Rabies is a worldwide fatal disease and represents a severe public health problem
in developing countries. The rabies is considered to be a reemerging zoonoses
in many parts of the world; among particularly in Asia, Africa and Latin America
where the disease is enzootic despite the availability of proven and control
tools. India is highly endemic country for rabies and tops the list regarding
contribution of human deaths in the globe (Sudarshan et
al., 2007). The rabies virus is a single stranded negative sense RNA
virus belonging to the genus Lyssavirus of the family Rhabdoviridae.
The virus causes fatal encephalitis both in humans and animals which still a
major public health problem in developing countries in Asia, Africa and Latin
America (Trabelsi et al., 2005). Ten million
post-exposure treatments are reported among humans (Martinzn,
2000). Production of vero cell based rabies viral vaccine in Roller poly
ethylene terephthalate bottles (Jagannathan et al.,
2009) and higher vero cell density was achieved in the Fibracel disks verses
Cytodex-1 in spinner flask culture for the production rabies virus (Hassanzadeh
et al., 2011) showed highly encouraging results. The production of
vaccines, diagnosis and further research will therefore continue in this field.
Inactivation of this highly neurotrophic virus is essential for the preparation
of vaccines, diagnostic reagent and research purposes. In addition to rendering
the virus noninfectious, the inactivation method should have the least adverse
effect on the vaccine's immunogenicity. Furthermore, the inactivating method
should not introduce a toxic substance into the vaccine, in the form of either
residual reagent or altered vaccine components which may be toxic to the recipient
(Wiktor et al., 1972).
βPL is widely used as an inactivatent; It is very considerable value in
the production of biological for virus inactivation and perhaps for DNA inactivation.
It has several advantages, It does not require a neutralizing agent to stop
its reaction since it is completely hydrolyzed after 3 h at 37°C (Murata
and Kanegawa, 1973; LoGrippo, 1960). It is still
more quickly degraded in the presence of cellular debris and cell culture media,
Its degradation products are not toxic. Vaccine manufacturing also assess the
efficacy of inactivation procedure by various in process control tests such
as tissue culture amplification test and inactivation kinetics. Inactivation
kinetics gives an idea of the predictable time of inactivation and also the
order of inactivation. There are many indeterminate factors which might extend
the longevity of inactivation kinetics like aggregation, adsorption to walls
of the vessel, or presence in aerosol droplets above the surface of the liquid.
One point of difference is the initial reactant concentration (Hiatt,
1964). The effective virus inactivation should achieve without any irreversible
loss of viral infectivity and with minimal loss of antigenic properties (Budowsky
and Zalesskaya, 1991). From the study of Sheng-Fowler
et al. (2009) the levels of clearance of HIV retroviral cell substrate
DNA that can be achieved with either benzonase digestion of DNA or chemical
inactivation by beta propiolactone. Furthermore, the inactivating method should
not introduce a toxic substance into the vaccine, in the form of either residual
reagent or altered vaccine components which may be toxic to the recipient.
Validation of the viral inactivation process is an crucial part of quality
assurance in the immunobiological production industry and time is also a crucial
factor because to get more inactivated viral harvest or yield in a short period
and which it has to subjected to further purification and down stream processing,
this findings make possible to diminish the time frame and to estimate the residual
βPL in various levels in the tissue culture derived anti rabies vaccine.
The inactivation process plays an important role in the viral vaccine preparation.
The βPL and formaldehyde are important inactivators that are commonly used
in biological preparations. Though βPL has more merits when compared with
formaldehyde (Perrin and Morgeaux, 1995). In the rabies
viral vaccine production, βPL has been used for inactivation and during
the inactivation process, for the presence of residual βPL may be decreased
as the time progress. The present study describes validation of the inactivation
strategies, usage of βPL at in different concentrations, inactivation time
MATERIALS AND METHODS
Cells: The Vero cell line was procured from the American type culture
collection (ATCC) at the 113th passage. At 125th passage level Vero cells were
received as monolayer and as frozen cells, from ATCC. Master cell seeds were
prepared at 130th passage level and the working cell banks were established
at 136th passage level. Quality control tests were performed as per requirement
and found complying.
