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EVALUATION OF THE IMMUNOGENICITY OF EACH OF L-AMINO OXIDASE- AND L-ASCORBIC ACID-
INACTIVATED HEPATITIS A VIRUS IN MICE AS POTENTIAL VACCINE CANDIDATES
Amal Osman Abdullatif1, Mahmoud Mohamed Tawfick*2,3, Abeer Khairy Abdulall1, Aly Fahmy Mohamed4
Address(es):
1Microbiology and Immunology Department, Faculty of Pharmacy (Girls), Al-Azhar University, Nasr City, Cairo, Egypt.
2Microbiology and Immunology Department, Faculty of Pharmacy (Men), Al-Azhar University, Nasr City, Cairo, Egypt.
3Microbiology and Immunology Department, Faculty of Pharmacy, October University for Modern Sciences and Arts, 6th October City, Giza, Egypt.
4The Holding Company for Biological Products, Vaccines and Drugs (VACSERA), Dokky, Giza, Egypt.
*Corresponding author: mahmoud_tawfick@azhar.edu.eg
ABSTRACT
Keywords: Hepatitis A, vaccine, virus inactivation, L-amino oxidase, L-ascorbic acid, alum
INTRODUCTION
Hepatitis A virus (HAV) is the most common cause of infectious viral hepatitis
which is acquired via the faecal-oral route. Hepatitis A is a common infection
worldwide that is associated with unsafe drinking water, inadequate sanitation
and poor personal hygiene (Wu and Guo, 2013). It is estimated that
approximately 1.5 million clinical cases of HAV infection occur worldwide per
annum. In addition, HAV infection is very common in underdeveloped countries
such as Africa, parts of South America, the Middle East and India. In high
endemic areas, hepatitis A occurs early in childhood; however, the development
in sanitary conditions has resulted in a shift of the age groups affected by
hepatitis A with increasing incidence in older age groups (Sartori et al., 2012).
In Egypt, a study that has been performed in 2008 revealed that the frequency of
HAV infection in children from low social class was high (81 %), while the
prevalence rate was low (27.3 %) in those of higher classes (Franco et al., 2012).
The disease is self-limited however HAV infection may result in acute liver
failure and death, while risk increases with age and the presence of chronic liver
disease (Wu and Guo, 2013). Prevention of HAV infection could be achieved by
avoidance of exposure to contaminated food and water, proper disposal of excreta
as well as administration of vaccine to those risk groups, such as health care
workers (Zuckerman et al., 2009). Indeed, universal immunization would
successfully control hepatitis A, although high costs and limited availability of
vaccines preclude such a recommendation (Tahaei et al., 2012).
Vaccines against viral infections consist mostly of live attenuated or inactivated
viruses (Stauffer et al., 2006). In 1992, two HAV formalin-inactivated
vaccines, namely Havrix (GSK) and VAQTA (Merck), were available in United
States and some other developed countries. These vaccines were developed by
growing the virus in human diploid cell lines and then inactivated by treatment
with formaldehyde (Karayiannis et al., 2004). Other formalin-inactivated HAV
vaccines include Avaxim (Sanofi Pasteur) and Epaxal (Crucell Switzerland)
(Glück et al., 1992; Vidor et al., 1996). These vaccines are administered
intramuscularly as a two-dose regimen, given at zero and six to 12 months.
They generally well tolerated, with occasional reports of mild local reactions or,
more rarely, fever and malaise (Karayiannis et al., 2004; Tahaei et al., 2012).
Immunity induced by these vaccines is achieved in approximately 100 % of
immunocompetent patients one month after receiving the recommended two
doses (Fiore et al., 2006).
Several inactivating agents have been described to successfully inactivate
viruses for vaccine purposes. Still, formaldehyde is the most widely used
inactivating agent in vaccine industry for decades (Madhusudana et al. 2004).
However, formaldehyde inactivation efficacy varies between vaccines
concerning formalin concentration, time of inactivation and temperature.
