Formulation and stabilization of recombinant protein based virus-like particle vaccines.

ADR-12688; No of Pages 14 Advanced Drug Delivery Reviews xxx (2014) xxx–xxx

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Advanced Drug Delivery Reviews journal homepage: www.elsevier.com/locate/addr

Formulation and stabilization of recombinant protein based virus-like particle vaccines☆ Nishant K. Jain 1, Neha Sahni, Ozan S. Kumru, Sangeeta B. Joshi, David B. Volkin, C. Russell Middaugh ⁎ Macromolecule and Vaccine Stabilization Center, Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66047, USA

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Article history: Accepted 18 October 2014 Available online xxxx Keywords: Virus like particles Vaccine Protein Formulation Stability

a b s t r a c t Vaccine formulation development has traditionally focused on improving antigen storage stability and compatibility with conventional adjuvants. More recently, it has also provided an opportunity to modify the interaction and presentation of an antigen/adjuvant to the immune system to better stimulate the desired immune responses for maximal efficacy. In the last decade, there has been a paradigm shift in vaccine antigen and formulation design involving an improved physical understanding of antigens and a better understanding of the immune system. In addition, the discovery of novel adjuvants and delivery systems promises to further improve the design of new, more effective vaccines. Here we describe some of the fundamental aspects of formulation design applicable to virus-like-particle based vaccine antigens (VLPs). Case studies are presented for commercially approved VLP vaccines as well as some investigational VLP vaccine candidates. An emphasis is placed on the biophysical analysis of vaccines to facilitate formulation and stabilization of these particulate antigens. Published by Elsevier B.V.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rationale, requirements and strategies for formulation development of vaccines . . . . . . . . . . . . . Case studies of the characterization, stabilization and formulation of recombinant protein based VLP vaccines 3.1. Approved VLP vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Human papilloma virus (HPV) vaccines . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. Hepatitis B vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3. Hepatitis E vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. VLP based vaccines under development . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Influenza vaccine candidates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Norwalk virus vaccine candidates . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3. Ebola and Marburg virus vaccine candidates . . . . . . . . . . . . . . . . . . . . . . 3.2.4. Hepatitis C vaccine candidates . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.5. Human immunodeficiency virus (HIV) vaccine candidates . . . . . . . . . . . . . . . . 3.2.6. Malaria vaccine candidates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction ☆ This review is part of the Advanced Drug Delivery Reviews theme issue on "Protein stability in drug delivery applications". ⁎ Corresponding author. E-mail address: [email protected] (C. Russell Middaugh). 1 Current address: Vaxess Technologies Inc., Cambridge, MA 02139, USA.

The human immune system is extremely complex involving an interplay among various T- and B-cells, different classes of antibodies as well as a large array of other proteins including receptors and cytokines. It is fair to say that despite our continuing increase in the

http://dx.doi.org/10.1016/j.addr.2014.10.023 0169-409X/Published by Elsevier B.V.

Please cite this article as: N.K. Jain, et al., Formulation and stabilization of recombinant protein based virus-like particle vaccines, Adv. Drug Deliv. Rev. (2014), http://dx.doi.org/10.1016/j.addr.2014.10.023

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N.K. Jain et al. / Advanced Drug Delivery Reviews xxx (2014) xxx–xxx

understanding of the immune response, our state of knowledge remains incomplete [1,2]. The existence of the memory response, however, serves as a basis for the construction of vaccines of prophylactic and therapeutic importance [3,4]. We do know that the immune system is exquisitely turned to recognize viral, bacterial, fungal and parasitic pathogens, even if the nature of this recognition is not always understood. It is therefore not surprising that our earlier vaccines as well as many modern ones attempt to mimic actual pathogens as closely as possible without causing disease [3,5]. Thus, attenuated, live and killed viruses and bacteria constitute many of our more effective vaccines. The advent of recombinant DNA technology, however, opened up the possibility of more easily creating a wide variety of highly specific, safer and more stable vaccines [1,6,7]. Unfortunately most recombinant protein-based vaccines at least in their monomeric forms are only weakly immunogenic despite the use of adjuvants to enhance their efficacy. The major exception to this disappointing result is the dramatic success of virus-like particles (VLPs), high organized oligomeric forms of recombinant proteins [8,9]. A general property in the development of recombinant proteinbased vaccines is that they can take advantage of recent advances in the formulation and stabilization of recombinant therapeutic proteins. In contrast, the formulation development of live, attenuated viruses and bacteria as stabilized vaccines for human use is essentially an empirical process due to their complexity. Typically, using either a cell based plaque assay or an immunogenicity assay in a test animal (usually mice), one stores a variety of formulations and dosage forms (i.e., liquid and lyophilized) at various temperatures for variable periods of time and monitors for loss of replicating virus/bacteria or fall of neutralizing antibody titers, respectively. Because of the relative instability of these complex viral and bacterial vaccines, they are often freeze-dried to obtain adequate stability during long term storage [10–12]. Nevertheless, a number of viruses (e.g., rotavirus, polio) are sufficiently stable that they are formulated in the solution state and can even be delivered orally [13]. In contrast, most recombinant protein vaccines, especially those in the form of VLPs offer the potential of convenient solution formulations as described below. Our new extensive knowledge of the formulation strategies for recombinant therapeutic proteins is a strong contributor to such efforts. The major difference in the design of stable formulations for recombinant protein-based therapeutic drugs versus vaccines is the frequent need in the latter for adjuvants which significantly complicates the formulation process [8]. Here we will focus on the formulation of VLPs due to their current success as vaccines but the principles involve apply equally to other recombinant protein antigens. 2. Rationale, requirements and strategies for formulation development of vaccines VLP based vaccines offer the potential for a safe and effective alternative to live, attenuated and inactivated vaccines [8,9]. It is clear in this case that maintaining the native conformation of the protective protein epitopes within a VLP antigen at all stages of vaccine manufacturing, storage and administration is crucial to the development of a successful vaccine product [14]. Conformational alterations and aggregation of an antigen may perturb the stability, efficacy and safety of a vaccine candidate [15]. Like other macromolecules being evaluated as pharmaceutical entities, an appropriate vaccine formulation program is required to stabilize these relatively fragile molecules against chemical and physical stresses encountered during development [16]. These requirements are usually addressed by incorporating a selective combination of different classes of excipients [14,17]. Traditionally, such vaccine formulation designs involved primarily a trial and error approach in which different combinations of excipients were added and their effects on product potency and stability were monitored. Due to the complex nature of most vaccine antigens, traditional stability indicating assays primarily relied on in vitro

