General features of virus-like particle [48].*

subunit vaccine has low immunogenicity and often combined with adjuvants to induce protective immunity. The adjuvants are capable to enhance vaccine effectiveness and stimulate immune responses [28].

Human alphaherpesvirus 3 (HHV-3) is known as Varicella-zoster virus (VZV), which is the causative agent of varicella (chicken pox) and herpes zoster (shingles). In 1978, the first commercial Varicella-zoster immune globulin, Vzig™ (Massachusetts Public Health Biologic Laboratories, Boston, Massachusetts) became available. However, the supply was removed from the U.S. market in 2006. The alternative preparation, Varizig® (Cangene Corporation, Winnipeg, Canada) was licensed by FDA in 2012 and has shown to be comparable to Vzig™ [Saol Therapeutics Inc. 2012]. Varizig® is supplied as a sterile solution containing human Varicella-zoster immune globulin (IgG) which showed for post-exposure prophylaxis in high-risk patients [51].

Zostavax® is the vaccine licensed for herpes zoster prevention in individuals above the age of 50. It is a lyophilized preparation which is given as subcutaneous injection [52]. A non-replicating liposome-based subunit vaccine (HZ/su) is the new development for zoster prevention. The HZ/su is a non-live recombinant VZV glycoproteins E with the adjuvant ASO1B [53]. A randomized placebo-controlled study has shown that HU/su poses age-independent defense against HZ and has better efficacy compared to Zostavax® in reducing the risk of HZ for immunocompromised adults with the age above 50. Unlike HZ/su, Zostavax® lose efficacy as age increase [54]. HZ/su vaccine is not yet approved by FDA.

**35**

*New Generation Peptide-Based Vaccine Prototype DOI: http://dx.doi.org/10.5772/intechopen.89115*

*5.8.2 Virus-like particle*

Liposomes are nano-carriers and they are useful in delivering vaccine antigen by forming liposome-based vaccine delivery systems. It is advantageous over other carriers due to its biocompatibility, non-toxic and biodegradable features [55]. Besides, liposomes can be customized to achieve desired immune profiles by optimizing their composition, antigen-loading strategies and the use of adjuvants system [28, 56].

Eculizumab (Soliris®) is a humanized monoclonal antibody (mAb) which function as a terminal complement inhibitor [57]. It was the first therapeutic agent approved by the FDA for atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH)-associated with thrombotic microangiopathy (TMA) in 2007 and 2011 respectively [58]. Soliris® (Alexion Pharmaceuticals, New Haven, Connecticut, USA) is in the form of sterile solution for i.v. injection. Eculizumab increases the patient's susceptibility to meningococcal infection (Neisseria meningitidis), all patients must be vaccinated against meningococcal

Virus-like particles (VLPs) is one of the alternative types of nanoparticles delivery system [59]. A recent research has shown that the development of autologous C5 vaccine in nanoparticle form is able to elicit strong humoral responses [60]. A peptide epitope (PADRE peptide) in the C5 vaccine is used to create a recombinant virus-like particles (VLPs). It showed a reduction in hemolytic activity and protect the mice from complement-mediated intravascular hemolysis [60]. Based on the study's result, it is showed that the recombinant VLPs could be used as an alterna-

VLPs is known as an emerging class of targeted delivery vehicles with potential

One of the limitations of VLPs are phagocyte-mediated clearance [59]. Besides, a recent study showed that ellipsoid nanoparticles can extravasate from the blood vessel more effectively than spherical nanoparticles. Meanwhile, the ellipsoid shape is possible for conventional polymeric NPs, but is not feasible for icosahedral VLPs. However, this limitation can be overcome through the modification of VLP surface by adding a variety of useful ligands [61]. VLPs may be able to efficiently extravasate from the vasculature of the blood vessels by showing multiple ligands with high

of overcoming the limitations of other nanoparticles [48]. VLPs is a potential delivery system due to their immunogenic nature, well-defined structure, ability to present a wide variety of potential epitopes, and ease of production [60]. They lack natural genome thus it is non-infectious. Besides, it can turn as self-adjuvant which

**6. Importance** *in vitro* **and** *in vivo* **experiments using peptide-based** 

After forming a synthetic vaccine prototype, the cytotoxicity of the bioconjugate of the peptides and biopolymers is first determined (generally we use MTT analysis). After the apoptotic effect of the prototype on living cells is measured by flow cytometric detection, the vaccine prototype with the most viable cell number should be selected for further study [62]. After all these methods, immunization is the next step. We immunize BALB/c mice with each one of the peptides biopolymer conjugates or peptides loaded nanoparticles following conventional immunization protocol. The goal is to identify the most antigenic vaccine prototype. The

infections prior to or at the time of initiating Eculizumab.

tive or supplement for Eculizumab.