Generally, the higher the formalin concentration and temperature the faster is
the inactivation, although this may adversely affect the antigenicity owing to
thermal degradation and destruction of important epitopes (Sanders et al.,
2015). As the integrity of the immunological epitopes of inactivated vaccines is
virus-inactivant related. Therefore, it is a matter of interest to evaluate cheap
and easily available alternative chemicals for fast and efficient inactivation of
viruses without affecting its antigenicity (Madhusudana et al., 2004). Some
studies have revealed that L-ascorbic acid (LAA) or vitamin C can be used as
an inactivating agent for both DNA and RNA viruses while retained good
antigenicity (White et al., 1986; Rawal et al., 1995; Madhusudana et al.,
2004; Abd El-Razek et al., 2011). In addition, several studies reported that L-
amino acid oxidas (LAO) present in animal secretary fluids, scorpion and snake
venoms have strong antimicrobial activities against various pathogenic bacteria
and viruses (Meenakshisundaram et al., 2009; Alyan et al., 2014; Kasai et
al., 2015). Izidoro (2014) reported the possible inhibition of HIV-1 replication
by LAO isolated from Trimeresurus stejnegeri venom. Based on the available
information on virus inactivation efficacy of both LAO and LAA, the objective
Hepatitis A virus (HAV) is one of the most common causes of acute viral hepatitis worldwide. Formaldehyde is the currently used
inactivating agent in HAV vaccine processing despite of its adverse effects. The current study aimed to evaluate both L-amino acid
oxidase (LAO) and L-ascorbic acid (LAA) as alternative inactivants for HAV and the immunogenicity of inactivated HAV in mice.
Vero cell line was used for cultivation of HAV. The cytotoxicity of LAO and LAA on Vero cells was evaluated using 3-(4,5-
dimethylthiazol-2-yl) 2,5 diphenyl tetrazolium bromide (MTT) assay. The immunogenicity of each LAO- and LAA-inactivated HAV
was examined in parallel with reference HAV vaccine in mice. Humoral (total IgG) and cellular immune responses (IFN-γ and IL-5)
were evaluated in mice sera using ELISA. Both LAO and LAA could efficiently inactivate HAV within 30 and 36 hrs post treatment,
respectively, at concentrations of 0.4 µgm/ml of LAO and 1.5 mg/ml of LAA. Inactivated vaccines were immunogenic to mice on both
the humoral and cellular levels. LAO prepared vaccines showed a more promising immune reactivity than LAA prepared ones and
alum-adsorbed vaccines were more immunogenic than non-adjuvanted ones. In conclusion, data recorded suggest that both LAO and
LAA can be used as inactivating agents for HAV grown in cell culture. LAA- and LAO-inactivated HAV can be potential vaccines as
they provide effective humoral and cellular immune responses comparable to that of the reference vaccine. The stability of test vaccines
is recommended to be traced at different thermal conditions, in addition to different stabilizers and different pharmaceutical
formulations must be tested trying to produce a lyophilized formula for long-term stability.
ARTICLE INFO
Received 18. 8. 2016
Revised 7. 10. 2016
Accepted 20. 10. 2016
Published 1. 12. 2016
Regular article
doi: 10.15414/jmbfs.2016/17.6.3.937-942
J Microbiol Biotech Food Sci / Abullatif et al. 2016/17 : 6 (3) 937-942
938
of our study was to evaluate them as alternative inactivants to HAV and related
immunogenic efficacy of both LAO- and LAA-inactivated HAV in mice.
MATERIAL AND METHODS
Hepatitis A virus strain and cell line
HAV strain HM175 was kindly supplied from water pollution
department, National Research Centre, Cairo, Egypt. African green
monkey kidney cell line [Vero cells, clone CCL-81] was kindly provided by
Research and Development Sector, the Egyptian Holding Company for
Biological Product and Vaccines (VACSERA, Giza, Egypt).