antigenicity measurements and/or in vivo immunogenicity studies. Such assays are usually time consuming and manifest high variability. This empirical approach combined with limited analytical tools can hamper the speed and success of formulation and stabilization of many vaccine candidates. More recently, it has been proposed that a more rational, physicochemical approach (preformulation characterization) should be included in the vaccine formulation development process, and that it should start in the early phases of vaccine manufacturing and clinical development [15,16,18,19]. With advancement in a wide variety of biophysical and mass spectrometry technologies, the paradigm has been shifted towards a more robust analytical approach for vaccine preformulation characterization [16,18,20–22]. These approaches are focused on designing a formulation based upon a more thorough understanding of the complex nature of a macromolecular entity at a physical/chemical level and their interaction with the different components of a formulation such as adjuvants [16,17,19,22]. More sophisticated biophysical and high throughput analytical tools are now available which can help to fill previous analytical gaps. For example, a macromolecular vaccine antigen of interest is first characterized using these analytical tools under various stress conditions such as temperature, pH, ionic strength variation, freeze thaw, and shaking. The physico-chemical characteristics of the degradation products, such as structurally altered antigens and/or aggregates, are studied and mechanisms are elucidated for the observed degradation pathways. Based on the collective information obtained, solution conditions (e.g., pH and ionic strength, etc.) are optimized and the effects of different excipients/additives on the degradation pathways are monitored. The effects of various combinations of excipients on the macromolecule's degradation pathways are then studied and the storage stability of candidate formulations is monitored under accelerated, stressed and real time conditions using appropriate stability indicating assays [16,17]. One general approach to vaccine preformulation characterization is based upon the empirical phase diagram (EPD) in which an antigen is first characterized using various high throughput biophysical techniques under different sets of solution conditions such as pH, temperature, and ionic strength, etc. The physical stability information obtained is mathematically modeled through a vector representation into a colored plot known as an empirical phase diagram (EPD) [23,24]. Changes in color reflect changes in the structure of the macromolecule with regions of similar color representing different states (non-thermodynamic) [23]. Information about optimum solution condition can be obtained directly from the EPD, and the EPD is then used as key starting point to develop a high throughput assay to screen a library of excipients/stabilizer for their effects on the physical stability of the macromolecule vaccine antigen. It should be noted that for the construction of an EPD, the physico-chemical data is collected by exposing the target molecule to accelerated environmental stress conditions. The macromolecules, due to their complex structure, may have different degradation pathways in real time storage than the accelerated stress conditions. Thus, the real time studies may also be required on a case by case basis to further validate the stabilizing conditions identified by the EPD approach. There are a number of papers from our laboratory describing the details of this approach with a wide variety of vaccine antigens [20,23–30]. Some specific examples of the preformulation characterization and formulation development of VLP based vaccines using the EPD approach are discussed in the case studies of the characterization, stabilization and formulation of recombinant protein based VLP vaccines section below. In the next stages of vaccine formulation development, not only are optimum buffer conditions and stabilizers selected to enhance structural/conformational stability and to maintain the potency of antigen [17,22,31], but also different dosage forms are evaluated (e.g., liquid and lyophilized) and then optimized to be most compatible with the route of administration of the vaccine to patients



(e.g., injection, and oral, nasal, etc). In some cases, especially with live, attenuated viruses and bacteria, liquid formulations of vaccines have limited shelf-life and are therefore formulated as freeze-dried dosage forms [10–12,32]. Drying techniques such as freeze and spray drying however, also involve exposure to a variety of additional environmental stresses which can perturb the structure of an antigen and consequently often require one or more excipients for stabilization against freezing and drying [10–12,32,33]. Formulation design of vaccines is focused not only on maintaining native structure and function over a long period of time, but also on enhancing immune responses by the use of adjuvants. Although various adjuvants can be used for boosting immune responses in vivo, there are only a handful of adjuvants that have regulatory approval for human use including aluminum salts (e.g., aluminum hydroxide and aluminum phosphate), oil-in-water emulsions (e.g. MF59 containing a squalene emulsion in citrate buffer with Span 85 and Polysorbate 80), and AS04 (a combination of monophosphoryl lipid A and aluminum salt) [34]. There are number of additional adjuvants which are in preclinical and/or clinical development [34,35]. Modern vaccine formulation design often considers a careful selection of adjuvants to provoke a particular type (Th1 vs. Th2) of immune response [35–38]. There are numerous mechanisms proposed for the immuno-potentiation offered by various adjuvants although, the exact mechanisms of action are not currently well understood as discussed in detail elsewhere [35,36,39,40]. Unfortunately, to date, immune responses are almost always suboptimal with isolated recombinant protein-based vaccine antigens except in the case of VLP and toxoid based vaccine antigens. VLP based vaccines offer relatively better immune responses presumably due to the presence of multiple epitopes which better simulate the surface of viruses and micro-organisms [8,9]. The efficacy of these vaccines however, can also be further enhanced by the presence of adjuvants. For example, the VLP based HPV vaccines developed by Merck and GSK include aluminum and the AS04 adjuvant, respectively [38]. Adjuvants are often an integral part of vaccine formulations and some of them are relatively unstable under certain environmental stress condition [41]. For example, vaccines containing aluminum salts as adjuvants can be susceptible to freeze thaw stress resulting in adjuvant agglomeration [35,42,43] as well as antigen conformational destabilization on aluminum salt surfaces [44,45]. Thus, vaccine formulation development should include a careful consideration of the stability not only the antigen, but also the adjuvant, and the antigen–adjuvant complex (if present), during manufacturing, storage and administration [41,46]. Most currently approved vaccines are administered by injection by the intramuscular (IM) or subcutaneous (SC) route. Novel dosage forms and alternative routes of administration can potentially enhance vaccine accessibility to patients by a more efficient delivery or increases in patient compliance. Novel delivery systems may also result in minimizing the magnitude of doses (i.e., dose sparing) and decreasing pain (although recent improvements in needle design have significantly decreased this problem). For example, mucosal immunization offers innate as well as systemic immunity and doesn't require needle based devices for vaccine administration [47–49]. This type of vaccination can also be helpful in the prevention of improper parenteral immunization related infections. Most mucosal vaccines are delivered via oral or nasal routes. VLPs, due to their particulate nature are an attractive target for this type of immunization [47]. Moreover, VLPs are usually relatively more stable than simple recombinant protein vaccines and may better withstand the adverse environment encountered during oral or nasal administration. Microneedle administration offers another approach for intradermal administration of vaccines without pain or fear from needles. These formulations have been reported to provide a significant dose-sparing effect by inducing protective immunity at lower doses [50,51]. There are number of studies in which VLPs have been employed using alternative routes and novel dosage form for efficient delivery [47]. Some of these are discussed in the case studies presented below.

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3. Case studies of the characterization, stabilization and formulation of recombinant protein based VLP vaccines 3.1. Approved VLP vaccines 3.1.1. Human papilloma virus (HPV) vaccines The human papilloma virus (HPV) vaccines are a recent example of a commercially available and dramatically successful recombinant protein-based VLP vaccine. HPV is a common virus with 80% of women encountering the virus at some point in their lives with most infections being asymptomatic. HPV infection, however, is the major cause of cervical cancer in women [52], producing 6.2 million new infections and 275,000 deaths annually worldwide [53]. HPV infection also appears to cause genital warts and penile and/or anal cancer in men. HPV is spread through sexual contact [54]. Vaccination results in near complete protection (N 90%), but only from the serotypes that compose the individual vaccines [55–58]. There are currently two HPV vaccines on the market: Gardasil® and Cervarix®, produced by Merck and GSK, respectively. Both vaccines are composed of the L1 major capsid protein of certain HPV serotypes. When produced in cell culture, in yeast for Gardasil® and insect cells using a baculovirus expression system for Cervarix®, the recombinantly expressed L1 proteins assemble into VLPs (Fig. 1). Gardasil® is a quadrivalent vaccine (0.5 ml/dose) directed against HPV serotypes 6 (20 μg/dose), 11 (40 μg/dose), 16 (40 μg/dose), and 18 (20 μg/dose), while Cervarix® (0.5 ml/dose) is directed against serotypes 16 (20 μg/dose) and 18 (20 μg/dose). In terms of adjuvants in the vaccines, Gardasil® is formulated with Merck aluminum adjuvant (225 μg per dose), while Cervarix® is formulated with AS04 (aluminum hydroxide, 0.5 mg per dose combined with 3-O-desacyl-4′monophosphoryl lipid A, 50 μg per dose) adjuvant [59–61]. Gardasil® and Cervarix® are both highly thermally stable, remaining potent during long term storage at 2–8 °C for ≥ 3 years. Both vaccines can also withstand transient exposure to elevated temperatures (see below). They are, however, both sensitive to freezing [62–64] presumably due to the presence of the aluminum salt, which is sensitive to the freezing process as described above. The Gardasil® production process requires not only purification of the VLPs, but also a VLP reassembly step to ensure proper formation of the VLPs that resemble the structure of wild type HPV [65–67] (Fig. 1). The dis/reassembly process was developed by following VLP size using a combination of methods including DLS, SV-AUC and TEM which enabled the analysis of the structure of the particle [65]. Early formulations of Gardasil® compared the disassembly and reassembly forms of the VLPs to the untreated particles, with the latter being significantly less stable and immunogenic (in mice) [65]. The initial formulation used HPV VLPs directly purified from yeast containing 0.15 M NaCl and was unstable even at 4 °C (80% potency loss after 12 months). The second, optimized formulation contained 0.32 M NaCl, 0.01% polysorbate 80, and 10 mM L-histidine (pH 6.2) and maintained 100% potency after 12 months at 4 °C [61,64]. This formulation was, however, still unstable at 25 °C for relatively short periods of time (40% potency loss after 1 month). After combining the optimized formulation with VLPs from the disassembly and reassembly steps, the vaccine exhibited excellent thermal stability and immunogenicity [64] (Fig. 2). In the case of Gardasil®, the key to developing a successful VLP based vaccine was a combination of proper formulation and assembly of the VLPs in a manner that resembles and maintains the structure of the wild type virus. The potency of HPV vaccines over time can be assessed using immunogenic responses in an in vivo-mouse assay and/or in an in-vitro relative potency (IVRP) assay that measures the presence of antigenic epitopes. These two assays correlate well, with the main advantage of the IVRP being that it does not require the sacrifice of live animals and takes only 3 days to complete instead of the 4–6 weeks of the animal method. Another advantage of the IVRP is