**vaccine prototypes**

is proficient in breaking the immune tolerance.

affinity for the tight connections between endothelial cells [59].

Liposomes are nano-carriers and they are useful in delivering vaccine antigen by forming liposome-based vaccine delivery systems. It is advantageous over other carriers due to its biocompatibility, non-toxic and biodegradable features [55]. Besides, liposomes can be customized to achieve desired immune profiles by optimizing their composition, antigen-loading strategies and the use of adjuvants system [28, 56].

### *5.8.2 Virus-like particle*

*Current and Future Aspects of Nanomedicine*

subunit vaccine has low immunogenicity and often combined with adjuvants to induce protective immunity. The adjuvants are capable to enhance vaccine effec-

Human alphaherpesvirus 3 (HHV-3) is known as Varicella-zoster virus (VZV), which is the causative agent of varicella (chicken pox) and herpes zoster (shingles). In 1978, the first commercial Varicella-zoster immune globulin, Vzig™ (Massachusetts Public Health Biologic Laboratories, Boston, Massachusetts) became available. However, the supply was removed from the U.S. market in 2006. The alternative preparation, Varizig® (Cangene

Corporation, Winnipeg, Canada) was licensed by FDA in 2012 and has shown to be comparable to Vzig™ [Saol Therapeutics Inc. 2012]. Varizig® is supplied as a sterile solution containing human Varicella-zoster immune globulin (IgG) which

Zostavax® is the vaccine licensed for herpes zoster prevention in individuals above the age of 50. It is a lyophilized preparation which is given as subcutaneous injection [52]. A non-replicating liposome-based subunit vaccine (HZ/su) is the new development for zoster prevention. The HZ/su is a non-live recombinant VZV glycoproteins E with the adjuvant ASO1B [53]. A randomized placebo-controlled study has shown that HU/su poses age-independent defense against HZ and has better efficacy compared to Zostavax® in reducing the risk of HZ for immunocompromised adults with the age above 50. Unlike HZ/su, Zostavax® lose efficacy as

showed for post-exposure prophylaxis in high-risk patients [51].

age increase [54]. HZ/su vaccine is not yet approved by FDA.

tiveness and stimulate immune responses [28].

*General features of virus-like particle [48].*

**34**

**Figure 4.**

Eculizumab (Soliris®) is a humanized monoclonal antibody (mAb) which function as a terminal complement inhibitor [57]. It was the first therapeutic agent approved by the FDA for atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH)-associated with thrombotic microangiopathy (TMA) in 2007 and 2011 respectively [58]. Soliris® (Alexion Pharmaceuticals, New Haven, Connecticut, USA) is in the form of sterile solution for i.v. injection. Eculizumab increases the patient's susceptibility to meningococcal infection (Neisseria meningitidis), all patients must be vaccinated against meningococcal infections prior to or at the time of initiating Eculizumab.

Virus-like particles (VLPs) is one of the alternative types of nanoparticles delivery system [59]. A recent research has shown that the development of autologous C5 vaccine in nanoparticle form is able to elicit strong humoral responses [60]. A peptide epitope (PADRE peptide) in the C5 vaccine is used to create a recombinant virus-like particles (VLPs). It showed a reduction in hemolytic activity and protect the mice from complement-mediated intravascular hemolysis [60]. Based on the study's result, it is showed that the recombinant VLPs could be used as an alternative or supplement for Eculizumab.

VLPs is known as an emerging class of targeted delivery vehicles with potential of overcoming the limitations of other nanoparticles [48]. VLPs is a potential delivery system due to their immunogenic nature, well-defined structure, ability to present a wide variety of potential epitopes, and ease of production [60]. They lack natural genome thus it is non-infectious. Besides, it can turn as self-adjuvant which is proficient in breaking the immune tolerance.

One of the limitations of VLPs are phagocyte-mediated clearance [59]. Besides, a recent study showed that ellipsoid nanoparticles can extravasate from the blood vessel more effectively than spherical nanoparticles. Meanwhile, the ellipsoid shape is possible for conventional polymeric NPs, but is not feasible for icosahedral VLPs. However, this limitation can be overcome through the modification of VLP surface by adding a variety of useful ligands [61]. VLPs may be able to efficiently extravasate from the vasculature of the blood vessels by showing multiple ligands with high affinity for the tight connections between endothelial cells [59].