Maintenance of Vero cell line
The 199 Eagle [E-199] medium (GIBCO, USA) supplemented with 200 mM L-
glutamine, 10 % foetal calf serum (FCS) (Sigma Aldrich, USA), 100 IU/ml of
penicillin and 100 mg/ml of streptomycin (Invitrogen, USA) was used to
maintain Vero cell line in tissue culture flasks (TPP, Switzerland) according to
Doyle and Bryan (1998) and Mather and Roberts (1998), where the growth
medium of the mother bottle was decanted. It was incubated at 37°C in 5 % CO2
incubator (Jouan, France) until monolayer was developed. The monolayer was
washed gently using sterile phosphate buffered saline [PBS] of pH 7.2. PBS was
decanted and the cell monolayer was washed with 10 ml pre-warmed 0.25 %
(W/V) trypsin-EDTA solution [Invetrogen, USA] and left in contact with cells
for 15 - 30 seconds. Trypsin was decanted, the TC flasks were incubated with the
trace trypsin at 37°C until the cells detach from the surface. Cells were dispensed
in number of flasks to maintain cell count of ~ 2 × 105/ml. TC flasks were kept
till monolayers were developed. The actual cell number in the suspension was
calculated by counting the cells using the haemocytometer and trypan blue dye
(Sigma Aldrich, USA) exclusion method.
Virus seed stock preparation.
Maintained Vero cells were inoculated with the HAV. The growth medium was
carefully decanted and HAV as 0.1 MOI (multiplicity of infection) was
inoculated onto Vero cells. HAV infected flasks were shaken for 15 minutes
intervals to assure well virus distribution. Maintenance medium (100 ml) was
then added to each infected flask. Infected flasks were incubated at 37°C and
examined microscopically every day till the development of cytopathic effect
(CPE). Flasks developed CPE were subjected to freezing and thawing three
times to extract both cell free and cell associated virus from cells (EL-
Karamany, 1987).
HAV seed stock infectivity titration
HAV harvest was titrated on Vero cells, where HAV seed was 10-fold serially
diluted using sterile E-199 medium. Dilutions of HAV were dispensed to Vero
cells pre-cultured 96-well plates as 0.1 ml/well. Plates were incubated at 37°C for
7 days with daily microscopic examination using inverted microscope (Hund,
Germany). Infectivity titre was determined according to Reed and Muench
(1938) equation:
PD Index = (A-50 %) / (A-B) × Log dilution (10)
Where A is the percentage of CPE at dilution immediately above 50 % and B is
the percentage of CPE at dilution immediately below 50 %. After that index was
applied to the dilution that produced the percentage of cytopathic effects
immediately above 50 %.
Chemical inactivants
L-amino oxidase (LAO) and L-ascorbic acid (LAA), used for HAV inactivation
in this study, were purchased from Sigma Aldrich, USA. They were prepared at a
concentration of 1 mg/ml and processed for evaluation of their safe
concentrations. LAA solution contained CuSO4 at a final concentration of 5
µg/ml.
MTT assay
Cytotoxic effects of both LAO and LAA was determined on Vero cells using 3-
(4,5-dimethylthiazol-2-yl) 2,5 diphenyl tetrazolium bromide (MTT), where Vero
cells were dispensed in 96 well plates. Plates were incubated till confluency. Test
inactivants were 2-fold serially diluted and 24 hrs post incubation at 37°C, dead
cells were washed out using PBS. Remaining viable cells were stained with MTT
stain as 50 µl (5 mg/ml)/well. Plates were incubated at 37°C for 3-4 hrs.
Developed crystals were dissolved using 0.4 % acidified iso-propanol or
Dimethyl sulphoxid (DMSO). Developed colour was read at 570 nm wave length
using Biotek- ELx-800 ELISA microtiter plate reader. The viable cell number
was calculated according the equation: Viable % = OD Test × 100 / OD of cont.
The safe concentration of test inactivant was determined.
Determination of inactivation kinetics of HAV
Inactivation kinetic relative to time post treatment with LAO and LAA was
determined according to Madhusudana (2004) and El-Karamany (1987) where
1 ml of chemically treated virus was collected at time interval of one hour. Virus
samples were 10-fold serially diluted 101 - 108 in 199-E medium. Prepared
dilutions were dispensed onto 24 hrs pre-cultured Vero cells in 96 well plates
(TPP-Swiss). Infected plates were kept at 37°C in 5 % CO2 (Jouan–France) with
daily microscopic observation using inverted microscope for detection of the
CPE. The 50 % end point induced CPE was determined according to Reed and
Muench (1938).
Acute toxicity of test inactivants
Acute toxicity of each test inactivants was performed according to Abd El-
Razek et al. (2011). Intraperitoneal acute toxicity was studied in Swiss Webster
male mice. The animals had free access to feed and drinking water. Mice were
allocated into groups (10/cage). Test chemicals safe concentrations were
administered intraperitonealy. General symptoms of toxicity and mortality were
observed for 24 hrs, after which the animals were left for further 7 days for
delayed toxicity.