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Fig. 1. The morphology of representative HPV VLPs expressed and purified from yeast before (left panels) and after disassembly and reassembly treatment (right panels) as determined by atomic force imaging (top panels) and transmission electron microscopy (bottom panels). The two micrographs (bottom panels) are shown at the same scale. Adapted and reproduced from reference [64] with permission of ASPET.

that it only exhibits 10% variability while the in-vivo mouse assay can vary by as much as 40% [68]. Shank-Retzlaff et al. conducted a related study examining the thermal stability of Gardasil® using an IVRP assay. The authors found the estimated half-life of Gardasil® to be 130 months at 25 °C, 18 months at 37 °C, and 3 months at 42 °C. Examination of the thermal stability of individual HPV VLP serotypes by differential scanning calorimetry (DSC) found subtle changes in the primary sequence of different serotype L1 proteins correlated with

relatively large differences in stabilizing intermolecular contacts within the HPV VLPs [62]. Cervarix® is formulated with 8 mM sodium dihydrogen phosphate dihydrate, 150 mM NaCl, 50 μg MPL, and 500 μg of aluminum hydroxide [69]. To illustrate the storage stability of Cervarix®, Le Tallec et al. performed a real-time, long-term stability study and subjected the vaccine to transient temperature excursions (25 °C and 37 °C) to simulate a possible type of stress a vaccine could encounter during

Fig. 2. The relative in-vitro potency monovalent HPV type 16 VLP formulations adsorbed to Merck aluminum adjuvant stored at different temperatures. The compositions the vaccine formulations shown in the figure are as follows: no. 001: 0.15 M NaCl that was directly purified from yeast; no. 002: 0.32 M NaCl, 0.01% polysorbate 80, 10 mM histidine, pH 6.2 that was directly purified from yeast; no. 003: no. 002 + 0.1% polyanions that was directly purified from yeast; no. 004: identical formulation to no. 002 with disassembled and reassembled HPV VLPs. All formulations contain 450 mg/ml aluminum adjuvant. Adapted and reproduced from reference [64] with permission of ASPET.



shipping and handling. The authors found no significant difference between the thermal stability, as measured by in vivo and in vitro potency tests (described above), in Cervarix® that had been stored continuously at 2–8 °C for up to 3 years and vaccines that were transiently exposed to 25 °C (for up to 1 month) or 37 °C (up to 7 days) [63]. New formulations and novel delivery methods of HPV vaccines may be required in the future. For example, HPV vaccines, which are adsorbed to aluminum salts, are freeze sensitive resulting in irreversible damage to the vaccine. As discussed in more detail below, some studies have been completed on the HBV vaccines in an effort to improve the freeze sensitivity of the vaccine through evaluation of different formulations. This was accomplished by the addition of polyols to the formulation to lower the freezing point [41]. Recent work suggests that disaccharides might also be used for the same purpose [70]. Similar approaches could be evaluated with HPV vaccine against freezing stress, although, to date there are no published studies. In terms of novel delivery of HPV vaccines, there has been some recent investigation into delivery of the VLPs by the intranasal and oral routes, but these vaccination strategies have yet to be approved for use in humans [71,72]. 3.1.2. Hepatitis B vaccines Hepatitis B vaccines were the first recombinant VLP based vaccines and were introduced for human use in 1986. Hepatitis B virus (HBV) is a 42 nm double-shelled DNA virus of the Hepadnaviridae family that replicates in the liver and can cause hepatic dysfunction, cirrhosis and cancer in chronically infected and symptomatic carriers [73]. Over 2 billion people worldwide are estimated to be infected with HBV and out of these approximately 400 million individuals are chronically infected and are at a risk of serious illness and death [74]. The virus is transmitted by exposure to infected blood and other body fluids. Vaccination against hepatitis B has proven to be the most effective measure for preventing diseases that could potentially be caused by HBV [75]. Licensed recombinant vaccines against hepatitis B contain the main viral envelope protein, hepatitis B surface antigen (HBsAg) [76,77]. This surface protein is a cysteine-rich, lipid bound protein of 226 amino acids. In the plasma of individuals chronically infected with HBV, 22 nm spherical non-infectious subviral particles containing only HBsAg and lipids are present in addition to the intact virion particles [76–78]. These 22 nm particles derived from the plasma of people infected with HBV were used in the first licensed vaccine against HBV in the late 1970s [79]. Recombinant hepatitis B vaccines gradually replaced the plasma derived vaccine in the mid-1980s but continue to contain the HBsAg as the key antigen. The HBsAg is produced using recombinant DNA technology in yeast and mammalian expression systems by inserting the gene sequence of HBsAg into the host cells [73,80,81]. The expressed HBsAg spontaneously self-assembles into highly immunogenic spherical VLPs containing lipids from the production cells. These recombinant VLPs closely resemble the natural 22 nm particles isolated from the plasma of patients with chronic HBV infections although they differ in their glycosylation pattern [9,74]. The recombinant HBsAg based VLPs contain a high content of host cell lipids and thus differ from the natural non-enveloped VLPs which do not contain host components [9]. The host cell lipids have been shown to strongly interact with the surface protein in the VLPs and play a role in maintaining the protein's conformational stability and antigenicity [82]. The expressed and purified HBsAg particles have also been shown to undergo spontaneous maturation to augment VLP stability and antigenicity while strengthening the inter-subunit interactions through disulfide bond formation/isomerization [83,84]. Following expression and production, HBsAg VLPs are purified by various techniques to remove other host-cell components and then adsorbed to aluminum salt adjuvants [76]. The purity of the final product is analyzed by SDS-PAGE as well as HPLC for the presence of residual host proteins [76]. The potency is confirmed by enzyme immunoassays (EIA)

