INDEX

Sr.No	Contents	Page No.
1	Abstract	vi
2	List of figures
3	List of Tables
4	List of Abbreviations
5	Introduction
6	Review of Literature
7	Materials and methods
8	Results and conclusion
9	Discussion
10	References

 

Abstract

Dengue is one of the most important vector borne diseases endemic to tropical and subtropical climates. There is an urgent need for an effective and stable Dengue vaccine. Vaccine stability studies were performed on three batches of lyophilized live attenuated Dengue Tetravalent vaccine (DTV) with a view to find out: 1. Behaviour of DTV during breaches in cold chain maintenance due to shipment delays or mishandling, which is especially important in developing countries, 2. Prediction of shelf life at storage temperature/overages required during formulation to maintain a therapeutic dose, 3. Cumulative effect of temperature over a time helps to find out discard point of vaccine with help of vaccine vial monitor (VVM) studies. Preliminary data obtained in this ongoing studies will help in decision making about vaccine formulation and assess vaccine quality under appropriate storage conditions.

List of figures

Figure No	Title	Page No.
1	Change in global distribution of dengue virus serotypes
2 (A)	Schematic model of DENV particle
2 (B)	3D structure of DENV
3	Dengue virus genome
4	Schematic diagram of dengue virus life cycle
5	Live attenuated chimeric vaccine based on the YFV-17D vaccine

List of tables

S.No.	Title
1	Stability schedule
2	Intra-Assay variability of plaque assay (n=20)
3	Percentage of Residual Moisture at 40⁰C
4	Rate of degradation of Dengue 1-4 at 40 ᵒC (n = 3 batches)
5	Rate of degradation of Dengue 1-4 at 25 ᵒC (n = 3 batches) up to 6 weeks
6	Percentage of Residual Moisture at 25⁰C
7	Rate of degradation of Dengue 1-4 at 15 ᵒC for 1 month (n = 3 batches)
8	Percentage of Residual Moisture at 15⁰C
9	Rate of degradation of Dengue 1-4 at 37 ᵒC (n = 4 batches)
10	Estimated ROD for dengue 1 at 5 ᵒC in DTV
11
12
13
14
15
16
17
18

List of Abbreviations

ROD	Rate of degradation
DENV	Dengue virus
PFU	plaque forming units

hapter1. Introduction

Contents

1. Exposition of problem
2. Project overview

1. Exposition of problem

Denguevirus represents a growing threat all over the world especially in tropical and subtropical regions with respect to its spread all over the world. More than hundred countries have been affected with three million people at risk (Hunsperger et al., 2009). Continuous spread of dengue virus over tropical and subtropical regions dominantly is a matter of concern and immediately requires development of an effective dengue vaccine. Since the spread of virus is mostly in subtropical and tropical regions with warm climate, it is necessary that vaccine should be stable at higher temperatures. Live attenuated viruses are intrinsically sensitive to temperatures; to maintain efficacy and stability of live vaccine formulation, temperature is a crucial factor. Vaccine formulations must be maintained within a specified refrigeration range that would render vaccine stable. Ambient temperatures in the dengue affected areas deviate from the temperature of refrigeration. Therefore, “Cold chain” system is adapted for successful delivery of active vaccine. Cold chain refers to a network which is distributed for maintaining optimal cold temperatures during the duration of transport, storage, handling of vaccine. The system involves continuous monitoring of temperature of shipment containers with the aid of data loggers and individual vaccine vials using vaccine vial monitors (VVMs). Thus, cold chain requirements add up to the major economic and logistic burden, particularly in the lower resource settings, where refrigeration and electricity is limited. The estimated cost of vaccination programs is 200-300 million per year (Das P et al.2004) where the financial cost for cold chain is 80% of the total cost of vaccine (Chen X et al.2011). Maintainace of cold chain is crucial for temperature sensitive compounds like vaccines.  Failure in cold chain maintenance results in wastage of vaccine or delivery of ineffective, sub therapeutic doses of vaccine delivery (Jeney Zhang et al. 2012). In addition, improper storage of vaccines leads to irreversible titre loss which accordingly affects vaccine potency. Hence stability of vaccine has a major impact on the successful immunization programs. Therefore, stability program is important to find out the vaccine storage requirements and vaccine behaviors during temperature excursions.

Live attenuated viruses (LAV) are widely used as a vaccine (Livengood, et al. 2011). These vaccines require storage and shipment at 2-8⁰C, (Kaufmann et al. 2011). Stability of vaccine is mainly based on the factors like type of virus, pH of vaccine, choice of stabilizer, vaccine container. Among environmental factors, temperature and humidity are important factors affecting quality of vaccine over time. Vaccines lose their potency over time and temperature is responsible for potency loss. Hence, it is important to know conditions in which potency of vaccine is maintained. So it is important to carry out stability studies of vaccine targeting factors like temperature and humidity over time, which will give fair idea about storage temperature range as well as help to predict shelf life of vaccine.

Objective of present study is to determine stability of live attenuated dengue vaccine (consisting of Dengue1-4 serotypes) at accelerated, stress, intermediate temperatures

to predict shelf life of vaccine at 2-8⁰C along with this to assess the losses of vaccine due to temperature excursion as well as to find out appropriate storage conditions.

1.2 Project overview

The Serum Institute of India Ltd. (SIIL) is working on live attenuated dengue vaccine and plan to market in the dengue endemic regions of the world. Dengue is prevalent in tropical and subtropical countries having temperature as a major environmental factor affecting stability of vaccine over time. This requires maintenance of cold chain during vaccine distribution and in immunization schedules. Therefore, a stable vaccine at elevated temperatures in tropical and subtropical countries is desirable as a part of WHO programmatic suitability for pre-qualification. This study is aimed at finding the stability profile of dengue vaccine quality and to find degradation kinetics of the vaccine potency with respect to temperature and humidity. This study will also assess losses during transportation in terms of potency due to temperature excursions as well as it will help to predict shelf-life of vaccine in given container closure system. As a part of stability studies, Vaccine vial monitor (VVM), a chemical label indicator on each vaccine vial, for monitoring vaccine quality during transportation will also be studied.

Approach

Standardization of plaque Assay

Method suitability of plaque Assay

Estimation of Reference titres

Exposing DTV (Dengue Tetravalent Vaccine) at different temperatures and humidity for different time periods

Virus titration by plaque assay for titre determination as per time points

Estimating degradation rate at different temperatures and humidity by Arrhenius plot

Assigning of Vaccine Vial monitor

Chapter 2.Review of Literature

Contents

2.1 About Dengue virus

2.1.1 Introduction to dengue virus

2.1.2 DENV epidemiology and global burden of dengue disease

2.1.3 DENV structure, classification and genome

2.1.4 DENV proteins and their functions

2.1.5 Mechanism of DENV infection

2.1.6 Clinical features of dengue disease

2.1.7 DENV replication scheme

2.2 Dengue vaccines

2.2.1 Introduction to DENV vaccines

2.2.2 Protective immune responses against DENV

2.2.3 Challenges in developing dengue vaccine

2.2.4 Technological approaches to dengue vaccine development

2.2.5 Ideal dengue vaccine

2.2.6 Current progress in dengue vaccines

2.3 Stability study

2.3.1 Introduction – stability study

2.3.2 Types of stability study

2.1 About Dengue virus

2.1.1 Introduction to dengue virus

2.1.2 DENV epidemiology and global burden of dengue disease

2.1.3 DENV structure, classification and genome

2.1.4 DENV proteins and their functions

2.1.5 Mechanism of DENV infection

2.1.6 Clinical features of dengue disease

2.1.7 DENV replication scheme

2.1.1 Introduction to dengue virus

The dengue virus is a mosquito born Flavivirus belonging to the family Flaviviridaewhich are distributed globally. Dengue virus has four serotypes (Dengue1-4), which are antigenically distinct (Gubler et al.1995). Dengue virus serotypes are found mainly in subtropical and tropical regions of the world, predominantly in Asia, Africa, and America (Bhatt et al.2013). Dengue virus is transmitted to humans by Aedes mosquito species – Aedes aegypti, and Aedes albopictus (Lambrechts et al.2010).

Infection with dengue virus varies in terms of pathological conditions ranging from a mild febrile illness ie.Dengue Fever (DF) to severe fatal Dengue Hemorrhagic Fever (DHF)/ Dengue Shock Syndrome (DSS) (Malavigeet al.2004). The infection with one of the four DENV serotype provides lifelong homotypic immunity with transient heterotypic immunity.

In last 50 years, dramatic increase in dengue incidence has occurred (Kyle et al.2008); Dengue global disease burden is390 million infections per year (WHO fact sheet, 2017; Bhatt et al.2013). Further, no effective drug against dengue infection is available till date and medical intervention include only supportive treatment. An effective Dengue vaccine is needed to curtain the rising cases of Dengue infection. The challenges in dengue vaccine development are existence of four serotypes, lack of an animal model, unclear understanding of the determinants of DHF in immunologically primed individuals. Hence an effective vaccine development is a priority by WHO against four serotypes.

2.1.2 DENV epidemiology and global burden of dengue vaccine

Currently more than 125 countries are affected with dengue; 50 million to 200 million infections have estimated globally (Murray et al.2013).  In last 50 years dengue infections have increased 30 times with rising geographical distribution from urban to rural areas. Transmission of dengue is highly affected by warming temperatures in addition to this, ratio of primary and secondary infections to rising temperatures have been increased (Jetten et al.1997). Change in epidemiology of dengue has been recorded from the year 1900 to 2003 (Gubler et al.2004)

Figure 1 shows that global distribution of dengue from year 2000-2007(Gubler et al.1997)

In 2012,  WHO declared dengue as most important mosquito borne viral disease in the world due to its remarkable geographic spread of the virus and its vector into previously affected areas and the subsequent burden of the disease (Simmons et al. 2012).