Aluminum phosphate (Alum) adjuvant
The solutions of each of 0.63 M AlCl3.6H2O and 0.3 M Na3PO4.12H2O were
prepared in 40 ml normal saline. Prepared solutions were 0.2 μm filtered.
Contents were stirred continuously during the procedure at 40 to 60 rpm. After
wards, 0.3 M Na3PO4.12H2O solution was added to the mixing bottle, and then
300 ml normal saline was added. The antigen was also added followed by
addition of 0.63 M AlCl3.6H2O solution to the mixing bottle. The pH was
maintained between 6.5 – 6.8. The final volume was adjusted with sterile normal
saline and the suspension was mixed for 2 hrs at 37°C (Gupta, 1998; Lindblad,
2004).
Mice immunization
Six to eight weeks old Swiss Webster male mice, housed at the animal facility of
VACSERA, Giza, Egypt, were used in this study according to the
recommendations of Animal Care and Use Committee (ACUC). Alum was used
as adjuvant at concentration of 0.35 mg/ml to enhance the immune response to
the injected inactivated virus. In this study, five groups of mice, 10 each, were
immunized subcutaneously. Four groups were immunised individually with
LAO- and LAA-inactivated HAV, alum adsorbed and none-adsorbed, while the
fifth group was immunised with the currently marketed HAV vaccine Havrix.
Havrix is an alum-adsorbed commercial vaccine (Glaxo SmithKline) containing
1440 ELISA units [EU] of formalin-inactivated HAV as a positive control.
Immunized mice were bled through retro orbital plexus. Immune sera prepared
from blood samples collected from each group at a two-week interval post-
immunization. Antibody level [IgG] and cytokines [IFN-γ and IL-5] produced
post-immunization were monitored.
Detection of HAV-specific antibodies in mice sera
Antibodies against HAV vaccine were detected in post-immunization sera
samples using enzyme-linked immunosorbent assay (ELISA) according to Abd
El-Razek et al. (2011), where sera samples were diluted as 1/100 in dilution
buffer (PBS + 1 % BSA) and added to HAV antigen pre-coated 96-well maxisorb
ELISA plates. Sera samples were serially diluted and plates were incubated for
an hour at 37°C. Plates were washed three times with 300 µl of 1× wash buffer
(PBS + 0.05 % Tween 20) using automated ELISA plate washer for better
washing performance. Then, 100 µl of anti-mouse IgG-HRP conjugate were
added to all wells leaving one empty for the substrate blank. Plates were mixed
gently for 5-10 seconds and then incubated for 60 minutes at 37oC. Plates were
washed as previous. Tri Methyl Benzdeine (TMB) substrate buffer (Sigma
Aldrich, USA) was added as 100 µl/well and plates were kept in dark for 20
minutes at room temp. The reaction was stopped using 100 µl of 2N H2SO4.
Optical density of developed colour was measured at 450 nm using BioTek-XL-
800 –USA) ELISA reader within 60 min.
Cytokines determination
The amounts of IFN-γ and IL-5 in mice immune sera were assayed using specific
sandwich ELISA kit for each cytokine (eBiosciences, USA) according to the
manufacturers’ instructions.
Statistical analysis
Results are expressed as mean values for three three independent experiments.
Comparison between the different groups was made using unpaired student 𝑡-
J Microbiol Biotech Food Sci / Abullatif et al. 2016/17 : 6 (3) 937-942
939
tests to assess significance using GraphPad Prism 5 software. Differences at P
values less than 0.05 were considered significant.
RESULTS AND DISCUSSION
Treatment with LAA or LAO could successfuly inactivate HAV
Testing the cytotoxic effects of test inactivants LAO and LAA showed that the
safe concentrations were 0.4 µg/ml and 1.5 mg/ml, respectively. These
concentrations were used to inactivate HAV, which showed no toxicity to mice
as all inoculated tmice were alive throughout the designed 7-day period of test.
Data recorded revealed that HAV was completely inactivated within 30 hrs and
36 hrs post-treatment with LAO and LAA, respectively, and no residual infective
virus was detected (Figure 1).