Revival of cells: Stored vials containing Vero cells were taken from
liquid nitrogen and immediately placed in a sterile beaker containing sterile
distilled water (DH2O) 37°C for rapid thawing. Cells were transferred
quickly into two roux bottles containing growth medium (100 mL each with 10%
serum). The cells were incubated at 37°C to form monolayer. To remove toxic
DMSO and dead cells, a medium change was done after 12-15 h.
Media used for cell culture: Eagles
Minimum Essential Medium (MEM) with non-essential amino acids, phenol red and
1.5 g L-1 of sodium bicarbonate, pH 7.0 to 7.5 (Himedia, Bombay)
was used for Vero cell culture. The dry powder was dissolved in water for injection
(WFI) to 1X concentration. For maintenance of Vero cells, the MEM with 2.2 g
L-1 of Sodium bicarbonate instead of 1.5 g L-1 pH with
a range of 7.4 to 7.6 was used. For pH adjustment CO2 was used. Required
quantities of Neomycin and New Born Calf Serum (NBCS) were added at the time
of batch culture.
β-propiolactone: β-propiolactone is a colorless stable liquid
with a slightly sweetish odour in its undiluted state but unstable in aqueous
solution. It is soluble in water and miscible with ethanol, acetone, diethyl
ether and chloroform. β propiolactone (FERAK Berlin GmbH; NMR 98.5%) is
a highly reactive chemical because of the moderate fire hazard when exposed
to heat or flame. The βPL has very high chemical reactivity due to the
presence of a strained four membered lactone ring and it is available in a grade
containing 97% minimum active ingredient (Jones and Feldman,
Virus: Fixed strain of Pasteur virus (PV-11) obtained from Institute
Pasteur France was initially adopted to primary cultures of fetal bovine kidney
cells and later adopted to Vero cells. The mother seed (PV-RAGE-01) was stored
in liquid nitrogen. Master seeds were prepared from the mother seed on Vero
cells by harvesting the viral fluid, stabilized with 10% New Born Calf Serum
(NBCS) and distributed in 5 mL quantity into 50 mL vial and stored at -80°C/Liquid
nitrogen. Working virus seed stocks were prepared from master seed as described
above and tests for sterility, titre and adventitious agent were carried out.
Propagation of vero cells: Growth medium from the bottles showing confluent
monolayer were removed and washed with warm PBS to remove dead cells, serum
component and to enhance the trypsin activity. Small quantities of 0.25% trypsin
in Dulbeccos saline A with sodium bicarbonate was added to each bottle
and incubated at 37°C for few minutes for the detachment of cells. Based
on cell count, cells were dispersed in growth medium and transferred to roux/roller
bottles, incubated at 37°C for 48 h (Jagannathan et
Dynamics of virus replication: Roller bottles were examined under microscope,
washed with PBS, infected with virus seed material having a virus titre of 10-5
(10 mL per roller bottle) and incubated at 35°C about 30 min for adsorption
of the virus. Then about 250 mL of maintenance medium was added. Samples of
the maintenance medium were taken daily for virus titration. For maximum yield
of virus from the cell culture, the exact multiplication of infection (MOI)
relating to the infective virus material and the number of viable cells in the
culture were analyzed and used. 5% of the bottles were kept as control culture
bottles. For the detection of extreaneous viral agents by heamadsorption test,
Titration of Virus infectivity: The virus infectivity in mice (LD50)
was done as per the technique described by Koprowski (1973).