Figure 1.Geographical distribution of dengue from 2000-2007 (Lo et al. 2012).

A recent study, that reviewed all nations in the America, estimated that an aggregate annual cost of dengue for the Americas at US 2.1 billion dollars along with these 12 countries of Asia showed an aggregate annual economic burden of US 950 million dollars (World Health Organization. “Global health situation and projections estimates.” (1992).

Due to low case fatality (CFR), poor disease surveillance, low level of reporting, difficulties in diagnosis, inconsistent comparative analysis and impact of dengue is significantly higher than that which is significantly reported. The true burden of dengue is and economic impact associated with this is still unknown.

2.1.3 DENV structure, classification and genome

Virus structure

Dengue virus has a structure similar to other Flaviviruses; it is spherical with diameter 50 nm approximately. Mature virion is composed of 6% RNA, 66% Protein, 9%Carbohydrate and 17% lipid.  The E (envelope), M (membrane), and C (capsid): These three proteins are associated with the virions (Kuhn et al. 2002).

At the core of the virus is a nucleocapsid, made of the viral genome along with C protein. The nucleocapsid is surrounded by a membrane called the viral envelope, which is a lipid bilayer. Embedded in the viral envelope there are total 180 copies of E and M proteins; that spam throughout the lipid bilayer. These proteins form a protective outer layer that controls the entry of the virus into host cells (White et al. 1994).The major surface protein of the virus is E protein and is responsible for viral attachment and viral entry into the cells. It maybe interacts with viral receptors and mediates virus cell membrane fusion. Neutralizing antibodies usually recognize this protein and mutations in this protein may affect virulence. Envelope is apparently originated from the host cell membrane at the time of virus bussing (Goncalvez et al.2010). The M protein is a small proteolytic prM protein which plays major in virus maturation into an infectious form (Yu et al.2008)..Figure 2 (A) Schematic model of DENV particle, (B) 3D structure of DENV

Figure 2(A) left figure shows immature virus and right shows mature. The spherical capsid contains viral RNA (vRNA) and multiple copies of C protein. The surface of immature particles is made up of 60 spikes composed of trimers of heterodimers between the membrane associated prM and E proteins. The smooth surfaced mature particles are formatted after prM cleavage and contain tightly packed 90E homodimers (Heinz et a.2012).

Figure 2(B) shows that unusual arrangement of E protein dimmers fitted into electron density map. E protein domains I, II, III and fusion peptides are colored red, yellow, blue, green respectively (Heinz et a.2012).

Classification

Dengue viruses belong to family Flaviviridae, genus Flavivirus. There are four serotypes: DENV1, DENV2, DENV3, and DENV4. They belong to the larger, heterogeneous group of arthropod viruses called arboviruses. This is an ecological classification, which implies that the transmission between vertebrate hosts including host is dependent upon blood sucking arthropod vectors. Genus Flavivirus consists of total 70 antigenically related viruses (Kuhn et al. 2002). These Flavivirus include several antigenic complexes mosquito borne encephalitis complex (e.g. DENV complex and Japanese encephalitis complex) and tick borne encephalitis complex (e.g. Kyasanur forest disease virus and louping ill virus). The four DENV serotypes make a unique complex within a genome (Lanciotti et al. 1992).

DENV genome

DENV have a positive sense, single stranded RNA genome. The size of genome is about 10.7 kb; it encodes single long Open Reading Frame (ORF) flanked by 5’-3’ noncoding regions (NCR) / untranslated regions (UTR) of ~100 and ~400-700 nucleotides respectively. This ORF regions codes for single polypeptide processed by both virus encoded and host protease resulting in three structural proteins [capsid (C), precursor membrane (prM), envelope (E)] and seven nonstructural proteins [i.e. NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5] (Figure 3). The nonstructural proteins are associated with virus replication and dengue disease pathogenesis. The coding of the viral proteins is organized in the genome as C-prM-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 (Rothman et al.2004; Guzman et al.2010)

Figure 3 Dengue virus genome

Figure 3 the dengue virus genome encodes 3[capsid (C), precursor membrane (prM), envelope (E)] structural and seven non structural proteins [i.e. NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5].

2.1.4 DENV proteins and their function

DENV genome serves as mRNA for translation of viral proteins. Translation of single ORF produces a large polyprotein that is co and post translationally cleaved into 10 proteins (3 structural and 7 nonstructural proteins). On fourth part of the polyprotein at its N-terminal encodes the structural proteins (C-prM-E), followed by nonstructural proteins (Deubel et al.1986).The host signal peptidase is responsible for cleavage among C-prM, prM-E, E-NS1 near the C terminus of the NS4A. While a virus encoded serine protease, is responsible for cleavage among NS2A-NS2B, NS2B-NS3, NS3-NS4A, NS4A-NS4B, and NS4B-NS5.

Structural proteins

1. Capsid protein

It is highly basic protein of ~11 KDa. The C protein stabilizes the viral RNA within the viral nucleocapsid. The N terminus of the C protein encodes a nuclear localization sequence which allows translocation of C protein into nucleus and interaction with nuclear riboprotein. The DENV C protein may interact with human death domain associated protein Daxx and induces apoptosis.

2.                Membrane protein

The glycoprotein prM is ~26 KDa in size which is translocated into endoplasmic reticulum (ER) by C- terminal hydrophobic domain of the C protein. The prM proteins act as a chaperone that helps the folding of E protein. An endopeptidase , furin, cleavage in the Golgi complex forms precursor form of prM which helps in formation of M protein. The conversion of immature virus particle to mature virions occurs in the secretory pathway and coincides with the cleavage of prM into pr and M by the Golgi resident furin or furin like enzyme (Keelapang et al.2004). Hence ‘pr’ segment of prM is assumed to stabilize E and keep it from undergoing rearrangement to the fusogenic form in the reduced pH environment of the early secretory pathway (YI et al. 2011).

3.         Envelope protein

Majority of envelope protein (E protein) is exposed on the surface of DENV. This ~55 KD a structural protein has been extensively characterized. It is composed of 3 distinct domains (DI, DII, and DIII). These domains have been characterized for DENV 2 and DENV 3 by Xray crystallography. Domain I is eight-stranded barrel structurally positioned between DII and DIII. DII contains a dimerization region, which is involved in membrane fusion and dimerization of E protein. Domain III contains immunoglobulin fold and is thought to be involved in receptor binding (Sukupolvi-Petty et al 2007).

Nonstructural proteins

NS1 Protein

NS1 is ~46 KDa in size expressed in three forms: 1. ER-resident form, 2.membrane-anchored form, and 3. secreted form.ER-resident form plays important role in virus replication during infection. In infected cells, NS1 exists as hexamers and is another dominant target of humoral immunity. Soluble NS1 binds to plasma membrane of uninfected cells. Soluble NS1 binds to plasma membrane of uninfected cells by interactions with heparin sulfate and chondroitin sulfate E. NS1 is expressed on the surface of infected cells as membrane anchored form, possibly by a glycosyl phosphatidylinositol (GPI) anchor or lipid raft association. The functions of surface expressed NS1 have been reported to include signal transduction and complement activation (Alcon et al. 2002).

NS2A Protein

It is relatively small (~22 KDa), hydrophobic protein. The N terminus of NS2A is generated through cleavage of NS1-2A by an unknown ER resident host enzyme, whereas C terminus is generated by viral serine protease cleavage in the cytoplasm. By protease activity, NS2A can cleave itself from NS1 (Xie et a al. 2013).

NS2B Protein

It is small (~14 KDa) membrane associated protein. It forms serine protease complex using cofactor NS3. The cofactor activity of NS2B is encoded in conserved central hydrophilic regions of 40 residues, flanked by hydrophobic regions that mediate membrane association (Falgout et al.1991).

NS3 Protein

NS3 is a large (~70 KDa), multifunctional protein with several enzyme activities that are involved in polyprotein processing and RNA replication. The enzyme activities of NS3 include N-terminal protease domain, RNA 5’ triphosphatase, RNA helicase and RNA stimulated NTPase Domain in the C terminal region. The protease activity is required to process the polyprotein precursor and it is important for viral replication (Arias et al.1993).

NS4A and NS4B protein

NS4A and NS4B are small (~16 KDa and ~27 KDa respectively), hydrophobic proteins, their functions are not known. They both involved in blocking IFN-/- induced signal transduction. However NS4B can modulate viral replication by its interaction with NS3. (Falgoutet al.1991).

NS5 Protein

It is (~103 KDa), multifunctional and most conserved protein, which encodes S-adenosyl methyltransferase that can methylate the 5’ end of viral RNA and RNA dependent RNA polymerase. It plays major role in RNA replication. It contains N terminal RNA cap processing activity and C-terminal RdRP activity.The N terminal region of NS5 was found to contain homology with S-adenosyl Methionine (SAM) dependent methyltransferase suggesting that this protein is involved in 5’ cap formation (Brooks et al. 2002).