Figure 1 Evaluation of inactivation kinetics of HAV post-treatment with each
LAO and LAA relative to time using cell culture assay.
Each of LAA- and LAO-inactivated HAV induced humoral and cellular
immune responses in mice
Regarding the humoral immune response, total HAV-specific IgG response was
monitored. Data recorded revealed that HAV-specific IgG antibodies were
detectable in the mice immune sera as early as two weeks and the peak was
detected at the 6th week and 8th week post-immunization with test LAO-
inactivated vaccines either alum adjuvanted or non-adjuvanted and LAA-
inactivated vaccines either alum adjuvanted or non-adjuvanted, respectively.
There was a significant difference (P < 0.05) in the level of the antibody
production, which was detected 14 weeks post-immunisation with LAO prepared
vaccine than those detected post-immunization with LAA prepared vaccines.
Alum adjuvanted vaccines showed a significant elevated antibody level (P <
0.05) than non-adjuvanted ones throughout the immunization course. Based on
the antigen content, the purchased reference vaccine showed the highest level of
antibody response (Figures 2, 3).
Figure 2 IgG immune response of mice immunised with alum adjuvanted and
non-adjuvanted LAA- and LAO-inactivated HAV vaccines, relative to time post-
immunisation.
Figure 3 IgG immune response in mice 14 weeks post-immunization with HAV
vaccines. Data presented as mean + SE of three replicates.
Concerning the cellular immune response, IFN-γ and IL-5 cytokines were
estimated in immunised mice sera. The production of these cytokines could be
detected as early as 3 days post-immunisation with HAV-inactivated vaccines.
The level of IFN-γ was significantly higher (P < 0.05) in case of both alum
adjuvanted LAA- and LAO-inactivated HAV vaccines than non-adjuvanted ones
until 28 days pos-immunisation. The alum adjuvanted LAO- and LAA-
inactivated vaccines showed high levels than that produced by purchased positive
control vaccine (P < 0.05) at 14 days and 21 days post-immunisation. At 28 days
post-immunisation, there was no significant difference (P < 0.05) between the
level produced by LAO-adjuvanted vaccine and that of the purchased vaccine (P
> 0.05), while that of LAA-adjuvanted vaccine was slightly higher at P < 0.05
indicated by one asterisk. LAO-inactivated vaccines produced significantly
higher levels of IFN-γ (P < 0.05) than that produced by LAA-inactivated ones at
14, 21 and 28 days post-immunisation denoted by four asterisks, except at 28
days, there was no significant difference (P < 0.05) between adjuvanted ones
(Figures 4, 6).
Figure 4 Estimation of IFN-γ concentration in mice sera post-immunisation with
LAO- and LAA-inactivated HAV vaccines using specific sandwich ELISA.
IL-5 level was significantly higher (P < 0.05) in both LAO- and LAA-inactivated
vaccines adjuvanted with alum than those non-adjuvanted ones at 14, 21 and 28
days post-immunisation. LAO-inactivated vaccines produced significantly higher
levels of IL-5 (P < 0.05) than that produced by LAA-inactivated ones. The IL-5
level produced by purchased vaccine was significantly higher (P < 0.05) than that
produced by test vaccines at 14, 21 and 28 days post-immunization except LAO-
adjuvanted one which was significantly higher (P < 0.05) than control vaccine at
21 days (Figures 5, 6).
L A O
LA O - a lu m a dj uv a n te d
LA A
LA A -a l u m a dj uv a n te d
R e fe r en c e v a ccin e
0
1
2
3
4
F ou rt ee n w ee ks p o st -im m u n is atio n
H AV v ac cin e c an did ate
O .D . m ea s ur ed a t 4 5 0n m
*P= 0.0 3 3 3
****P < 0 .0001
***P = 0 .0001
**P= 0.0 0 6 5
**P = 0 .0078
****P < 0 .0001
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940
Figure 5 Estimation of IL-5 concentration in mice sera post-immunization with
LAO- and LAA-inactivated HAV vaccines using specific sandwich ELISA.
Figure 6 IFN-γ and IL-5 concentrations in mice sera at 28 days post-
immunization with LAO- and LAA-inactivated vaccines.