Groups of 10 mice were inoculated intra cerebrally with 10 fold dilution of
the viral harvest, each mouse receiving 0.03 mL. The mice were observed for
14 days and the number that died from rabies was recorded after the first 5
days from inoculation. Any mice showing signs of fixed-virus rabies infection
(paralysis, convulsions) between 5th day and 14th day were also included and
the titer was calculated by the method of Reed and Muench
Viral harvests: The supernatant fluids containing virus material were
harvested into large sterile bottles and the same bottles containing infected
cells used for another 4-5 harvests by replenishing with maintenance medium
at an interval of 72 h. The rabies virus comes out in the fluid by budding out
process and it does not produce any cytopathic effect (CPE) in the infected
Concentration and purification of SVH: Based on titer value, viral harvests
were pooled and sterile filtered using prefilter 0.45 μm membranes and
collected in 100 L stainless steel pressure vessels, as Single Viral Harvest
(SVH) the SVH were ensured for sterility further subjected to concentrated by
the technique namely Tangential Flow ultra Filtration (TFF) using the TFF system
(Millipore JMBM 0137) with A molecular weight cut off 100, 000 polysulfone membrane
(BIOMAX) was used for concentration. The SVH was concentrated to 20X level by
volume. Both retentate and filtrate materials were subjected to mouse titration
test to check the integrity of the system and the run. After the concentration
the material is Diafiltered with required volume of Phosphate Buffer Saline
(PBS) pH 7.2 and ensured the residual BSA. The concentrated, inactivated rabies
viral harvests are purified by chromatographic technique.
Virus inactivation kinetics experiments: Concentrated virus stored at
+4°C was dispensed in to 25 mL flasks. The purity, concentration of the
βPL were rechecked before has been started the inactivation, the concentration
was analyzed by the method of Tyler and Beesing (1952).
The following conditions are followed for all experiments, before addition of
βPL stock solution of 1:10 was prepared, double addition of βPL. First
half of addition the volume of βPL, incubated at +22°C for 2 h followed
by 2nd addition of the remaining volume of βPL incubated at the experimental
temperatures (+37, +22 and +4°C). After addition of all βPL all the
samples are under shaking up to 48 h for enhancing the inactivation.
The βPL were added as a two additions for obtain a final concentration
of 1:1000, 1:2000 and 1:3000. These suspension are incubated at +37, +22 and
+4°C with continuous stirring for 2 h. After 2 h all the samples are kept
in +4°C. Samples were collected from each flask 2,24,48,96 and 168 h after
addition of βPL. Prior to start the experiments the control βPL are
hydrolyzed in the water by the inactivation ratio likes 1:1000, 1:2000 and 1:3000,
the inactivation temperature +37°C, with the duration time of 2 h.
Avirulance test: The test was performed with undiluted inactivated sample.
The 0.03 mL of sample was inoculated intracerebrally into a group of 10 mice
weighing 12-15 g. Those four week old Swiss albino mice were procured from the
Laboratory Animal facility department of Pasteur Institute of India, Coonoor.
The animals were housed and looked after as per the regulations and permission
to conduct the study was obtained from the Institutional animal ethics committee
for experimentation of animals The Mice were observed for 14 days. Any deaths
occurring during the first 4 days were disregarded. Simultaneously those samples
are inoculated into the mice for conformation of the virulence factor.
Experimental vaccine formulation: The inactivated and purified rabies
antigens were used for the formulation of inactivated rabies vaccine. Totally
six formulation of inactivated tissue culture rabies vaccine (VARV A/08, B/08,C/08,D/08,E/08,F/08
and G/08) was taken. Vaccines were formulated using antigens with virus titer
of log 10-5.1 before inactivation. human albumin and maltose were
added as additives to a final concentration of 1 and 5%, respectively in the
single human dose.
Immunization: Two sets of Guinea pigs each containing 10 numbers was
immunized with three doses of each 1.0 mL of each of A/08,B/08,C/08,D/08,E/08,F/08
and G/08 formulations on days 0, 3 and 7 by intramuscular inoculation respectively.
The serum samples from immunized guinea pigs were collected on the 90th day
after the primary inoculation and titrated for the estimation of rabies specific
antibodies by Rapid Fluorescent Focus Inhibition Test (RFFIT) method.