2.1.5 Mechanism of DENV infection

Primary DENV infection incubation is 4-7 days, during which virus replicates in dendritic cells in close proximity to the mosquito bite and infect lymphocytes and macrophages followed by entry into the blood stream. Dendritic cells (DC) and Antigen Presentation Cells (APC) are integral part in inducing immune response. The dengue virus preferentially targets DCs; specifically monocytes derived DCs and Human skin Langerhans cells (LCs). Cell surface heparins sulfates are also involved in cell surface are also involved in cell surface attachment of dengue virus to mammalian cells (Halstead et al.1988).

The co receptors for viral entry into DCs have been identified and analyzed. C type lectin, CD209/DC-SIGN are one of the receptors which are thought to bond to viral E proteins aiding the entry into DCs. The mannose is also identified as a receptor present in macrophages (Miller et al.2008). The dermal macrophages act as first innate immune cells responding against virus infection which are capable of protection against the dengue virus after mosquito bites (Kwan et al. 2008). C type lectin domain family 5 A (CLESC5A) can serve as a pattern recognition receptor (PRR) for macrophages interacting with dengue viruses to mediate inflammatory cytokine release.. Dengue infection is an immune pathological disease in which an immune response may aggregate DENV infection to damage host. This phenomenon is thought to be caused b antibody dependant enhancement (ADE) and was propose as an underlying pathogenic mechanism of DSS/DHF as early as 1960 (Halstead et al.1988). It is thought pre existing sub neutralizing antibodies and DENV form complexes which bin to FC-γ receptor bearing cells and results in increased viral infection and replication. This increase in viral replication directly increases the amount of virus in the blood contributing to DSS. A strong association between severe secondary infection and host genetics along with this virulence of different virus strains may also play important role in DHF pathogenesis. ADE is also accepted theory although there is still much to be proven regarding the direct cause of enhancement of viral replication (Murrell et al.2011).

2.1.6 Clinical features of dengue disease

Dengue viruses have distributed globally having wide spectrum of clinical illness. Clinical presentation of dengue is often unpredictable as compared to past evolution of disease. Large proportions of population recover from self-limiting non-severe illness whereas small population advances towards severe illness and shows plasma leakage with or without hemorrhage. Dengue virus infections are classified into three categories: 1.undifferentiated fever, 2. Dengue Fever (DF), and 3.Dengue Hemorrhagic Fever (DHF). There are four grades of DHF while grade 3 and grade4 has been defined as Dengue Shock Syndrome (DSS) (Dengue Guidelines for diagnosis, treatment, prevention and control by World Health Organization 2009).

Dengue Fever (DF)

3-14 days is incubation period for classic DF but it is generally 4-7 days. Sudden onset fever accompanied by headache, generalized myalgias, retro orbital pain, rash, abdominal pain, and nausea these symptoms are present with Dengue Fever. When virus enters into the skin through mosquito bite, virus replicates in local dendritic cells with later systemic infection of monocytes or macrophages followed by viral entry into the blood (Palucka et al.2000). Liver is the main target of virus while in case of febrile illness virus shows high titres(~10^{5 –}10⁶ infectious units/ml) to produce viremia (Whitehead et al.2007).Rash is mostly seen in DF which is present mostly in 80% cases. Macular, macula popular, petechial these are reported observation of rash which is common in trunk inside of the arms and thighs. Thrombocytopenia, leucopenia, elevation in serum transaminases are common laboratory abnormalities reported in symptomatic infections (Murphy et al.2011).

Dengue Hemorrhagic Fever (DHF)/ Dengue Shock Syndrome (DSS)

Hemorrhage is not most peculiar manifestation in case of DHF though DHF is characterized by fever, thrombocytopenia, hemorrhagic manifestation, increased vascular permeability with leakage of intravascular fluids (Gubler et al.1998). Vascular permeability and the loss of intravascular fluid contribute significantly to disease severity than hemorrhage. This severe form can be seen more frequently in secondary infection than primary infection. Viremia is generally 10-100 folds higher in DHF than DF (Whitehead et al.2007). DHF is mostly observed in children in areas where all four serotypes are present. Manifestations include capillary fragility, ecchymoses, bleeding from mucosa, gastrointestinal tract or other sites. Around the time of hiatus, patient’s condition can suddenly deteriorate with a narrowing of pulse pressure. Increased vascular permeability results in loss of plasmid fluid into the interstitial spaces and can result in pleural effusion and ascites which are reported in more than 50% in DHF patients examined by ultrasound procedure.

Hypovolemic shock ensues when sufficient leakage of plasma fluids into interstitial spaces has occurred which leads to compromised cardiac output. The course of the shock is life threatening in which patients recover or succumb within 24 hrs depending on appropriate intervention (Murphy et al.2011).

2.1.7 Dengue virus replication scheme

Attachment, penetration, uncoating

Dengue viruses attach to susceptible cells by mainly two mechanisms:

1. DENVs complexed to non-neutralizing, but anti-virion, immunoglobulin (IgG) antibodies may attach to monocytes or macrophages via Fc-γ receptors found at cell surface. When Fc-γ receptor- bearing cells are infected in the presence of serologically cross-reactive antisera diluted beyond the neutralization endpoint, a greater number of infected cells and higher virus titres are obtained (Porterfield et al.1986). This is known as immune infection enhancement, which contributes to the pathogenesis of DHF or DSS.

OR

2. Dengue viruses may attach to the cells including monocytes using Trypsin sensitive receptor (Henchal et al.1990). Hypothesized structure and composition of host receptor denotes that binding of host receptor to distinct regions of E glycoprotein.

There are two methods by which attached infectious virus may penetrate the host cells:

1. The virion envelope may fuse with plasma membrane (PM) with immediate deposition of the nucleocapsid into the cytoplasm.

OR

2. The PM may invaginate forming an endosome around the enveloped virus.

Similar to other enveloped viruses, the fusion of the DENV envelope with host cell membrane appears to be pH dependant (van der Schaar et al.2008). Acidic conditions activate a fusion protein which causes fusion of accumulation of the nucleocapsid within the cytoplasm though activation mechanism of fusion protein is yet unknown.

Existence of evidence of E protein being a fusion protein comes from following experiments that show:

• The anti-E antibodies inhibit fusion
• At acidic pH, E protein undergoes an irreversible conformational change

(Kampmann et al. 2009)

Primary translation and early RNA replication

DENV genome is a positive sense RNA; it must first to be translated for making RNA polymerase which is required for replication. The polymerase must then transcribe negative strand RNA that acts as template for additional positive strands.

During the long eclipse period (12-16 hrs) which triggers the formation of first virus progeny in which RNA must act as a template for replication and translation but not in the case of encapsidation. While each positive sense viral RNA churns out single polyprotein, negative sense RNA is used for synthesis of more positive sense viral RNAs.The whole viral machinery is synchronized to produce positive and negative strands at nearly equal rates for exponential RNA amplification. Positive strand RNA is diverted to virus assembly during the late eclipse phase (Henchal et al.1990).

Synthesis and proteolytic processing of viral proteins

Large precursor polyprotein which are encoded by single ORF help to obtain structural and Non-Structural (NS) proteins but this precursor protein is not seen in infected cells. The translation initiates at the first AUG (start codon) codon of theRNA genome and individual viral proteins are formed by co and post-translational proteolytic processing precursor peptides. The proteases corresponding to polypeptide processing appear to be virus and host encoded.

The host signal peptidase is responsible for cleavage among the C-prM, prM-E, E-NS1 and near the C-terminus of the NS4. While a virus encoded serine protease, is responsible for cleavages among NS2A-NS2B, NS2B-NS3, NS3-NS4A, NS4A-NS4B, and NS4B-NS5. Enzyme which is responsible for NS1-2A cleavage is presently not known (Henchal et al.1990).

RNA replication

The RNA replication can be detected within 3 hrs post infection and appears to occur in the peri-nuclear region of the infected cell in association with smooth membrane (Takedan et al. 1978). Three forms of RNA can be extracted from dengue virus infected cells and isolated by sedimentation through sucrose gradients: 1) RNase-resistant 20S to 22S RNA are known as replicative form;2) partially RNase resistant,, 20S to 28S RNA called replicative intermediate; 3) RNase sensitive replicative intermediate is converted into 42S RNA and many smaller (Wengler et al.1978).

The studies suggest that replicative form is a full-length, dsRNA containing one positive strand completely annealed to one negative strand. Replicative intermediate RNA may act as precursors to positive strand 42S viral RNA (Wengler et al.1978).

It is unknown that how RNA replication is regulated, but it is possible that early and late RNA polymerase complexes have distinct affinities for positive and negative strand templates. Late replication processes favor synthesis of the infectious positive strand. Increasing concentrations of C protein late in infection may begin the assembly of nucleocapsid, removing positive strands as a substrate for replication. It has been proposed that binding of C protein to a site at the 3’ end of positive strand RNA prevents it from being recognized by RNA polymerase but not by ribosome’s which bind at the 5’ end. This would help for its continued translation and explain the predominance of positive strand RNA later in infection (Liu et al.1997).