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using monoclonal antibodies and mouse potency tests using animal models. The bulk product is then aseptically filled and released in final dosage forms after appropriate testing for pH, sterility, general safety and potency. A number of hepatitis B vaccines are commercially available. Almost all of these vaccines are administered with aluminum adjuvants, although one of the recombinant HBV vaccines (Fendrix from GSK which is intended for use in adult patients with renal insufficiency) contains MPL (3-O-desacyl-4′-monophosphoryl lipid A) in addition to an aluminum adjuvant. Two FDA approved hepatitis B vaccines (RECOMBIVAX HB® from Merck and ENGERIX-B® from GSK) are available for use in the United States. Both of these vaccines are produced in yeast cells and formulated with aluminum adjuvant. It is worth noting that RECOMBIVAX HB® from Merck was the first approved human vaccine made by recombinant DNA technology. Multiple WHO qualified vaccines are available worldwide and are produced either in yeast or mammalian cells [9]. All commercial vaccines are liquid formulations and contain 5–40 microgram HBsAg protein per dose. The quantity of antigen per dose that induces protective immunity in infants, children and adults varies by manufacturer, presumably due to the differences in manufacturing processes and type of aluminum adjuvants. The vaccine is typically administered in three doses by intramuscular injection. Hepatitis B vaccines are available as monovalent formulations or in fixed combinations with other vaccines including, for example, diphtheria-tetanus-whole-cell or acellular pertussis (DTwP or DTaP)hepatitis B vaccine and DTaP-Haemophilus influenza type b conjugate (Hib)-inactivated poliovirus vaccine (IPV)-hepatitis B vaccine, among many others. Combination vaccines not only have helped decrease the number of immunizations but have been beneficial in decreasing disease prevalence [85,86]. Hepatitis B vaccine and combination vaccines containing hepatitis B vaccine should be stored and transported between 2–8 °C for optimal shelf-life and potency and must not be frozen. Freezing of the vaccine causes clumping of the aluminum adjuvant, dissociation of the HBsAg antigen from the adjuvant surface, structural damage to the antigen and thus loss of immunogenicity and potency of the vaccine [43,87]. Heat stability studies of recombinant hepatitis B vaccines have shown that suboptimal conditions of storage for 1 week at 45 °C and for 1 month at 37 °C did not alter the immunogenicity of the vaccine [88]. Exposure to elevated environmental temperatures, however, should be avoided. Thus, a cold chain is required for storage and shipment of this vaccine [87,89]. Studies by Braun et al. have shown that addition of propylene glycol and some other stabilizing excipients result in

Fig. 3. Effect of freeze thaw stress conditions on the immunogenicity of different hepatitis B vaccine formulations in mice. The formulations were subjected to three cycles of repeated freezing (− 20 °C, 20 h) and thawing (22 °C, 4 h) and stored for 12 months with appropriate controls at the indicated temperatures. “Original” represents Shanvac-B aluminum adjuvanted formulation (Shantha Biotech, India) transferred to a new vial. “Stable” is a new formulation that contains HepB antigen and adjuvant containing propylene glycol (20 mM), phosphate (40 mM) and histidine (40 mM) at pH 5.2. “F/T” represents samples that undergone freeze-thaw treatment prior to storage. The data are represented as the geometric mean titer with standard deviation in Balb/C mice (*p b0.001, compared to the original formulation stored at 4 °C and n = 8). Adapted and reproduced from reference [41] with permission of Elsevier.


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successful development of a freeze-stable and heat-stable hepatitis B vaccine formulation (Fig. 3). Addition of polyols was shown to protect the vaccine against several freeze-thaw cycles which helped prevent loss of potency due to freezing stress (Fig. 3) [41,90]. Approximately 5–10% of vaccinated patients fail to elicit protective levels of antibodies even after completing a three dose regimen [91]. Continued efforts towards development of better vaccines with improved immunogenicity are thus essential. Multiple research areas for development of more efficacious hepatitis vaccines are being currently explored. One method of interest includes formulation of HBsAg antigen with novel adjuvants such as oil in water emulsions, along with lipid A and CpG based immunostimulatory adjuvants among others [92,93]. Other areas of significant interests involve improving the antigenic quality of HBsAg protein through modifications of purification procedures [94,95], exploring novel antigens such as synthetic peptides and lipopeptide particles, mutants of HBsAg protein, a DNA sequence that contains part of HBV and the hepatitis B core protein HBcAg [79] as well as exploring alternate routes of delivery (mucosal) and novel dosage forms such as freeze dried or powder formulations [79,96]. 3.1.3. Hepatitis E vaccine Hepatitis E infection affects ~ 20 million people worldwide and causes ~ 57,000 deaths annually [97,98]. It is a key reason for acute viral hepatitis with over three million cases appearing every year [97]. The virus is transmitted via the fecal-oral route from contaminated food and water. The disease is prevalent in developing counties where adequate sanitization facilities are often not available [97,98]. There have been a number of outbreaks of hepatitis E reported in the literature primarily in these counties [98,99]. An increase in the number of cases of HEV infection has also been observed in the more developed world as well [99,100]. Analysis of natural and experimental HEV infections found the presence of antibodies against HEV even several years after infection [100]. This provided a clue for the development of a vaccine against hepatitis E. HEV is a ssRNA containing non-enveloped virus containing approximately 72,000 bases with three open reading frames (ORFs) [99]. These ORFs encode a number of different proteins involved in a variety of biological functions. Four genotypes of HEV have been reported with genotype 1 the causative agent for most human disease [101]. It has been established however, that all genotypes belong to a common serotype [100]. This has strengthened the possibility of a broad spectrum hepatitis E vaccine. A 72 kDa capsid protein encoded by ORF 2 is associated with assembly of the virion and shown to elicit antibodies against HEV [102,103]. This protein has been identified as a potential antigen for the development of a hepatitis E vaccine [104]. A number of full length and truncated versions of the capsid protein have been expressed in different expression systems and tested as potential vaccine candidates. Among the different candidates tested, a 56 kDa protein expressed in baculovirus showed efficacy in phase II clinical trials [105]. The vaccine formulation consisted of rHEV (20 μg) adsorbed on aluminum hydroxide (0.5 mg) in buffered saline (0.5 ml) [105]. No further formulation development has been reported for this vaccine. Another vaccine candidate, HEV 239 consisting of icosahedral VLPs expressed in Escherichia coli has been successful in eliciting protective and neutralizing antibodies in a phase III clinical trial [106]. In this vaccine formulation, purified antigen (30 μg) was adsorbed to aluminum hydroxide (0.8 mg) in buffered saline (0.5 ml). This formulation was administered intramuscularly in 16–65 year old healthy volunteers at 0, 1 and 6 months [106]. After three doses, the vaccine (HEV 239) was found to be 100% efficacious with few and mild adverse effects [106]. This VLP based vaccine was recently approved by the State Food and Drug Administration of China and launched in the Chinese market as Hecolin™ [104,107,108]. This is the world's first approved hepatitis E vaccine [104,108,109]. Novel delivery methods including mucosal delivery of the HepE VLP based vaccines has also been investigated in various animal models. Studies in experimental animals showed the capability of HepE VLPs