RFFIT: The standard rabies virus was titrated in neuroblastoma cells
for the assessment of immunogenicity. The vaccine immunized guinea pig sera
were diluted in cell culture medium subsequently the serial dilutions of serum
and a fixed amount of rabies virus were mixed in sterile 96 well culture plates
and incubated at 37°C for 1 h. A suspension of neuroblastoma cells (0.05
mL) containing 25,000 cells was added to each well. Positive and negative controls
were incorporated and the plates (as a reference) were incubated at 37°C
for 48 h. The cell culture fluid was then removed, the cells fixed with acetone
and kept at -20°C for 1 h. The monolayers in the plates were stained with
fluorescin isothiocyanate (nutritional Biochemical Corporation Cleveland OH)
conjugate at 37°C for 1 h. The conjugate was removed, the plates gently
washed with distilled water to all the wells and 1 drop of mounting buffer was
added and observed under UV fluorescence microscope (Smith
et al., 1996). The immunogenicity of the formulated materials was
determined by the inhibition of infective particles.
Safety: A double dose of the vaccine was administered to two guinea
pigs from each formulation. They were observed for local as well as systemic
reaction for a period of 14 days.
RESULTS AND DISCUSSION
The vero cells monolayers were inoculated and infected with PV-11 (Pasteur
rabies virus) The virus was harvested from infected cultures from third day
after inoculation as viral harvest, five times at intervals of 72 h. and the
virus titers was 105.2 . Furthermore the pooled SVH are concentrated
by the pellicon ultrafiltration system (Jagannathan et
Inactivation: β propiolactone (FERAK Berlin GmbH; NMR 98.5%) is
highly reactive chemical because of the moderate fire hazard when exposed to
heat or flame. β propiolactone is available in a grade containing 97% minimum
active ingredient (Jones and Feldman, 1981).
The βPL are hydrolyzed in the water at the concentration of 1:2000 are
gradually to compare with the other ratios, after 2 h the residual βPL
are more than 50% in the 1:1000 ratio and under gone minus in the 1:3000 (Table
1). The residual βPL in +4°C in all ratio are similar linear gradient.
When the single addition of the entire calculated volume of βPL gets hydrolyzed
before all the viruses are inactivated. The same volume of βPL as two additions
can complete its process of inactivation within the same period of time.
The 1:2000 ratio was taken for the subsequent experiment, since all the three
various temperatures based inactivation experiments. The level of residual βPL
was not higher then other experiments those were conducted at various temperatures.
The result of 1:2000 was showed linear others are not linear (Table
2). The βPL lot. Nos 18/97 and 20/97 is also showed the repeative results
in all inactivation temperatures and the purity of βPL are varied from
1% only (Table 3).
Rabies virus has been inactivated by various chemical and physical agents for
vaccine production. βPL has been used extensively for the production of
rabies vaccines (Peck et al., 1951).
|| Remaining (%) of βPL in water for injection
|Common factors: -: Inactivation temperature-+37°C,
Inactivation time: 2 h
||The inactivation of concentrated rabies viral harvests in
different dilutions, temperature, time and the remaining % of βPL in
|Common factors:- Inactivation time:- 2 h
||The inactivation of concentrated rabies viral harvests in
1:2000 dilution, temperature, time and the residual βPL in different
interval, it containing the concentration of the βPL and their lot
|Common factors:- Inactivation ratio 1:2000, Agitation:-Yes,
Inactivation time:-48 h
The βPL action on the cell substrate DNA has been extensively studied
there has been very little analysis of its effects on chromosomal DNA and especially
on the residual fraction co-purifying with virus during vaccine preparation
(Morgeaux et al., 1993). The treatment with
βPL of continuous cell lines can reduce the hazards associated with such
containing residual cellular DNA (Perrin and Morgeaux,
Avirulance test: The entire different samples those are inoculated to
intracerabrally and also different interval. Animals are not showed any symptoms;
few test animals are observed upto 42 days, upto those days that animals are
not showed any rabid symptom. These results are represents the titre of the
virus. It states that all active virulence is inactive in 1:2000 ratios by the
process of βPL inactivation.
Immunogenicity: For the analysis of neutralizing antibody the guinea
pigs were immunized with vero cell derived material and the sera were analyzed
by the RFFIT method. The antibody titre in International units of the serum
samples for Vero cell derived material was found above required level. A two
fold dose of the vaccine formulations when injected to susceptible sero negative
guinea pigs did not produce any adverse reactions.