Virus assembly and release

Assembly and release of dengue viruses has the following phases:

1. Assembly of nucleocapsids from C protein and RNA
2. Budding of nucleocapsids through membrane containing integral E and prM proteins to acquire an envelope
3. Exit from the cell, or due to budding process or afterwards in exocytic vesicles
4. Cleavage of the prM protein, leading in a reorganization of the virion surface and virion maturation

On the contrary to this, alpha viruses which posses their envelope as nucleocapsids in the cytoplasm and bud through the plasma membrane (PM), the Flaviviruses appear to mature in a different manner. With few exceptions, Flavivirus nucleocapsides are not seen in cytoplasm freely while it appears as a envelope like particles (VLP) associated with intra-cytoplasmic vacuoles, golgi vesicles and also within the cisternae of rough endoplasmic reticulum (RER). The enveloped particles which appear to be derived from intracytoplasmic membrane are sometimes larger than mature virions and may represent precursors. Flavivirus nucleocapsids assemble from C protein that contains a hydrophobic stretch of amino acids at the C terminus which may anchor it to the membrane of RER and is removed by proteolytic cleavage during the maturation of virions (Henchal et al.1990).

It is reported that PR-159 strain of DENV-2 matures in mosquito cells by budding at both intra-cytoplasmic and plasma membrane (van der Schaar et al.2009). Morphogenesis pathway will differ depending on the host or viral strain. Nevertheless of how development occurs, the source appears to intra cytoplasmic rather than at the PM. Lipid composition of virion envelope more closely resembles that of cytoplasmic membrane (Eckels et al.1980).

Release of virus from infected cells presumably occurs via secretary exocytosis as virus containing secretary vesicles fuse with PM. Released virus contains little, prM; therefore cleavage of prM must occur during or before exit from the cells. Cleavage of prM is accompanied by reorganization of the virion envelope from one containing prM-E heterodimers to one containing E-protein trimers. Immature, prM containing flavi-virions are about 60 fold less infectious than the mature virus. The prM protein may maintain the virion in a highly stable but relatively inert state. Final cleavage step makes the virus competent for infection but more labile (Henchalet al.1990).

Figure 4: Life cycle of Dengue virus in infected cell

DENV bind to cells through interactions between E proteins and cellular receptors on target cells. In addition to this, viruses may also bind to and enter cells as an immune complex in a process called ADE of infection after binding DENVS are internalized into cells via clatherin mediated endocytosis and traffic into late endosomal compartment in which virus mediated fusion occurs in a pH dependant fashion. The positive stranded genomic RNA is then released into the cytoplasm in which translation occurs. Replication of viral RNA occurs in context of complex 3D networks of membranes induced by the viral non structural proteins. Virus assembly occurs on membranes derived from the ER. Virions bud into the ER as immature virus particles that incorporate sixty trimeric spikes of prM and E proteins. During egress, prM is cleaved by the cellular serine protease furin. A relatively smooth, infectious, mature virus particle is then released into the extracellular space.

 

2. Dengue Vaccine
   1.      Introduction to DENV vaccines
   2.      Protective immune responses against DENV
   3.      Challenges in developing dengue vaccine
   4.      Technological approaches to dengue vaccine development
   5.      Ideal dengue vaccine
   6.      Current progress in dengue vaccines

1.   Introduction to DENV vaccines

In tropics and subtropics rapid spread of dengue infection represents as rapidly growing public health problem. As per the reports 2-5 billion people are at the risk of dengue. In recent years spread of dengue has increased dramatically. Hence to control this emerging disease development of safe and effective vaccine is required (Webster et al.2009).

Obstacles in development of dengue vaccines are lack of an animal model which reproduces human disease, existence of four serotypes, and risk of enhanced disease upon subsequent natural infection if antibody to one or more serotypes diminishes over time which implies development of vaccine which must fulfill the criteria of protection and immunity against all four serotypes (Durbin et al.2010). Live form of vaccine is preferred as it gives long lasting protection. Ideal vaccine would have ability to elicit broad and durable immune responses against all four serotypes in both children and adult along with that it would have good safety profile (Lang et al.2012).

Although development process of vaccine is dealing with many obstacles, a DENV vaccine has made great progress in recent years. For development of candidate vaccine many approaches have been used including live attenuated tetravalent vaccine, chimeric tetravalent vaccine based on attenuated dengue virus or Yellow fever 17D, vector ( Flavivirus and Non-Flavivirus)  born recombinant vaccines (Murrell et al.2011). Two tetravalent candidate vaccines are in phase II clinical trials and several live candidates in phase I while other DNA, Subunit vaccines are in preclinical stages of development (McArthur et al.2013). Live attenuated tetravalent dengue vaccine based on yellow fever backbone 17D produced by Sanofi Pasteur is in phase III efficacy trials (Guy et al.2010).

2.2.2Protective immune responses against DENV

Cellular and humoral immunity contribute together to DENV clearance and protection. The main target of antibody (Ab) responses against the DENV is E protein which is an important component on the surface of dengue virus. During viral entry, E protein binds to cellular receptors and mediates fusion of the viral envelope with cellular membrane (Tsai et al.2013). NS1 protein is not a virion component; it is circulated through expression on the surface of virus infected cells. In case of acute infection, it acts as a diagnostic marker as it expresses in the sera of affected individuals. (Chuang et al. 2013).

The antibodies against NS1 can trigger complement mediatedlysis of DENV infected cells in vitro and protect mice from DENV challenge. Monoclonal antibodies against prM/M have been shown to provide protection against DENV challenge (Lu et al. 2013).  Heterogeneous dengue virus-specific CD4+ cytotoxic T cells are stimulated in response to infection with a dengue virus and that a nonstructural protein, NS3, contains multiple dominant T-cell epitopes (Kurane et al.1991).

2.2.3 Challenges in developing dengue vaccine

Four serotypes of virus are responsible for infection. Antibodies produced against a particular serotype are specific. Hence in the endemic regions chances of sequential infections are more where multiple serotypes exist. Infection with one serotype gives lifelong immunity but previous infection is a risk factor for more severe form of dengue upon subsequent infection with remaining serotypes (Monath et al.2007). Therefore it is a need to develop a tetravalent vaccine which will provide lifelong immunity against all four serotypes. The exact mechanism of protective immunity is not yet clear. Antibody mediated Dengue virus neutralization plays major role in protective immunity formation against dengue virus infection (Chen et al.1997).

Another challenge for vaccine development is a harmful effect of immune enhancement in dengue pathogenesis. Antibody dependant enhanced (ADE) is reasonablephenomenon in development of immune response by neutralization, which consists of increased rate of viral replication by immune sera (Halstead et al.2003). ADE also supports to produce immune response against all four serotypes for large number of population. On the contrary to this, elevated incidences of dengue may result due to increased levels of ADE in case of ADE enhanced and ADE non enhanced serotypes (Cummings et al.2005). Increased replication in Fc-receptor bearing cells due to ADE is seen in secondary infections. Antibodies against prM do not neutralize the infection by showing cross reactivity in addition to this it promotes ADE. Hence partially cleaved prM from viral surface supports ADE formation by raising antibody against prM. This will give new approach to develop vaccine (Dejnirattisai et al.2010).

Main challenge in   development of dengue vaccine is lack of an animal model. Normal mice do not display when infected with human DENV isolates although mice adapted DENV strains produces a paralysis phenotype which is used to evaluate efficacy of candidate dengue vaccine(Wilder-Smith et al. 2010). The non-human primates which are susceptible for infection do not show significant clinical signs of the infection (Cassetti et al. 2010).

2.2.4 Technological approaches to dengue vaccine development

Development of tetravalent vaccine supports 3 facts that primary infection with one serotype provides long term immunity against reinfection for that particular serotypes which lasts for years. Considering this fact it is not able to give long lasting immunity against other 3 serotypes. Second fact is that in dengue endemic areas there is a fair chance of existence of all 4 serotypes and if seasonable change in serotype occurs then monovalent vaccine is not sufficient to produce immunity as compared to tetravalent vaccine. Third fact is that in case of ADE and secondary infection hypothesis to avoid future incidence of dengue a vaccine a vaccine must induce protective immunity against all four serotypes (Edelman et al.2007).

A vaccine should induce antibody levels that imitate which are associated with natural infection. A vaccine should provide immunity within short dosages and it should be cheap as well as readily available.  Live vaccines can fulfill these criteria than recombinant subunit, nonreplicating killed whole subunit vaccine (Edelman et al.2007).In case of attenuated vaccines it will provide better immunity than inactivated, subunit vaccines. As inactivated, subunit vaccines need multiple dosages, which will provide short-term immunity. Hence it will make cost of vaccine higher than attenuated vaccine. Development of live, attenuated vaccine is still facing difficulties. To develop a successful vaccine there should be balanced ratio of immunogenicity and reactivity (Edelman et al.2007).

Recombinant subunit vaccines have advantages over live attenuated vaccines. These vaccines are safe because proteins used are not as dangerous as compared to live attenuated virus it will also balance immune response. Hence the risk of incomplete immunity and adverse effects of ADE can be avoided. Recombinant E proteins can be produced using suitableE.coli, insect cells, mammalian cells. As E protein has majority of epitopes, which have capacity to neutralize (Advances in Virus Research, Volume 88).

Other approach is use of chimeric viruses which can be developed using recombination DNA technology.  By using cDNA of DENV4 prM, C, E genes were replaced with DENV1 OR DENV2 structural genes. Therefore chimeric virus DENV1/DENV4 can be produced. This was experimented in rhesus monkeys which produced neutralizing antibodies against DENV1/DENV4 (Lai et al.1996).

Use of ionizable cationic lipid nanoparticles in formulation of subunit tetravalent dengue vaccine has induced significant immune response in non human primates. Dengue virus enveloped protein (DEN-80E) was formulated with nanoparticles in all four serotypes. Nanoparticles were able to boost CD4+ and CD8+ T cell response (Swaminathan et al. 2016). But subunit vaccines have problem of low reactogenicity and immunogenicity.