to induce both local as well as systemic immune responses [110,111]. Oral immunization with recombinant HepE in mice showed an increase in titer of HEV-specific antibody in serum [110]. Another study in cynomolgus monkeys showed that oral immunization with purified rHEV VLPs can induce the secretion of serum IgM, IgG, and IgA [111]. The animals were protected from HepE infection after challenging with native infectious virus. There are a number of other studies in which chimeric HepE VLPs have been explored for use in oral delivery of HepE vaccine antigens [112,113]. These studies indicate the future possibility of mucosal immunization with a HepE VLP based vaccine. 3.2. VLP based vaccines under development 3.2.1. Influenza vaccine candidates Influenza is a respiratory infection caused by the influenza virus which belongs to the Orthomyxoviridae family. Based upon the antigenic properties of their major proteins, three different types (A, B, C) of influenza viruses are recognized [114] with types A and B being the basis for seasonal epidemics in the United States. Depending on the presence and nature of the surface antigens, hemagglutinin (HA) and neuraminidase (NA) type A influenza viruses are further subdivided into different subtypes [114]. Seasonal influenza infects approximately 500 million people and causes ~ 250,000 to 500,000 deaths annually. A number of trivalent and quadrivalent influenza vaccines have been licensed in U.S. for the 2013–2014 influenza season [115]. These include some novel recently approved influenza vaccines such as (1) quadrivalent inactivated influenza vaccines: Fluarix Quadrivalent (GlaxoSmithKline), Fluzone Quadrivalent (Sanofi Pasteur) and Flulaval Quadrivalent (ID Biomedical Corporation of Quebec/GlaxoSmithKline), (2) quadrivalent live attenuated influenza vaccine for nasal administration: Flumist Quadrivalent (MedImmune, Inc.), (3) trivalent inactivated influenza vaccine based on cell culture: Flucelvax (Novartis Vaccines and Diagnostics) and (4) trivalent recombinant hemagglutinin (HA) influenza vaccine: FluBlok (Protein Sciences Corporation) [115]. Considered a significant morbidity/mortality threat with unpredictability arising from emerging variant strains of influenza virus, vaccination with recombinant protein and VLP based vaccines is a promising alternative to egg based vaccines [116]. Because of the variation in antigenic structures in influenza viruses (antigenic drift and shift), vaccines for influenza cannot be stockpiled and require responsive, rapid manufacturing, particularly in the case of pandemic events. One of the distinct advantages of recombinant protein and VLP based vaccines over conventional egg based inactivated vaccines is the ease/speed of production [117]. FluBlok (Protein Sciences Corporation) is a recently approved recombinant vaccine against seasonal influenza [117–119]. It is a trivalent vaccine produced using a baculovirus based expression platform. In addition, a number of influenza vaccine candidates based on expression of VLP antigens are also under investigation and have shown promise in preclinical and clinical trials [8,9,117,120]. These VLP based influenza vaccines primarily consist of the hemagglutinin (HA) and/or neuraminidase (NA) proteins along with one or two matrix proteins (M1, M2) [117,120,121]. New formulation approaches for improvements in the stability of VLP influenza vaccines are being investigated. In one approach, an empirical phase diagram (EPD) was developed to facilitate the visual recognition of changes in the physical structure of H1N1 influenza VLPs under a variety of stresses. The data were used to identify appropriate solution conditions for the screening of potential excipients for use as stabilizers (Fig. 4) [122]. Physical changes in influenza VLPs were studied as a function of temperature (10 to 85 °C) and pH (4 to 8) using various biophysical techniques. The EPD manifested multiple phases suggesting the presence of multiple states of the VLPs under different solution pH and temperature conditions (Fig. 4) [122]. It was observed that the influenza VLPs were relatively unstable at acidic pH values as indicated by lower melting temperatures (Tm) and higher aggregation propensities. The most stable, native-like phase of the VLPs was



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Fig. 4. Visualization of effect of temperature and pH on the structural integrity and physical stability of influenza virus-like particles using an empirical phase diagram (EPD). Temperature dependent biophysical stability data were collected utilizing different techniques over the pH range of 4 to 8. The measurements included effective diameter, polydispersity, static light scattering, circular dichroism signal at 227 nm, intrinsic fluorescence peak position and relative intensity at 330 nm, and extrinsic fluorescence using different probes including ANS (8-anilino-1-naphthalene sulfonate) fluorescence monitored by peak position and relative intensity at 485 nm, and generalized polarization of laurdan fluorescence. Adapted and reproduced from reference [122] with permission of Wiley-Blackwell.

observed at lower temperatures in the pH range of 6–8. Based upon the EPD, a stability indicating assay was developed and a library of generally recognized as safe (GRAS) compounds was tested to identify compounds which enhance thermostability of these VLPs under selected formulation conditions. Trehalose, sorbitol and glycine produced the greatest stabilizing effect on the structure of the VLPs under stress conditions [122]. Based on these preformulation characterization studies, these compounds were proposed for design of improved formulation of the flu VLPs for long term storage. VLP based influenza vaccine candidates have in some cases shown better protection against influenza infection than vaccines based on inactivated viron or recombinant HA in animal models [123,124]. Superior protective immunity by VLPs comprising H5 HA from A/Vietnam/ 1203/04 or its mutant was observed compared to an H5 HA recombinant protein vaccine in mice [124]. A better induction of Th1 type immune responses, hemagglutination inhibition titers, and plasma and memory B cells were identified as possible factors for the better protective immunity by H5 HA VLPs. Multivalent influenza VLP based vaccines could be useful in widening protection against influenza due to variation in antigenic structure. In one such study, a trivalent VLP formulation of influenza A (H1N1 and H3N2) and influenza B VLPs displayed wide protection against influenza virus in mice and ferrets [125]. Novel formulations employing alternate routes of administration have also been explored in various animal models. A formulation of a VLP based influenza vaccine was investigated for intradermal delivery through painless piercing of microneedles coated with VLPs containing M1 and HA of A/PR8 [51]. The coating of microneedles with VLPs resulted in ~94% loss in hemagglutinin activity whereas incorporation of trehalose extensively preserved the activity (Fig. 5). Mice immunized with the trehalose stabilized VLP vaccine formulations by microneedles showed higher antibody titers than unstabilized formulations (Fig. 5) [51]. Formulations administered through an intradermal route were more effective than intramuscular administration indicating a significant effect of route of administration on efficacy of the flu VLP vaccine. A similar effect of stabilization by trehalose and route of administration was observed in the case of microneedles with influenza H5 VLPs containing HA from H5N1 (A/VN/1203/04) [126] and M1 and HA from H1N1 (A/PR/8/34) viruses [127]. Recently, a study showed that patches coated with microneedles and influenza (H1N1 A/PR/8/34) VLPs stabilized with trehalose can induce longer term protective immunity

Fig. 5. Effect of trehalose on the stability and immunogenicity of a micro-needle formulation of an influenza VLP vaccine. (A) Measurement of percent hemagglutination activity in VLPs dissolved from the microneedles containing equal amounts of protein without (H1 VLPs) and with trehalose (H1 VLPsT). (B) Virus (influenza A/PR8)-specific total IgG responses in mice after immunization of groups of mice (n = 12) with a single dose of influenza VLPs. Virus-specific antibody responses were measured by ELISA in blood samples collected at weeks 1, 2, and 4 after vaccination. (C) Survival rates of mice after a lethal dose of virus (A/PR8 virus, 100 LD50) at week 7 after a single vaccination. The different samples include microneedle vaccine formulations without (MN) and with (MNT) trehalose administered intradermally, intramuscular injection of reconstituted vaccine formulation without (IM) and with (IMT) trehalose; intramuscular injection of intact VLPs (IM-intact) and coating solution only (mock). Adapted and reproduced from reference [51] with permission of ASM.