However, inactivation with βPL is considered to be the result of alkylation
of imidazole functional groups in the viral ribonucleic acid The total number
of hours required for inactivation also decreases with an increase in the use
of biological (therapeutic proteins or vaccines) produced from Continuous Cell
Lines (CCLs) has raised problems of safety. In particular, the presence of residual
cell nucleic acid (rcDNA) is associated with hypothetical risk of oncogenicity.
The fundamental reason for viral infectivity inactivation by propiolactone is
carboxyethylation of nucleic bases within the viral genome (Chen
et al., 1981). βPL treatment reduced the heamagglutination titer
to one-half of the control value after 4 h of treatment; however, no further
loss in hemagglutinin titer was detected during the subsequent 20 h of treatment.
Complement fixation by the rabies virus concentrate remained at 100% of the
control value, even after exposure to βPL for 24 h (Wiktor
et al., 1972).
Base carboxyethylation reactions are kinetically single-step and irreversible
under storage conditions for inactivation antiviral vaccines. Therefore the
infectivity of the inactivated virus cannot be restored by genome demodification
during storage of vaccines prepared with the aid of propiolactone, which ensures
the stable safety of the preparation. The kinetics of inactivation of RNA-containing
viruses reflects the kinetics formation of the first inactivating lesion in
the genome. (Budowsky et al., 1991).
Those samples were inactivated by 1:2000, 48 h with agitation are gave the
better result, to compare with the other test samples. The residual βPL
also decreased after 48 hrs that are very less to compare with other samples.
This experiment was done only in the ratio of 1:2000 and the time of the inactivation
also very less to compare with the other different samples. The results also
indicate that the rate of inactivation is directly proportional to the concentration
of βPL used in the process (Aarthi et al.,
2004); the total number of hours required for inactivation also decreases
with an increase in the concentration of βPL. All of the inactivating experiments
are in which the effect is time-dependent. As per the regulatory norms the ratio
of the βPL and viral harvest ought to not above the limit.
The titer of the virus was decreased at the inactivation temperatures like
+22 and 37°C, the hydrolysis of βPL also occurring at 37°C temperature.
Exposure at the 37°C helps in hydrolyzing the βPL which helps in avoiding
the step of addition of thiosulphate for neutralization. And also the βPL
does not require any neutralizing agent to stop its reaction since it is completely
hydrolyzed. It is more quickly degraded in the presence of cellular debris and
cell culture media. The rate of βPL hydrolysis increases in the presence
of proteins. These decreasing viral titers directly reflect the yield that seems
in the final potency of the vaccine. During the experiments the pH of the samples
are not increased drastically but slightly and maintained as the neutral pH,
the inactivation rate decreased linearly with increase in pH, whereas the 99%
viral inactivation time increased proportionately with increase in pH. the rate
of the viral inactivation is pH dependent (faster in acid than in alkaline medium),
having a fractional order with respect to both [H+] and [OH¯];
When BPL was used as the inactivating agent, infectivity was exponentially reduced
100,000-fold during the first 6 hr, whereas complement-fixing activity remained
similar to that of the untreated vaccine (Wiktor et
al., 1972). All the inactivated antigens are formulated with 1% human
albumin and 5% maltose. The proteins present in the liquid rabies vaccine are
not an influential factor for the efficacy of vaccines. Only its immunogenicity
of inactivated, purified rabies viral proteins plays the main role in the efficacy
(Jagannathan et al., 2011). The immunogenicity
of the vaccines is above the prescribed limit. And also the experimented vaccines
are not produced any adverse reaction in guineas pigs it seem the safe of the
In the vaccine manufacturing industry the time is a crucial factor because
to get more inactivated viral harvest are yield in a short period and they are
processed to further purification and downstream processing, this findings are
to reduce the time frame and the residual βPL in various level are influenced
the tissue culture derived anti rabies vaccine. Since inactivated vaccines are
more stable and easier than other attenuated virus vaccines. These results are
reflecting the safety, potency and efficacy of the Vero cell culture derived
rabies vaccine. These results are also encouraging and more studies are needed
for the shortening the time duration during inactivation.
The authors sincere thanks to the Director, Pasteur Institute of India,
Coonoor, for providing facilities to carry out the study and acknowledged.