2.2.5 Ideal dengue vaccine

An ideal dengue vaccine must provide balanced reactogenicity and immunogenicity (Edelman et al.2007). To produce complete protection against all four serotypes neutralizing antibodies have to be produced. To design a vaccine that is protective against all four serotypes molecular mechanism of pathogenesis must be considered. An ideal vaccine should be safe, cheap and readily available (Webster et al.2009). .It should induce high neutralizing antibodies against all four serotypes (Lai et al.1998). Vaccine should induce immunity within short dosage. As past infection by particular serotype induce immunity against homologous serotype while it also enhance disease severity against heterologous serotype therefore a vaccine should induce protective immunity as well as it must obtain antibodies against DSS or DHF. High virus titres may produce infection by biting mosquito and allow further replication of virus hence vaccine should have good safety profile. Vaccine should be effective for all age groups. It should give lifelong immunity (Guy, Bruno et al.2008).

2.2.6 Current progress in dengue vaccines

Currently no licensed vaccine is available for dengue in spite of this some dengue vaccine candidates are under development. These include live attenuated virus (LAV) vaccines, live chimeric virus vaccines, inactivated virus vaccines, live recombinant, DNA and subunit vaccines. The live viral vaccines have advanced to clinical trials but they have shown unequal immunogenicity of the four DENV serotypes and viral interference among them in tetravalent formulations. The non-viral vaccines also have been developed for safety issues which consist of subunit vaccines consist of E protein or its derivatives. To keep balanced neutralizing activity is major challenge for four serotypes. NS1 is another subunit vaccine having advantage that it is not a virion associated protein and hence it has no ADE effect (Lim et al. 2013).

1. Live attenuated virus (LAV) vaccine

It consists of weakened viruses which can induce adaptive immune response to both nonstructural and structural proteins though replication should be restricted in such a way to avoid pathological effects. 17D strain of YFV was used for live attenuated vaccine as it is a member of Flavivirus family. But search for equally successful attenuated vaccine has proven much more difficult. In preclinical study, the live attenuated viruses derived from serially passaged DENV in PDK cells were inoculated in rhesus monkey. Mahidol University at Bangkok, Thailand and Walter Reed Army Institute of Research (WRAIR) group in the USA independently developed attenuated vaccine candidates by passage in tissue culture for each serotype of dengue virus (Ishikawa et al.2014). Mahidol group produced tetravalent vaccine formulations which were used phase I and phase II clinical trials in Thai and Adult children. Some volunteers showed unacceptable reactogenicity while all of them were not seroconverted to all four serotypes. Hence further testing was stopped .unbalanced immunogenicity and reactogenicity were major issues for vaccines produced by Walter Reed Army Institute of Research (Wan et al. 2013).

2. Live chimeric virus vaccine

ChimeriVax dengue tetravalent vaccine (CVD1-4) is so far successful vaccine produced by Sanofi Pasteur which is YFV-17D backbone based expressing E and prM genes for all four serotypes. Pre clinical studies showed that tetravalent vaccine is genetically and phenotypic ally stable than YFV-17D. in phase I CVD appeared to be safe but phase II studies have shown that overall 30% effectiveness and efficacies against DENV serotypes 1,3 and 4. Hence CVD need more testing, modification for maintaining safety level (Guy et al. 2011).

Another vaccine is developed by Takeda .it is based on Dengue2 attenuated backbone which is attenuated by mutations in 5’ UTR regions, NS1, NS5 and by chimerization, prM and E genes of DENV-2 was replaced by DENV-1, DENV-3, and DENV-4.phase I trail in USA and Colombia showed good safety profile. Vaccine induced neutralizing antibody for all four serotypes after 2 doses. Currently vaccine is in phase II clinical trial in Colombia, Singapore, and Thailand (Wan et al. 2013).

NIAID-NIH used another approach on site directed mutagenesis of the viral genome to cause attenuation in the virus. A deletion of 30 nucleotides in 3’ untranslated region (UTR) of DENV-4 was first demonstrated to attenuated DENV-4 named as DEN4Δ30 , further it was used in phase I clinical evaluation. This helped in attenuation for DENV-1 and DENV-4 with retained immunogenicity, but it was less successful for DENV-2 and DENV-3.hence alternative strategy was designed for DENV-2 and DENV-3 using the DEN4Δ30 as a genetic for DENV-2 and DENV-3. These monovalent vaccines have been tested for attenuation and immunogenicity in animal models, humans whereas attenuated tetravalent DENV vaccine formulations are currently in phase II clinical studies (Blaney et al. 2006).

Figure 5. Live attenuated chimeric vaccine based on the YF17D vaccine

(https://www.researchgate.net/publication/261290114/figure/fig4/AS:272618456612883@1442008713403/Figure-2-Sanofi-Pasteur-CYD-chimeric-Yellow-fever-dengue-Dengue-Vaccine-The-CYD-TDV.png)

3. Inactivated virus vaccines

Over live vaccines it has two advantages i.e. no possibility of reversion into virulent strain and the relative ease of including balanced immune responses. Some obstacles in development are still their like lack of immunity to NS proteins and a requirement of adjuvants for increasing immunogenicity (Wan et al. 2013).

4. Live recombinant, DNA and subunit vaccines

Recombinant E proteins expressed from yeast and insect cells have been used to test immunogenicity and protective efficacy were tested in animal models. Adenovirus, alphavirus, vaccinia virus are designed for direct administration into the host which have been engineered to express E proteins.

For inducing immunogenic immune response Domain III of DENV E protein (EDIII) is considered as receptor binding domain. In mouse and non human primates it has been produced neutralizing antibodies against DENV.

To develop a tetravalent subunit vaccine, a consensus EDIII (cEDIII)  protein by aligning amino acid sequences from different isolates of the four DENV serotypes which showed that novel cEDIII induced cross neutralizing antibodies against all four serotypes in mouse model and DENV-2 in non human primate model. Heterologus Lipoprotein (recombinant lipo-EDIII) which modified for protein with EDIII protein was reported to produce enhanced immunogenic response than alum adjuvant formulated EDIII protein. Current studies showed that EDIII specific antibodies contribute to small proportion of neutralization in vitro (Wan et al. 2013).

3.1. Materials

3.1.1 Virus stocks: DENV Serotype 1, Serotype 2, Serotype 3 and serotype4

3.1.2 Cell Lines: Vero cell line (ATCC)

3.1.3 Media and reagents for cell cultures:

• Dulbecco’s Minimum Essential Media – Gibco® by life technologies
• Fetal Bovine Serum (FBS)
• Trypsin -EDTA Solution (0.01% W/V)
• True Blue Peroxidase solution
• 80% Methanol in WFI
• 70% (V/V) Isopropanol in WFI(70% IPA)
• Appropriate disinfectants (Dis-N-Det ,Virosi l,Minncare)

3.1.4 Consumables

• Tissue Culture Flasks(175 cm²)
• 24  well Tissue Culture Plates- corning costar
• Disposable Pipettes (10ml ,25ml)- corning costar
• Disposable sterile syringe with needle- BD Biosciences
• Sterile glass vials

3.1.5 Equipments:

• PPE (Sterile gloves, head cap, shoe cover, laboratory coat etc.)
• Biological Safety Cabinet(BSC)
• Inverted Phase contrast Microscope
• CO2 incubator
• TECAN –Infinite 200 PRO Multimode reader
• Water bath
• Micropipette and Pipette gun
• 0.1µ filtration assembly
• Newbauer’s  chamber

3.2 Methods

3.2.1 Approach

• The aim of present study was to find out following vaccine characteristics: 1. loss in vaccine potency upon long-term storage at suitable temperature based on degradation kinetics of vaccine considering parameters such as temperature and humidity, 2. Degradation profile of vaccine for which vaccine vial monitor (VVM) is applicable, and 3) Vaccine potency after reconstitution.
• Three batches of lyophilized dengue tetravalent vaccine (live, attenuated) consisting of all four serotypes were used to establish stability parameters.
• Batches were exposed to 40 ⁰C ,37 ᵒC,  25 ⁰C , and 15 ⁰C respectively; the samples in duplicates were withdrawn at various time points according to the sample pullout plan and stored at -20 ⁰C
• The samples were titrated for virus content using plaque assay
• Using the data obtained at various time points, the rates of degradation (ROD) for all temperatures were calculated and analyzed by Arrhenius equation.
• Arrhenius equation-

The Arrhenius equation explains the effect of temperature on rate of a reaction. According to Arrhenius, for every 10⁰C increase in temperature, the speed of reaction rises about 2-3 times

K = A e^-Ea/RT (Log K = log A –Ea/2.303 RT)

Log K: Rate of degradation, log A: Intercept on Y-Axis and (Ea/2.303 RT): slope of the line

• If the rates of degradation at various temperature follow the Arrhenius  equation then the data can be used to estimate the ROD at any other temperature
• Following the Arrhenius equation the data 40⁰C, 37 ᵒC, 25⁰C, 15⁰C was used to estimate the ROD and also the half life of vaccine at 5 ⁰C ± 3 ᵒC.
• Data of ROD at 5 ⁰C was available over a period of three months. This data was used to check whether the estimated ROD value matches with the observed value

3.2.2 Subculture of adherent cell lines

Aim and principle

In a given culture conditions, an adherent or anchorage dependent cell lines would actively grow until either the surface area is available or the medium is depleted of nutrients. Upon depletion of free surface or nutrients, the cells cease to multiply; this is generally referred as lag phase. At this point the cell lines should be sub-cultured in order to prevent the culture from dying. To culture the adherent cells, they need to be brought into suspension. The degree of adhesion varies from cell line to cell line but in the majority of cases proteases (e.g. Trypsin) are used to release cells from the culture flask. However, this may not be appropriate for some lines where exposure to proteases is harmful or where the enzymes used remove membrane markers/receptors of interest.  Cells should be mechanically brought into suspension in a small volume of medium using cell scrapers.