[128]. Mice showed 100% protection against influenza A/PR/8/34 lethal challenge after 14 month of vaccine immunization. High antibody titers were also observed over a year after immunization. Stabilization of the VLPs by trehalose was identified as a major contributing factor for a long lasting efficacy of the vaccine [128]. There are a number of other systemic studies which have looked at different aspects of microneedle formulation of influenza VLP based vaccines [50,129]. Mucosal delivery of a VLP based influenza vaccine has also been reported. In one such study, nasal immunization with influenza A/PR8 (H1N1) VLPs incorporating flagellin produced significant mucosal immunity [130]. Membrane-anchored flagellin (a TLR agonist) was used as a novel mucosal adjuvant [131]. 3.2.2. Norwalk virus vaccine candidates Norwalk virus is the primary cause of viral gastroenteritis in humans of all ages. More than 20 million cases of acute gastroenteritis have been reported each year in the United States [132]. This virus can easily spread from one person to another in closed spaces such as cruise ships, restaurants, schools, day care and residential settings [133]. This


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virus usually does not pose any serious illness in healthy populations, but can be fatal in children and people with weak immune system. The severity of symptoms results from dehydration that can eventually lead to death. Norwalk virus belongs to the family of Caliciviridae, genus Norovirus that has been classified into five genogroups [134]. Out of these five genogroups, types I and II are the most prevalent ones [135]. A new strain of Norovirus designated as GII.4 Sydney has been recently detected in Australia [136]. Norwalk virus contains single stranded, plus sense RNA with three open reading frames (ORF) and a poly A tail [137,138]. It consists of a capsid that is 38 nm in diameter and arranged in T = 3 icosahedral symmetry [139]. These three ORFs encode a non-structural polyprotein, major capsid protein and minor capsid protein [138]. This virus is genetically very diverse and there is no vaccine currently available. A major limitation associated with development of a vaccine for this virus is that it cannot be grown in cell culture. Several expression systems; however, have been developed such as insect [139], bacterial [140] and plant cells [141] for the expression of the major Norwalk capsid protein. This recombinant capsid protein can self-assemble into virus like particles (VLPs). These VLPs are similar in structure to the native virus capsid protein as shown in Fig. 6. These VLPs also possess immunogenic properties that can elicit systemic and mucosal responses in mice and humans [142,143]. It has also been established that the ABH histo blood-groups act as a ligand for the attachment of recombinant Norwalk VLPs to mucosal cell surfaces [144]. The preformulation characterization and stabilization of a Norwalk virus (NV) VLP, using recombinant major capsid protein (VP1) expressed in insect cells, have been examined as a function of temperature and pH [145]. These VLPs were incubated in the pH range that is found in gastrointestinal tract. Ausar et al. employed the empirical phase diagram (EPD) approach to characterize these VLPs based on biophysical stability data. The EPD was used as a tool to visualize changes in the physical properties of NV-VLPs as a function of pH and temperature stress. The NV-VLPs were found to be stable up to 55 °C at neutral and acidic pH. These VLPs were, however, very sensitive to alkaline pH which caused destabilization of the Norwalk virus [145]. At temperatures above 60 °C, extensive visible aggregation was observed. These observations led to the screening of a GRAS library of excipients in an attempt to increase the conformational stability and prevent aggregation of NV-VLPs. Excipients such as chitosan glutamate, sucrose, trehalose, sorbitol and mannitol prevented aggregation [146]. Norwalk virus VLPs formulated at pH 5.0 with chitosan glutamate (concentration: 0.5%) showed increased conformational stability as measured by differential scanning calorimetry (Fig. 7). The chitosan was also used to increase interaction of the VLPs with mucosal surfaces [147].

NV-VLPs were also formulated at pH 7.0 with different sugars and polyols. Sucrose, at 20% concentration was also able to enhance the thermal stability of NV-VLPs (Fig. 7). These two excipients served as the basis for the development of stable formulation of Norwalk virus VLPs. It should be noted that some of the GRAS excipients used in the screening experiments have not been approved by regulatory agencies for parenteral and vaccine formulations. They are included in the excipient screening study to widen screen and provide an opportunity to understand the nature of any interaction of target molecule with various compounds of a different chemical nature. A variety of different routes of administration, expression systems, and formulations have been evaluated with NP-VLPs. For example, the insect cell derived VLPs were administered orally to mice without adjuvant to evaluate their potential as a vaccine. The administration of a dose of 50 μg caused the production of Norwalk virus specific IgG and mucosal IgA in mice [148]. The oral dose of 50 μg produced a substantial amount of humoral and mucosal antibodies against Norwalk virus. These initial studies suggested that recombinant VLPs might be safely employed as an oral immunogen to prevent infection. The recombinant Norwalk virus capsid protein has also been expressed in transgenic tobacco leaves and potato tubers [141]. This protein when expressed in plant cells also assembles into virus like particles, which potentially provides a less expensive means of large scale production. Finally, recent studies have shown that dry powder formulations of Norwalk virus VLPs are as effective as liquid formulations. Dry powder formulations with mucoadhesives might increase the uptake of the antigen on mucosal surfaces. One such dry powder formulation is based on the GelSite® polymer that is extracted from Aloe vera L. polysaccharide [149]. GelSite®, delivered with Norwalk virus VLPs intranasally, can form gelatin like material when it comes in contact with body fluids. This in situ formation of gelatin can increase the retention time on mucosal surfaces and elicit a protective immune response against Norwalk infections. There have been significant developments in improving immune responses using novel adjuvants with Norwalk virus VLPs. Several TLR7/8 mucosal adjuvants such as murabatide and imidazoquinoline have been used to enhance the immunogenicity of the NV-VLPs [150, 151]. These adjuvants are co-delivered with Norwalk virus VLPs and are found to increase immune responses. The two other adjuvants recently used are monophosphoryl lipid A (GSK) and the mucoadhesive chitosan (ChiSys®) [152]. The vaccine candidates adjuvanted with these two candidates have completed Phase 1 clinical trials and were administered intranasally as a dry powder to healthy volunteers. It was observed that adjuvanted vaccine was able to induce high IgG and IgA responses and protected subjects from a viral challenge.

Fig. 6. Structure of recombinant Norwalk virus like particles. The images show an original electron micrograph (A) and surface representation of icosahedral symmetry (B) of the Norwalk VLPs assembled from NT20 mutant of the full length capsid protein. Samples were stained with 1% ammonium molybdate and the image was recorded by cryoelectron microscopy with a resolution of 22 Å. The bar in (A) represents 50 nm scale, and the numbers in (B) show the five and threefold axes of symmetry. Adapted and reproduced from reference [137] with permission of ASM.



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Fig. 7. The thermal stability of Norwalk virus VLPs as measured by DSC in the presence and absence of the excipients sucrose (pH 7.0) and chitosan glutamate (pH 5.0). The excipients were tested at increasing concentrations. Formulations containing 20% sucrose produced an increase in the Tm values of both peaks observed by DSC at pH 7.0. At pH 5.0, chitosan glutamate at a concentration of 0.5%, showed an increase in both structural transition temperatures (Tm and Tm1) peak areas. Adapted and reproduced from reference with permission of Wiley-Blackwell [146].