In the past two decades, the HAV cases have declined in several parts of the
world due to partly the developed vaccines (Kanyenda et al., 2015). The
commercially available HAV vaccines contain formalin-inactivated HAV, grown
on human cell lines and purified, that has been adsorbed with alum to enhance its
immunogenicity. While they are considered efficacious, often multiple boosters
are required to confer protection against HAV. These vaccines are also very
expensive to produce, available in limited quantities, and must be kept cold until
they are administered by injection (Mitchell and Galun, 2003). Still in
developing countries, such as Egypt, HAV vaccination may be considered on the
basis of epidemiological and cost-effectiveness concerns. Thus, the available
HAV vaccines are not yet commonly used (Franco et al., 2012). Viral
inactivation is an important procedure in vaccine development against viral
infections (Stauffer et al., 2007). However, the inactivating agent may have an
effect on the viral epitopes pattern to which the antigenicity and/or
immunogenicity of vaccine is related (Blackburn and Besselaar, 1991). Thus,
the inactivation procedures may drastically impair induction of neutralizing IgG
antibodies responses for most viruses (Bachman et al., 1994). Formaldehyde is
the inactivating agent used for HAV vaccines (Chowdhury et al., 2015).
However, it was observed that for formalin inactivation concentration, pH,
temperature and medium composition are extremely critical factors. Formalin
was found to require at least 24 – 96 hrs at 4°C –37°C for inactivation. In
addition, the higher concentrations of formaldehyde and temperature used to
speed up the inactivation may harmfully affect the immunogenicity of inactivated
virus (Chowdhury et al., 2015; Sanders et al., 2015). Seriously, an incomplete
formaldehyde inactivation procedure may be fatal for public health. For example,
incomplete inactivation of the Venezuelan equine encephalitis vaccines, prepared
by formalin inactivation, was the cause of the outbreak of the disease during the
1969 – 1972 pandemic in Central America (Brown, 1992). Furthermore,
formaldehyde which is an alkylating agent inactivates viruses via chemical
reaction with viral capsid proteins and nucleic acid (Budowsky et al., 1991), and
it has been classified by International Agency for Research in Cancer (IARC)
under group 2A. However, there is no epidemiological data referring to the
carcinogenic risk of the alkylating agents on humans (Chowdhury et al., 2015).
Owing to previous data, there is a need for available, cheap and efficient
alternatives for fast inactivation of HAV without affecting its immunogenicity
allowing HAV vaccines to be less expensive to be manufactured and more
amenable to mass vaccination programs. Thus, the main objective of the present
study was to evaluate the efficiency of LAO and LAA as inactivants of HAV and
examine the immunogenicity of each LAA- and LAO-inactivated HAV
compared with the currently available formaldehyde prepared vaccine.
In the present study, both test inactivants could successfully inactivate HAV
using safe concentrations (0.4 µgm/ml of LAO and 1.5 mg/ml of LAA), within
30 hours and 36 hours post-treatment, respectively. LAO has previously showed
remarkable virucidal activities to viruses such as varicella-zoster virus and herpes
simplex virus type 2 (Zedan et al., 2003; Alyan et al., 2014). In addition, data
recorded of HAV inactivation by LAO in this study is consistent with results of
Aly Mohamed and his colleagues (unpublished data), despite the use of another
virus model namely Rift Valley fever virus that was completely inactivated with
LAO within 6 hrs, and the immune potential of this vaccine was better than that
of the known β-propiolactone (BPL)-inactivated vaccine. Recently, snake venom
LAO has been recognized as a multifunctional protein with promising biomedical
applications because of its antimicrobial, anti-HIV, anticoagulant, and inducing
of platelet aggregation. LAO enzyme is acting specifically on L-amino acids and
generally on hydrophobic amino acids (Du et al., 2002). The inactivation of
HAV by LAO is possibly due to the oxidizing stress of LAO attributed to the
ability of the enzyme to localize H2O2 to the target cells through channels in its
structure that would direct the H2O2 product to the exterior surface of the protein,
near the glycan moiety. Thus, the glycan moiety is thought to be involved with
LAO-target cell interaction. Accordingly, the virucidal activity of LAO was the
base on which it was used as virucidal agent to prepare an improved HAV
vaccine (Shebl et al., 2012).