Procedure

1. Observe cultures using an inverted microscope to assess the degree of confluency and confirm the absence of bacterial and fungal contaminants
2. Remove the spent medium and wash the cell monolayer with appropriate volume of Trypsin –EDTA solution pre warmed in 37 ⁰C water bath
3. Pipette Trypsin on to the washed monolayer using 1 ml per 25 cm² of the surface area. Rotate the flask to cover the monolayer with Trypsin and decant excess of Trypsin
4. Keep the flask in incubator and keep it for 2-3 minutes.
5. Examine the samples using inverted microscope to ensure detachment of cells. Side of the flask may be gently tapped to release any remaining attached cells
6. Resuspend the cells in a small volume of 10% FBS containing DMEM (DMEM10%) to inactivate the Trypsin and mix the cells
7. Top up the volume of cell suspension to 100 ml( per 175 cm² flask) and perform cell count
8. Transfer the required number of cells to new labeled T-175 flask containing prewarmed medium
9. Incubate the tissue culture flask at 37 ᵒC

1. Infection with DENV

24 well cell culture plates were checked for confluency and absence of contamination. 90-95 % confluent cells were suitable for infection.  DMEM 10% was replaced by DMEM1X (without FBS) for washing purpose. Dengue virus vaccine was diluted (10^-1, 10^-2, 10^-3) in 2% DMEM. Cells were infected according to dilutions. Plates were incubated at 34⁰C for virus adsorption (90 minutes). After 90 minutes cells were overlaid with overlay media composed of methyl cellulose and plates were incubated for 6 days in CO2 incubator at 34⁰C.

2. Immunostaining

After 6 Days of incubation staining was performed. Remove residual overlay medium by washing 3 times with PBS. PBS is used for maintaining tonicity of infected cells. Fix cells using 80% Methanol for 15 minutes at room temperature. Dengue serotype specific primary antibodies were added and kept at 37 ⁰C in CO₂ incubator for 2 hours. After incubation cells were washed 3 times with PBS and blocking was carried out (1% Milk in PBS) for 10 minutes followed by addition of secondary antibody (1% Milk based) for 1 hour. After incubation, cells were washed 3 times with PBS and precipitable TMB substrate (True Blue Peroxidase, KPL) was added. After plaques were visible substrate was removed and plates were dried by tapping and plaques were manually counted in each well..

3.2.3 Aliquot preparation/

Three batches of live, attenuated, lyophilized vaccine were prepared containing DENV-1, DENV-2, DENV-3, DENV-4.SG (Sucrose Glycine) was used as a stabilizer which was added to the aliquots of the clarified harvest. Then these preparations were distributed into cryovials for each preparation. Vaccine was filled in glass vials with flip off seal caps with rubber stoppers

3.2.4 Exposing aliquots at 37 ᵒC, 40 ᵒC, 25 ᵒC, 15 ᵒC, 2-8 ᵒC

After lyophilzation, zero day samples were pulled out and freezed at -20 ᵒC and other aliquots are subjected to different temperatures (i.e. 37 ᵒC, 40 ᵒC, 25 ᵒC, 15 ᵒC, and 2–8 ᵒC). Samples were loaded into stability chambers which are maintained with following condition as per ICH guidelines:  After subjecting the aliquots at different temperatures and at different time points the aliquots were pulled out and freezed after respective time points according to the sample pull out plan.

Table 1. Stability schedule

Temperature	Time points
37 ᵒC	Day 1	Day2	Day3	Day5	Day7	Day14	Day30
40 ᵒC	Day 1	–	Day3	Day5	Day7	Day14	Day30
25 ᵒC	2 weeks	4weeks	6weeks	8 weeks	12 weeks	24 weeks	–
15 ᵒC	1 month	2months	3months	6months	–	–	–
2 -8 ⁰C	1 Month	2 months	3months	6months	9months	12months	18months
2 -8⁰C	24 months	36months	48 months	–	–	–	–

3.2.5 Plaque assay (Titration method)

Principle

The plaque assay helps to find out the number of infectious units in a given virus suspension. Plaque assay is a quantitative method. Plaques are discrete foci of infection originated from single infected virion. A plaque indicates spot of cell lysis or cytopathic effect (CPE).  In plaque assay the virus dilution is added to confluent cell monolayer for virus infiltration. Before incubation an overlay medium, composed of methyl cellulose (2%) is added to cell monolayer. This overlay medium prevent the development of secondary plaques by forcing released virus particles to infect only neighboring cells. The plates are then incubated at temperature until plaques are visible. Finally the plates are fixed stained with vital stain and observed either with the naked eye or with the microscope. The infectivity titre is expressed as the number of plaque forming unit per ml in Log scale (Log₁₀ PFU/mL).

Procedure

24 well cell culture plates (Costar) were plated with 0.15×10⁶ cells/ ml cell suspension per well and incubated in CO₂ incubator at 34 ⁰C for one day. After incubation the cells were washed first with DMEM (1X). Cells were infected with 10 fold dilutions of the virus made in 2% FBS containing DMEM (DMEM2%) with 200µl virus suspension/well and the cells were overlaid with the overlay medium composed of methyl cellulose and plates were incubated at 34⁰C for 6 days. After 6 days infected cells were stained. Then the plaques were counted manually and titre was determined as the average of pfu/ml titres of the single plate

3.2.6 Titration by plaque assay

The stability study samples pulled out and freezed according to the sample pullout plan were titrated by using plaque assay

3.2.7 Estimation of ROD at 37⁰C, 25⁰C, 40⁰C, 15⁰C

The virus titres of stability study samples were subjected to the linear regression analysis and ROD for each sample is determined.

3.2.8 Estimation of ROD at 2-8⁰C by Arrhenius plot

For Arrhenius plot the log of ROD at 37⁰C, 25⁰C, 40⁰C, 15⁰C were plotted against absolute temperature in ⁰k. Based on the equation of straight line obtained the ROD at 2-8⁰C were determined.

3.2.9 Vaccine Vial Monitor (VVM) studies

Principle

During transportation, each vaccine is monitored for cumulative heat exposure over a time and reveals its discard point. This is done by relevant vaccine vial monitor (VVM), a class of labels containing heat sensitive material placed on vaccine vial. Type of VVM is assigned to each vaccine as per degradation kinetics of a particular vaccine. Effect of temperature and time causes inner square of VVM to darken gradually and irreversibly. The VVM is a circle with a square inside it. It can be printed on a product label. The inner square of the VVM is made up heat sensitive material that is light at the starting and light after exposure of heat.  At discard point Inner Square is the same color as the outer square. Hence this study was done to find out product degradation profile of dengue tetravalent vaccine at 37 ⁰C in order to assign relevant VVM.

Procedure

Samples were incubated at 37 ⁰C for multiple time points (2 days, 3 days, 5 days, 7 days, and 14 days). After incubation titrations were carried out.

3.2.10 Container closure integrity of vaccine

To find out leakage this test was performed in which 20 psi pressure was applied with 6 in Hg vaccum. Test was performed to check dissolution of vaccine check, presence of water. Within 60 days there was no change in container closure integrity for all temperatures.

Results

Vaccine potency of live attenuated vaccine is proportional to the virus titers. Therefore, for practical purposes vaccine release specifications for a batch are considered as virus titers that will remain till end of expiry period that would confer protection after immunization. Thus calculation of minimum release specification of a final product (vaccine) must take into account stability losses upon storage at a given storage temperature. Upon storage the vaccine quality must remain within acceptable limits. Calculation of expiry period is normally based on real time/real storage temperature however, during developmental stage, predictions are made on the basis of data post exposure at accelerated temperatures. Thus, facilitating the decision making on choice of stabilizers/excipients/ approximate shelf-life and overages required during formulation of final product.

In the current study, 3 identical batches of 10,000 vials were filled which corresponds to approximately 10% of full scale commercial batch as per WHO recommendations. The container closure system was carefully chosen for its dimensions, color and material of construction (MOC). To support vaccine licensure, stability data is required that would support vaccine efficacy and quality.  Full scale stability program was initiated that comprises of long term stability study (2-8 oC), stress study, accelerated study and intermediate study. The program was designed such as to include shorter time points towards the beginning. For instance, samples for accelerated study was exposed at 15 days interval for 2 months followed by 1 and 3 months interval respectively.

Standardization of plaque assay

Multiple batches of antibodies were tried and suitability of each detection antibody was tested for plaque assay. For each antibody, serial dilutions were done and tested for reactivity during immunodetection of plaques. Following dilutions were found suitable:

1:2000- diluted in 1% BSA (Dengue 1)

1:500- diluted in 50% glycerol (Dengue 2)

1:50- diluted in 50% glycerol (Dengue 3)

1:100- diluted in 50% PBS (Dengue 4)

Estimation of intra-assay variability of plaque assay

Accurate estimation of virus potency is of crucial during stability studies; slight variations affect overall conclusion drawn during analysis. Therefore, to assess level of variability in our plaque assay for each dengue virus serotype, Dengue tetravalent vaccine was chosen as reference standard. Set of 20 tests were done on multiple days in order to find an average virus titers and associated variability with the testing procedure. Reference titers were established prior to start of stability studies (Table 2) Standard deviation was used as measure of assay variability for each serotype. The assay showed low variability with coefficient of variation below 10%. Thus, for each testing time point, reference was put in parallel to the test. Based on this data, assay validity criteria were set and fiducial limit was calculated (confidence interval 95%, dF). Therefore, the assay is considered valid only if the reference sample titers are within fiducial limit, failing which the titration was repeated.