The results established the safety and efficacy of the vaccine in Phase 1 clinical trial. A recent Phase 1 clinical study has also been conducted for the evaluation of a bivalent Norwalk virus VLP vaccine adjuvanted with MPL and aluminum hydroxide adjuvants. The two genotypes evaluated were GI.1 and GII.4 and encompass broader protection against Norwalk virus [152] (ClinicalTrials.gov NCT01168401). Four different dosage levels were administered intramuscularly (IM) and high serum antibody levels were reported. 3.2.3. Ebola and Marburg virus vaccine candidates The Ebola and Marburg virus belong to the family of Filoviridae that consists of single stranded, negative sense RNA viruses. These two viruses are the most lethal pathogens that infect both human and non-human primates [153] and cause fatality within 7–10 days of exposure time [154]. They have caused several outbreaks of severe hemorrhagic fevers and have been associated with 90% mortality rates [155]. There is also a bioterrorism threat (biosafety level 4, category A) posed by these viruses that has led to increased interest in developing vaccines against them [154,155]. The onset of disease is sometimes associated with the consumption of monkey meat [156]. The Ebola virus is divided into five species based on the location of their outbreak. These are Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Case d'Ivoire ebolavirus (CIEBOV), Bundibugyo ebolavirus (BEBOV) and Reston ebolavirus (REBOV) [157]. Ebola viruses are closely related to Marburg viruses in terms of morphology and structure [158]. The latter contain a larger RNA viral genome of ~19 kb [25]. The genome encodes seven structural proteins while Ebola virus also encodes one additional non-structural protein [159,160]. These are nucleoprotein (NP), viral protein (VP)30, VP35, VP24, VP40, glycoprotein (GP), polymerase (L) protein and secreted (s)GP (non-structural protein, specific to Ebola virus). This secreted protein has been found in large amounts in infected animals and humans [161]. There are no licensed vaccines available for protection against Ebola and Marburg viruses. Only one candidate, a recombinant adenovirus serotype 5 (rAd5) vector vaccine, has reached phase I clinical trial. The results from early clinical trials showed the vaccine to be safe,

immunogenic and tolerated by 23 healthy adults [157,162]. Several strategies have been applied to the development of vaccine antigens against Ebola and Marburg viruses such as attenuated vaccines, DNA and vector based vaccines [157,163]. The attenuated vaccines are generally not preferred due to their potential reversion to wild type. Viral based vaccines expressing structural proteins have shown some promising results but further evaluation is required [164]. Due to the limited success of the above mentioned vaccines, virus like particle (VLP) based vaccines have been examined as an alternative and promising method to protect against infections [165]. The ability of these VLPs to trigger humoral and cellular responses makes them excellent vaccine candidates against these viral infections [166]. A preformulation characterization study was performed to provide a better understanding of the formulation conditions required for a stable VLP based vaccine against Ebola and Marburg viruses. The physical stability of Ebola and Marburg VLPs was examined as a function of temperature (10 to 87.5 °C) and pH (3 to 8) [25]. Several biophysical techniques such as circular dichroism (CD), intrinsic and extrinsic fluorescence were used to study the conformational stability of these VLPs. Their aggregation behavior was also studied by static light scattering and dynamic light scattering. The data from the biophysical methods were incorporated into empirical phase diagrams (EPD) that provided a comprehensive overview of the structural stability of the VLPs. Both Ebola and Marburg VLPs were found to be thermally more stable at pH 7 and 8. At pH 3 and 4, both VLPs showed alterations in structure and increased aggregation at higher temperatures. This behavior may reflect properties of the VLPs that could cause decreased biological activity and shelf life of the vaccine. This work serves as a basis for the development of appropriate formulations of the two VLPs. In terms of ongoing optimization of vaccine antigens and adjuvants for Ebola and Marburg VLP vaccines, studies have shown that the matrix protein VP40 alone is sufficient for the production of the Ebola and Marburg VLPs in mammalian cell cultures. Other viral proteins such as the NP, GP and VP24 proteins may also be required for these vaccines [167,168]. These VLPs were shown to activate dendritic cells, which in


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turn, enhanced the proliferation of T cells. Saponin-derived QS-21 and MPL-like adjuvants have also been added to Ebola VLP formulations. The addition of these adjuvants protected mice against viral infection after only a single inoculation at low dose [169]. Hybrid VLPs containing Ebola virus GP and Marburg virus VP40 proteins have been constructed and expressed with morphology similar to the wild type VLPs [169,170]. The hybrid VLPs provided protection against Marburg viral infections, but not against Ebola virus, in guinea pigs [169]. In contrast, vaccination with mixtures of Ebola and Marburg VLPs in mice and guinea pigs, have produced high levels of virus specific neutralizing antibodies [169,171,172]. 3.2.4. Hepatitis C vaccine candidates Hepatitis C virus (HCV) is a relatively small (55–65 nm in diameter) RNA virus that contains a ~9.6 kb genome [173]. Acute infection with HCV usually results in chronic infection that can lead to liver cirrhosis or hepatocellular carcinoma. In the United States, hepatitis C is the most common blood-borne infection since about 3.2 million people are chronically infected with the virus [174,175]. Although 15–25% of people that develop acute infections are able to clear the virus and avoid chronic infection, the current treatment strategies use a combination of PEG-interferon α-2a with ribavirin and are only 20–60% successful and expensive [176]. There is currently no approved vaccine for HCV. This is complicated by the significant number of HCV genotypes that arise from the high error rates of the low-fidelity viral RNA polymerase [177,178]. Additional challenges include limited availability of small animal models and the nature of the immune response for effective clearance [179]. The HCV genome encodes a ~ 3000 amino acid polypeptide which, through post-translational proteolysis, is cleaved to 10 different proteins including the nucleocapsid core protein and two envelope glycoproteins (E1 & E2) [180]. Several studies have utilized the E1 protein, assembled as a VLP, as the basis for a potential candidate for a vaccine against HCV [181–183]. Lorent et al. found that a truncated form of the E1 glycoprotein self-assembled into VLPs after expression in yeast or mammalian cells [184]. In terms of VLP stability, these VLPs dissociated to E1 monomers in the presence of certain detergents. A combination of biophysical techniques including CD/FTIR, fluorescence, and DLS was used to characterize the secondary structure, tertiary structure, and hydrodynamic radius of the E1 VLP, respectively [184]. He et al. conducted a followup study of yeast-derived E1 (E1y), in which the authors further characterized VLPs as a function of temperature, pH, and dissociative detergents [30] using the same biophysical methods. Based on the results obtained using the aforementioned techniques, HCV E1y was found to be most thermally stable as an assembled VLP (no detergent present) at pH 7 and 8. This work also demonstrated improvements in the stability profile of the assembled E1 VLP over the monomeric E1 protein [30]. These types of studies provide the foundation for continued formulation development in hopes of improving immunogenicity and storage stability. 3.2.5. Human immunodeficiency virus (HIV) vaccine candidates Human immunodeficiency virus (HIV) is an enveloped single stranded positive sense RNA containing virus of genus Lentivirus and family Retroviridae [185,186]. It is responsible for causing Acquired Immunodeficiency Syndrome (AIDS). The current global burden of HIV is around 34 million with 24 million deaths and emergence of about 2.6 million new cases [187,188]. Therapeutic interventions such as antiretroviral therapy and various prevention methods have successfully reduced the incidences of AIDS (N30% decrease between 2001 and 2012) and decreased its mortality as indicated by prevention of around 700,000 deaths in 2012 [187,189]. The complete eradication of HIV infection, however, requires a major breakthrough such as discovery of safe and highly efficacious vaccine [189]. In the last three decades, a number of attempts have been