Regarding the cytotoxicity of LAO, our findings were found to be in agreement
with Shebl et al. (2012) study which revealed that LAO showed significant
cytotoxicty to Vero normal cells at concentrations higher than 0.4 µgm/ml. The
LAO cytotoxicity may be attributed to the released H2O2 enhancing the oxidative
stress on cell nucleus causing cell death and progressive apoptosis. Concerning
the use of LAA as an inactivant for HAV, our findings are supported by
Madhusudana et al. (2004) that ascorbic acid can be used as an inactivating
agent for viruses grown in cell line although the authors used it for rabies virus.
In addition, earlier experiments used LAA as an inactivating agent for both RNA
and DNA viruses, confirmed its efficiency as inactivating agent for number of
viruses including vaccinia virus (Turner et al., 1964) HIV virus (Rawal et al.,
1995) and in vitro infectivity of herpes viruses and paramyxoviruses (White et
al., 1986). Abd El-Razek et al. (2011) study revealed also the complete effective
inactivation of Rift Valley fever virus using LAA for vaccine development. The
mechanism whereby ascorbic acid inactivates viruses is not fully understood.
Although, it was partly explained by the presence of oxygen which is essential
and ascorbic acid undergoing auto-oxidation results in the formation of OH
groups that could bring about the inactivation of the cell free viruses (Murata et
al., 1986).
The mice immunisation experiments performed in this study revealed that both
inavtivated HAV vaccines induced a good antibody (humoral) immune response
as indicated by the results of the HAV-specific IgG ELISA. Regarding the
immunogenicity of LAO-inactivated vaccine, the results revealed that HAV-
specific antibodies were detected 14 days post-immunization and this was in
agreement with Keeffe et al. (1989), despite their trial was conducted on the
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941
clinical level. Whilst, the immune response peak was on the 8th week post-
immunization with LAO or LAO alum adjuvanted vaccine, similar to the
antibody response produced by HAV reference vaccine, and on the 6th week post-
immunization for LAA or LAA alum adjuvanted vaccine. Subsequently, the level
of antibody production starts to decrease. Natural infection with Hepatitis A virus
leads to life long detectable antibody in most individuals, whereas vaccine
induced antibody levels wane over time. In healthy adults, vaccine induced anti-
HAV has been observed to decrease rapidly from one month after the booster
vaccination until six month later, followed by a rather constant decrease over the
subsequent two years, approximately 14 % per year (Van Damme et al., 1994).
Thus, in most countries, booster-vaccination policy is guided by manufacturers'
recommendations, national authorities, or both. Data showed that after a full
primary vaccination course, protective antibody amounts persist beyond 10 years
in healthy individuals, and underlying immune memory provides protection far
beyond the duration of anti-HAV antibodies (Van Damme et al., 2003).
One of the major cellular effectors is the CD4+ helper T cells that elaborate
cytokines which enhance both antibody and cellular immune responses. For
investigating the cellular immune response in immunised mice and the
predominant phenotype, cell-mediated (Th1) or humoral (Th2) immune
responses elicited by immunization with LAO- or LAA-inactivated HAV, the
level of two cytokines IFN-γ (Th1 cytokine) and IL-5 (Th2 cytokine) were
estimated in the post-immunisation sera. The cytokine-specific sandwich ELISA
results revealed that the mice immune response generated with both LAA- and
LAO-inactivated HAV immunisation was a mixed Th1/Th2 immune response
profile similar to that produced by HAV reference vaccine. However, the
quantitative measurement of the level of each cytokine indicated that the
concentrations of Th1 cytokine IFN-γ (cellular immunity) was higher than that of
Th2 cytokine IL-5 (humoral immunity). These results indicated that the extent of
up-regulation of IFN-γ production was the highest when compared to that of IL-
5, confirming a significant Th1 immune response. Accordingly, there was distinct
humoral and cellular immune responses post-immunisation with both LAA- and
LAO-inactivated HAV. Consistent with our results, Schmidtke et al. (2005)
study results which revealed that both distinct B and T cell responses were
determined within 14 days after immunisation with formalin-inactivated vaccine
(Havrix) in humans. However, the cellular immune response in the current study
might be the predominant one which is fitting for vaccination against viral
infections by HAV. That is because the cellular immune response is very vital for
combating viral infections (Lappin and Campbell, 2000). In addition, clinical
and experimental evidence proved the hypothesis that HAV hepatocellular
damage and the efficient elimination of virus-infected hepatocytes are mediated
by virus-specific, proliferating T lymphocytes derived interferon (Schmidtke et
al., 2005). The data recorded in this study concerning type of immune responses
was also in agreement with Cederna et al. (1999), despite their trial was on the
clinical level, which revealed that HAV antibody and proliferative effective T
cell response were elicited by a formalin-inactivated HAV vaccine in the
immunized subjects. However, antibodies produced against HAV are present
over an extended period in the human sera, indicating the likely importance in
maintaining providing a long-term immunity (Wang et al., 1996). These results
indicated the high and good immunogenicity of each of LAA- and LAO-
inactivated HAV in Swiss Webster mouse strain. Although, there was no
significant difference in the immunogenic potential between the LAO- and LAA-
inactivated HAV; both showed equivalent antigenic potency as measured by
indirect ELISA.