Table2. Intra-Assay variability of plaque assay (n=20)

	Dengue1	Dengue2	Dengue3	Dengue4
Mean Titres  (Log10 PFU/mL)	4.010	4.972	4.797	4.293
Fiducial limit	4.000-4.142	4.929-5.064	4.705-4.908	4.428-4.437
Standard deviation	0.136	0.171	0.156	0.234
% coefficient of variation	3.4	3.4	3.3	5.5

Linear regression analysis of stress studies

Stress study was performed to evaluate impact of environmental factors like temperature and moisture which may affect quality of vaccine. Stress study was performed by exposing 3 batches of lyophilized tetravalent vaccine at 40 ᵒC for predetermined time points. Samples were loaded in stability chambers maintained at 40±2 ᵒC and relative humidity of 75% ±5%. Samples were pulled out at predetermined time points as per stability protocol and virus titration was performed for each batch During formulation, set titer of each vaccine batch was kept at 4.700 log₁₀ PFU/ml; all batches had uniform composition of excipients. Figure 1-4 shows degradation of Dengue1, Dengue2, Dengue3, and Dengue4 respectively at 40 ᵒC within 30 days for all 3 batches of DTV. Titration was performed using plaque assay to calculate potency in Log₁₀ pfuml over a period of 30 days. Data showed that average ROD of per day at 40 ᵒC calculated form 3 batches for dengue 1-4 is 0.064, 0.063, 0.050 and 0.084 log₁₀ PFU/ml/day respectively. Temperature is important factor that directly affect potency of a vaccine. Interestingly, pH and moisture content of the vaccine did not change while virus titers dropped, dengue 1 for example, from 4.010 to 2.000 Log₁₀ PFU/mL within 14 days. The data suggest that temperature had direct effect on live viral vaccine and not an indirect effect because of breach in container closure system attributable to thermal expansion and contraction of container at high temperature.

Figure (1) Degradation of Dengue 1 for 3 batches at 40 ᵒC for 30 days

Figure (2) Degradation of Dengue 2 for 3 batches at 40⁰C for 30 days

Figure (3) Rate of degradation of Dengue 3 for 3 batches at 40 ᵒC

Figure (4) Degradation of Dengue 4 for 3 batches at 40 ᵒC for 30 Days

Table 6 shows Rate of degradation (ROD) for Batch1, Batch2 and Batch3 respectively at 40 °C for 30 days. R²valuewas used as a measure of goodness of fit for the data analysis of linear regression analysis. pH and moisture content of individual vaccine vials were tested at initial and final time points (1 month). Data showed change in moisture content. (Table 3).  pH was constant at initial time point and at final time point. (Batch 1-7.7, Batch 2-7.69, and Batch3-7.65). Sterility was checked for bacterial contamination and all batches were sterile at initial and final time point.

Table 4. Rate of degradation of Dengue 1-4 at 40 ᵒC (n = 3 batches)

		Dengue1	Dengue2	Dengue3	Dengue4
Batch 1	ROD	0.080	0.061	0.053	0.068
	R2	0.689	0.717	0.641	0.880
Batch 2	ROD	0.036	0.058	0.041	0.049
	R2	0.842	0.686	0.500	0.700
Batch 3	ROD	0.075	0.070	0.056	0.135
	R2	0.610	0.822	0.681	0.777

Table 3. Percentage of Residual Moisture at 40⁰C

Time point	Batch 1	Batch 2	Batch 3
Day 0	2.070	2.211	2.464
1 month	2.188	2.216	1.997

 

Linear regression analysis of Accelerated studies

3 batches were loaded on 25 ᵒC ±2 ᵒC stability chambers with 60 ±5 % relative humidity. Samples were withdrawn in two weeks interval for first 2 months as per stability protocol. Virus titration was performed on 2, 4, and 6 weeks by plaque assay as mentioned before with standardized protocol. Figure (5-8) shows degradation profile of 3 batches of Dengue1 to 4 respectively at 25 ᵒC up to 6 weeks with 4 time points.

Figure (5) Degradation of Dengue 1up to 6 weeks for 3 batches

Figure (6) Degradation of Dengue2 up to 6 weeks (n=3)

Figure (7) Degradation of Dengue3 up to 6 weeks for 3 batches

Figure (8) Degradation of dengue 4 up to 6 weeks for 3 batches

Table 8 shows rate of degradation (PFU/ml/day) of 3 batches at 25 °C along with their respective R² values from linear regression analysis. pH and moisture content of individual vaccine vials were tested at initial and final time points (6 weeks). Data showed change in moisture content. (Table 6 ). pH was constant at initial time point and at final time point. (Batch 1-7.7, Batch 2-7.69, and Batch3-7.65) Sterility was checked for bacterial contamination and all batches were sterile at initial and final time point

Table 5. Rate of degradation of Dengue 1-4 at 25 ᵒC (n = 3 batches) up to 6 weeks

		Dengue1	Dengue2	Dengue3	Dengue4
Batch 1	ROD	0.021	0.0171	0.022	0.021
	R2	0.422	0.711	0.758	0.493
Batch 2	ROD	0.019	0.027	0.023	0.033
	R2	0.974	0.825	0.556	0.758
Batch 3	ROD	0.0154	0.017	0.021	0.025
	R2	0.103	0.149	0.362	0.671

Table 6. Percentage of Residual Moisture at 25⁰C

Time point	Batch 1	Batch 2	Batch 3
Day 0	2.070	2.211	2.353
1 month	2.053	2.206	2.053

Linear regression analysis of Intermediate stability studies

Intermediate stability studies are performed when the product is not stable at accelerated temperatures for 6 months. Since DTV is currently in developmental stages and the stability profile is not known, therefore stability at intermediate temperatures were also perused in parallel. Lyophilized DTV vaccines in secondary cardboard containers (packaging containers) were loaded on to stability chamber as for other temperatures mentioned above. Intermediate temperature stability chambers are maintained at 15 ᵒC ±2 ᵒC. Samples were withdrawn as per predefined schedule and titrated by plaque assay immediately after removal. Figure (9-12) shows degradation profile of Dengue1-4 respectively at 15 ⁰C up to 1 month containing two time intervals. Plaque assay results are represented as Log₁₀ pfuml. pH and moisture content of individual vaccine vials were tested at initial and final time points (1 month). Data showed change in moisture content. (Table8). pH was Constant at initial and final time point.

( (Batch 1-7.7, Batch 2-7.69, and Batch3-7.65) Sterility was checked for bacterial contamination and all batches were sterile at initial and final time point

Figure (9) Degradation of Dengue1 up to 1 month for 3 batches

Figure (10) Degradation of Dengue up to 1month for 3 batches

Figure (11) Degradation of Dengue3 up to1month for 3 batches

Figure (12) Degradation of Dengue4 up to 1month for 3 batches

Table 7. Rate of degradation of Dengue 1-4 at 15 ᵒC for 1 month (n = 3 batches)

		Dengue1	Dengue2	Dengue3	Dengue4
Batch 1	ROD	0.037	0.0154	0.0157	0.0251
	R2	1.000	1.000	1.000	1.000
Batch 2	ROD	-*	0.018	0.044	0.103
	R2	-*	1.000	1.000	1.000
Batch 3	ROD	0.0248	0.007	0.169	0.126
	R2	1.000	1.000	1.000	1.000

* Data not obtained because of technical error

Table 8 Percentage of Residual Moisture at 15⁰C

Time point	Batch 1	Batch 2	Batch 3
Day 0	2.070	2.211	2.353
1 month	2.235	2.154	1.963

VVM studies and linear regression analysis

To find out the applicability of appropriate VVM type (temperature monitors present on individual vials, i.e. VVM2, VVM 7, VVM 14 or VVM30). Four lyophilized batches packed in secondary container were loaded onto stability incubator maintained at 37 ᵒC ±1 ᵒC. VVM protocol consisted of sample pull out plan up to 14 days. Figure (13-16) shows Dengue virus seortypes 1-4 degradation profiles at 37 ᵒC. For VVM studies acceptance criteria was kept at drop of not more than -0.7 Log₁₀ PFU/mL titers compared to initial titer. The value was chosen based on data from previous stability studies. Titration was performed using plaque assay to calculate potency in pfuml over a period of 14 days.

Figure (13) Degradation of Dengue1 within 14 days for 3 batches

Figure (14) Degradation of Dengue2 within 14 days for 3 batches

Figure (15) Degradation of Dengue3 within 14 days for 3 batches

Figure (16) Degradation of Dengue4 within 14 days for 3 batches

Table 12 shows Degradation profile of 5 batches at 37°C in terms of Rate of degradation and associated R² values from linear regression analysis.