made to develop a vaccine against HIV [190,191]. To date, more than 230 clinical trials have been conducted to evaluate various vaccine candidates against HIV [192]. None, however, has demonstrated the desirable protection [191,192]. In 2009, a phase III study on vaccine candidate RV144 showed a partial success and provided hope for the development of a prophylactic vaccine against HIV [193]. Some of the major challenges in the development of a vaccine against HIV include the high variability of the viral genome which helps in its evasion of the host immune system, the presence of poorly immunogenic conserved epitopes which fail to elicit broadly neutralizing antibodies and the lack of a suitable animal model among many others [190]. VLP based vaccines represent a promising and safe strategy for the development of an effective vaccine against HIV due to their noninfectious and replication deficient nature [192,194,195]. The inherent advantages include the ability to invoke both types of immune response, particulate nature and possibility of expression of multiple epitopes to elicit broad immune responses [194]. The HIV genome codes for a Gag precursor protein which has ability to self-assemble to form virus like particles of 100–120 nm [186,192,195,196]. VLPs based on an HIV Gag polyprotein (Pr55gag) have shown promise as a vaccine candidate in animal models including non-human primates [192]. These VLPs closely mimic native HIV particles in morphology and size. The HIV-VLPs can express particular epitopes or whole protein and can also be expressed with chimeric antigens to effectively elicit both humoral and cellular immune responses [192,195]. Various approaches have been explored to develop an efficient VLP based vaccine against HIV [192]. These include expression of gp120/160 in trimeric form [192], expression of a Gag-RT fusion protein [197], expression of the entire oligomeric gp120 molecule [195,198], and co-expression of HIV structural genes with DNA or live viral vectors such as vaccinia virus Ankara (MVA) [199]. Obtaining highly pure material in a sufficient quantity for human clinical trials without compromising immunological properties represents an important technical challenge in the development of VLP based HIV vaccines [192]. Preservation of the structural/conformation integrity of HIV-VLPs during production and storage is very important for optimum potency. Various macromolecule formulation approaches can provide solutions for these stability related issues. In one study, the storage stability of HIV-1 Pr55gag VLPs produced in baculovirus was evaluated for one year [200]. Various formulations of the VLPs containing trehalose, sucrose and sorbitol at 5 and 15% were stored for 3, 6 and 12 months at 4 °C, − 20 °C and − 70 °C and their structural integrity monitored by transmission electron microscopy (TEM). Osmolytes at 5% concentration were unable to stabilize the VLPs under various storage conditions. Trehalose at 15% concentration conferred the highest stabilizing effect. The VLPs stored at − 70 °C for 12 months in 15% trehalose showed intact morphology and also produced protection against two freeze thaw cycles [200]. 3.2.6. Malaria vaccine candidates Malaria is another example in which a VLP based vaccine candidate has recently shown promising results in a phase III clinical trial [201,202]. Malaria is an insect born disease transmitted by female Anopheles mosquito and caused by Plasmodium parasites. In 2012, approximately 207 million people were affected and 627,000 died primarily in sub-Saharan Africa [203,204]. Several prophylactic and therapeutic approaches have been adopted to decrease malaria infection and avert deaths. Resistance to anti-malarial drugs and insecticides, however, is still one of the biggest challenges in prevention and treatment of malaria [205]. The development of a safe, effective, and affordable vaccine is probably an ultimate solution to completely eradicate malaria [206,207]. Different approaches have been undertaken to develop a malaria vaccine including pre-erythrocytic to protect at early stages of infection, blood stage targeting to decrease the severity of disease by decreasing the number of parasites in the blood and transmission blocking to disrupt the life-cycle of the malaria parasite [205]. Several vaccine candidates based on various phases of the life cycle of



the malaria parasite are at various stages of preclinical and clinical development [205]. WHO has complied most of the malaria vaccine projects as a spreadsheet called rainbow table [208]. The GlaxoSmithKline (GSK) vaccine candidate RTS,S is the most advanced malaria vaccine candidate [204,209]. In a recent phase III clinical trial, it has shown some protection against clinical and severe malaria in 6–12 week old infants and 5–17 month old children in Africa [201,202]. The RTS,S is a VLP based vaccine targeted against Plasmodium falciparum which is the most deadly form of the malaria parasite [210]. It targets the pre-erythrocytic phase and induces both humoral and cellular immune responses [202]. The vaccine includes a portion of the circumsporozoite protein (CSP) of P. faciparum fused to the N-terminus of the hepatitis B surface antigen (HBsAg) [209,210]. RTS,S is formulated with the AS01 adjuvant which is a liposome based proprietary adjuvant system and contains liposomes, 3′-O-desacyl-4′monophosphoryl lipid A (MPL) and Quillaja saponaria 21 (QS21). GSK has recently approached the European Medicine Agency for a marketing license for RTS,S [211]. This vaccine is a collaborative effort among GSK, the malaria vaccine initiative of PATH with funding from the Gates Foundation. 4. Conclusion The success of VLPs as recombinant vaccine antigens has been spectacular including effective prophylactic vaccines to protect against hepatitis B (HBV) and human papillomavirus (HPV) infections. It seems likely that this approach will see increased use as illustrated above for new VLP vaccine candidates including influenza and Norwalk virus vaccines. Because they are physically and chemically more well-defined entities than classical vaccines, VLP vaccines can be subjected to a rigorous preformulation characterization and formulation development process. This will involve formulations containing both conventional and novel adjuvants and delivery systems. This should lead to the successful development of a series of new, highly stable, efficacious VLP based vaccines in the near future. References [1] W.C. Koff, D.R. Burton, P.R. Johnson, B.D. Walker, C.R. King, G.J. Nabel, R. Ahmed, M.K. Bhan, S.A. Plotkin, Accelerating next-generation vaccine development for global disease prevention, Science 340 (2013) 1232910. [2] P. Oyston, K. Robinson, The current challenges for vaccine development, J. Med. Microbiol. 61 (2012) 889–894. [3] D.A. D'Argenio, C.B. Wilson, A decade of vaccines: integrating immunology and vaccinology for rational vaccine design, Immunity 33 (2010) 437–440. [4] F. Zepp, Principles of vaccine design—lessons from nature, Vaccine 28 (Suppl. 3) (2010) C14–C24. [5] B.G. De Geest, M.A. Willart, B.N. Lambrecht, C. Pollard, C. Vervaet, J.P. Remon, J. Grooten, S. De Koker, Surface-engineered polyelectrolyte multilayer capsules: synthetic vaccines mimicking microbial structure and function, Angew. Chem. Int. Ed. Engl. 51 (2012) 3862–3866. [6] D.M. Smith, J.K. Simon, J.R. Baker Jr., Applications of nanotechnology for immunology, Nat. Rev. Immunol. 13 (2013) 592–605. [7] R. Rappuoli, A. Aderem, A 2020 vision for vaccines against HIV, tuberculosis and malaria, Nature 473 (2011) 463–469. [8] A. Roldao, M.C. Mellado, L.R. Castilho, M.J. Carrondo, P.M. Alves, Virus-like particles in vaccine development, Expert Rev. Vaccines 9 (2010) 1149–1176. [9] N. Kushnir, S.J. Streatfield, V. Yusibov, Virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development, Vaccine 31 (2012) 58–83. [10] C.J. Burke, T.A. Hsu, D.B. Volkin, Formulation, stability, and delivery of live attenuated vaccines for human use, Crit. Rev. Ther. Drug Carrier Syst. 16 (1999) 1–83. [11] G.D. Adams, Lyophilization of vaccines, Methods Mol. Med. 4 (1996) 167–185. [12] S. Zhai, R.K. Hansen, R. Taylor, J.N. Skepper, R. Sanches, N.K. Slater, Effect of freezing rates and excipients on the infectivity of a live viral vaccine during lyophilization, Biotechnol. Prog. 20 (2004) 1113–1120. [13] H.F. Clark, C.J. Burke, D.B. Volkin, P. Offit, R.L. Ward, J.S. Bresee, P. Dennehy, W.M. Gooch, E. Malacaman, D. Matson, E. Walter, B. Watson, D.L. Krah, M.J. Dallas, F. Schodel, M. Kaplan, k.P. Heaton, Safety, immunogenicity and efficacy in healthy infants of G1 and G2 human reassortant rotavirus vaccine in a new stabilizer/buffer liquid formulation, Pediatr. Infect. Dis. J. 22 (2003) 914–920. [14] J.O. Josefsberg, B. Buckland, Vaccine process technology, Biotechnol. Bioeng. 109 (2012) 1443–1460.

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