The concentrations of Th1 cytokine IFN-γ in alum adsorbed LAA- and LAO-
inactivated HAV was higher in those non-adsorbed ones. This finding is
consistent with the practically established concept that alum adjuvant is
administered with antigens in experimental murine and rabbit immunization
studies to enhance immunity (Gupta, 1998; Lindblad, 2004). Indeed, all HAV
commercially available formalin-inactivated vaccines are adsorbed onto alum as
an adjuvant (Karayiannis et al., 2004; Tahaei et al., 2012). Previous published
studies concerned the cellular immune response to vaccine prepared using LAO
as a natural product is very rare as the majority of inactivating agents are
chemicals in nature not natural derivatives. However, the use of LAA as an
inactivating agent showed similar immune response, and particularly the cellular
immune response pattern as in case of inactivation of rabies virus reported by
Madhusudana et al. (2004) and Abd El-Razek et al. (2011). Thus, it was hardly
to compare HAV vaccine potentials to other prepared vaccines using another
inactivating agent but using the same technique except in case of preparing Rift
Valley fever virus vaccine inactivated with both BPL and LAO despite the
previous reporting of LAO as a virucidal agent to Rift Valley fever Virus,
Vesicular Stomatitis Virus (VSV), Herpes and Adeno viruses (Abd El-Razek et
al., 2011; Alyan et al., 2016). Accordingly, data recorded regarding the immune
response is in agreement with one of the authors of this study, Aly Mohamed
and his colleagues (unpublished data), that LAO-inactivated Rift Valley fever
vaccine was high immunogenic than the BPL-inactivated one. The use of alum-
adjuvanted vaccines has showed a better immune response than non-adjuvanted
ones.
CONCLUSION
It can be reported that both LAA and LAO are promising and equally efficient for
inactivation of HAV. Each of LAA- and LAO-inactivated HAV appears to
provide potentially effective anti-HAV vaccines owing to the finding that they
induced both good humoral and cellular immune responses. The immune
potentials of both LAA- and LAO-inactivated vaccines are almost equally to that
of formalin-inactivated HAV vaccines currently available in the Egyptian market.
There was no distinguished difference in the values of cellular immune response
parameter of interest. Both HAV-inactivated vaccines were biologically of near
bio-reactivity to immune system. Alum as adjuvant is useful to enhance the
immune potentials of inactivated HAV on the experimental level. However, the
present study is limited by showing the immune response only for days and/or
weeks, thus long-term tracing of immune response on both the cellular and
humoral levels is warranted. The HAV-inactivated vaccines in the current study
were not challenged in experimental animals as human is the only reservoir host
of HAV and the lack of animal model that mimics the human infection. An
extensive investigation of the HAV vaccine-induced immune response should
include the analysis of circulating HAV-specific T lymphocytes. As storage,
handling and the heat stability of vaccines are consequently matters of great
concern. Thus, studying the stability either real time or accelerated one and
tracing the accumulative effect of residual LAO is recommended on the
biochemical pathological level. Both LAO and LAA should be further compared
with other inactivating agents along with testing different inactivation agents
and/or vaccine adjuvants to maximize the immune response and formulate the
best suitable method for HAV vaccine preparation.
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