Table 9. Rate of degradation of Dengue 1-4 at 37 ᵒC (n = 4 batches)

		Dengue1	Dengue2	Dengue3	Dengue4
Batch 1	ROD	0.126	0.128	0.124	0.127
	R2	0.929	0.890	0.953	0.928
Batch 2	ROD	0.115	0.113	0.143	0.115
	R2	0.687	0.964	0.972	0.513
Batch 3	ROD	0.045	0.136	0.098	0.047
	R2	1.000	0.940	0.922	0.515
Batch 4	ROD	0.177	0.103	0.078	0.068
	R2	0.935	0.901	0.728	0.237

 

Prediction of rate of degradation and shelf life at storage temperature (5 ᵒC) by arrhenius plot

The kinetics of degradation of a product is a function of temperature and time. With increase in temperature rate of degradation (ROD) is increases in a given time. The relation between ROD and time has been defined by Arrhenius equation (see equation below).

Arrhenius equation:

Ln (K) = Ln(A) – Ea/2.303 RT

Where:

K-  Rate of reaction (ROD),

A = Arrneius factor (constant)

T- Temperature in degree Kelvin,

R- Gas constant (constant, 8.314 kJ/mol)

Ea- Activation Energy of a reaction

Therefore, ROD is directly proportional to 1/T. Graph plotted (Ln (ROD) vs. 1/T (Kelvin) is used for extrapolating straight line to refrigerated temperature (5 ⁰C) one can able to predict ROD and thus shelf line at 5⁰C.

In the table given below, T represents temperature, ROD represents rate of degradation from linear regression analysis. Estimated Ln (K) was calculated from graph using Arrhenius equation.

Estimation of ROD at 5 ᵒC for Dengue 1

Rate of degradation of Dengue 1 at multiple temperatures were compiled for 3 batches of DTV for arrhenius analysis (Table 10). Arrhenius plot was used for estimation of relative rate of degradation at 5 ᵒC (Figure 17).

Table 10. Estimated ROD for dengue 1 at 5 ᵒC in DTV

T (°C)	T (°K)	1/T  (°K)	Batch 1			Batch 2			Batch 3
			ROD (K)	Ln(K)	ELn(K)	ROD (K)	Ln(K)	ELn(K)	ROD (K)	Ln(K)	ELn(K)
40	313	0.0032	0.08	-2.526	-2.384	0.036	-3.324	-3.647	0.075	-2.5902	-2.684
37	310	0.0032	0.126	-2.071	-2.545	0.115	-2.163	-3.645	0.045	-3.1011	-2.980
25	298	0.0034	0.021	-3.863	-3.223	0.019	-3.963	-3.637	0.015	-4.1997	-4.220
15	288	0.0035	0.03	-3.507	-3.832			-3.630			-5.333
5	278	0.0036			-4.484			-3.622			-6.526

ELn(k) = estimated Ln (k)

Figure 17. Arrhenius plot of Dengue 1 in DTV vaccine (n= 3 batches)

Estimation of ROD at 5 ᵒC for Dengue 2

Rate of degradation of Dengue 2 was calculated by arrhenius analysis as for dengue 1 (Table 14, Figure 18).

Table 14. Estimated ROD for dengue 2 at 5 ᵒC in DTV

T (°C)	T (°K)	1/T  (°K)	Batch 1			Batch 2			Batch 3
			ROD (K)	Ln(K)	ELn(K)	ROD (K)	Ln(K)	ELn(K)	ROD (K)	Ln(K)	ELn(K)
40	313	0.0032	0.061	-2.797	-2.422	0.061	-2.8473	-2.0421	0.070	-2.659	-2.208
37	310	0.0032	0.137	-1.988	-2.644	0.137	-2.0326	-2.5203	0.14	-1.966	-2.5361
25	298	0.0034	0.017	-4.075	-3.577	0.017	-3.612	-4.5293	0.018	-4.017	-3.9140
15	288	0.0035	0.015	-4.200	-4.414	0.015	-6.908	-6.3313
5	278	0.0036			-5.312			-8.2631			-6.4747

ELn(k) = estimated Ln (k)

Figure 18. Arrhenius plot of Dengue 2 in DTV vaccine (n= 3 batches)

Estimation of ROD at 5 ᵒC for Dengue 3

Rate of degradation of Dengue 3 was calculated by arrhenius analysis (Table 15, Figure 19).

Table 15. Estimated ROD for dengue 3 at 5 ᵒC in DTV

T (°C)	T (°K)	1/T  (°K)	Batch 1			Batch 2			Batch 3
			ROD (K)	Ln(K)	ELn(K)	ROD (K)	Ln(K)	ELn(K)	ROD (K)	Ln(K)	ELn(K)
40	313	0.0032	0.059	-2.830	-2.453	0.041	-3.1942	-2.4002	0.049	-3.01593	-2.57454
37	310	0.0032	0.124	-2.087	-2.660	0.143	-1.94491	-2.72097	0.1	-2.30259	-2.84226
25	298	0.0034	0.022	-3.817	-3.530	0.023	-3.7723	-4.0688	0.017	-4.07454	-3.96705
15	288	0.0035	0.015	-4.200	-4.310	0.004	-5.5215	-5.2778
5	278	0.0036			-5.146			-6.5737			-6.05748

ELn(k) = estimated Ln (k)

Figure 19. Arrhenius plot of Dengue 3 in DTV vaccine (n= 3 batches)

Estimation of ROD at 5 ᵒC for Dengue 4

Rate of degradation of Dengue 3 was calculated by arrhenius analysis (Table 16, Figure 20).

Table 16. Estimated ROD for dengue 4 at 5 ᵒC in DTV

T (°C)	T (°K)	1/T  (°K)	Batch 1			Batch 2			Batch 3
			ROD (K)	Ln(K)	ELn(K)	ROD (K)	Ln(K)	ELn(K)	ROD (K)	Ln(K)	ELn(K)
40	313	0.0032	0.068	-2.688	-2.429	0.049	-3.016	-2.4658	0.144	-1.938	-1.7571
37	310	0.0032	0.127	-2.064	-2.596	0.115	-2.163	-2.6848	0.154	-1.871	-2.0987
25	298	0.0034	0.022	-3.817	-3.298	0.033	-3.411	-3.6052	0.028	-3.576	-3.5338
15	288	0.0035	0.025	-3.689	-3.927	0.01	-4.605	-4.4307			-4.8211
5	278	0.0036			-4.602			-5.3156			-6.2010

ELn(k) = estimated Ln (k)

Figure 20. Arrhenius plot of Dengue 4 in DTV vaccine (n= 3 batches)

pH and moisture content of DTV was tested during initial and final time points at respective temperatures. No change in pH and moisture content was noticed at 2-8 ᵒC for 2 months (representative figure 21 is shown). Similar results were obtained after exposure of DTV vaccine at 40 ᵒC for up to 1 month. This also suggested that no breach in container closure system occurred and the degradation profiles inherent to the virus.

Figure (21) shows the pH at all stability temperature within 60 Days for all 3 batches. This graph shows that there is no effect of temperature on the pH of vaccine for 60 Days.

 

Conclusion

Plaque assay was standardized as a quantitative method for virus potency estimation in stability studies. Arrhenius plot was used to estimate overages at shelf life of 2 years at 5 ᵒC (using data for 40 ᵒC, 37 ᵒC, 25 ᵒC, 15 ᵒC).

Shelf life estimation for 5ᵒC

For estimation of rate of degradation at 5 ᵒC, natural log of degradation rate, Ln (k), was calculated by extrapolation of slope of Arrhenius equation to 5 ᵒC. To calculate rate of degradation at 5⁰C antilog of Ln (k) was used. Assuming expiry of 2 years (730 days) expiry with acceptable potency of 3.0 log₁₀ PFU/dose.

Following equation was used is used for calculation of overage required during formulation:

Ln (P) = Ln (I) + KT

P = Final remaining potency after given time and temperature

I = Initial potency

K= estimated rate of degradation,

T= Temperature (5 ᵒC),

Therefore, amount of overages required during formulation is calculated as difference between Final potency and initial potency (i.e. Overage = P-I)

Overages required (Log ₁₀ PFU/ml)  = K.T

Table 17. Estimation of overages required during formulation  Batch1 Estimated Rate of degradation Rate of degradation Overages required Mean Dengue11 Batch 1 -4.484 0.0112 8.2 9.60 Batch 2 -3.622 0.0266 19.5 Batch 3 -6.526 0.001 1.06 Dengue2 Batch 1 -5.312 0.0049 3.60 1.63 Batch 2 -8.263 0.0002 0.18 Batch 3 -6.474 0.001 1.12 Dengue3 Batch 1 -5.146 0.0058 4.26 2.33 Batch 2 -6.573 0.0013 1.01 Batch 3 -6.057 0.002 1.70 Dengue4 Batch 1 -4.602 0.0100 7.33 4.13 Batch 2 -5.315 0.0049 3.59 Batch 3 -6.201 0.002 1.47

By considering overages for 2 years at 5 ᵒC for all batches it can be concluded that Batch 3 is more stable than Batch1 and Batch 2. The results of shelf life prediction need to be confirmed with complete data for stability studies as well as with further data of real time studies. Critical parameters like pH, container closure integrity, sterility, moisture content did not show any prominent change at all temperatures and at every time point though loss in titres was seen hence it can be concluded that temperature is the important factor which affect stability of vaccine. For stability studies, ±0.3 was kept as fiducial limit considering standard deviation.

For VVM , –0.7 was kept as acceptable range in such a way that the loss between the titres at each time point

In case of VVM, considering all results it can be concluded that all three batches are least stable as they can lie within an acceptable range of -0.7 for 2 days at 37 ᵒC and 225 days at 5 ᵒC.