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Characteristics and viral propagation properties of a new human diploid cell line, Walvax-2, and its suitability as a candidate cell substrate for vaccine production

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Human diploid cell strains (HDCSs), possessing identical chromosome sets known to be free of all known adventitious agents, are of great use in developing human vaccines. However it is extremely difficult to obtain qualified HDCSs that can satisfy the requirements for the mass production of vaccines. We have developed a new HDCS, Walvax-2, which we derived from the lung tissue of a 3-month-old fetus. We established primary, master and working cell banks successfully from reconstituted frozen cells. Observations during the concurrent propagation of Walvax-2 and MRC-5 cells revealed differences in terms of growth rate, cell viability and viral sensitivities. Specifically, Walvax-2 cells replicated more rapidly than MRC-5 cells, with Walvax-2 cells attaining the same degree of confluence in 48 hours as was reached by MRC-5 cells in 72 hours. Moreover, Walvax-2 cells attained 58 passages of cell doublings whereas MRC-5 reached 48 passages during this period. We also assessed the susceptibility of these cells to rabies, hepatitis A, and Varicella viruses. Analysis of virus titers showed the Walvax-2 cells to be equal or superior to MRC-5 cells for cultivating these viruses. Furthermore, in order to characterize the Walvax-2 cell banks, a series of tests including cell identification, chromosomal characterization, tumorigenicity, as well as tests for the presence of microbial agents, exogenous viruses, and retroviruses, were conducted according to standard international protocols. In conclusion, results from this study show that Walvax-2 cell banks are a promising cell substrate and could potentially be used for the manufacturing of HDCVs.
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Characteristics and viral propagation properties of
a new human diploid cell line, walvax-2, and its
suitability as a candidate cell substrate for vaccine
production
Bo Maab, Li-Fang Heb, Yi-Li Zhangb, Min Chenb, Li-Li Wangb, Hong-Wei Yangb, Ting Yanb,
Meng-Xiang Suna & Cong-Yi Zhenga
a College of Life Sciences; WuHan University; Wuhan, Hubei, PR China
b Yunnan Walvax Biotechnology Co. Ltd.; Kunming, Yunnan, PR China
Accepted author version posted online: 24 Mar 2015.
To cite this article: Bo Ma, Li-Fang He, Yi-Li Zhang, Min Chen, Li-Li Wang, Hong-Wei Yang, Ting Yan, Meng-Xiang Sun & Cong-Yi
Zheng (2015) Characteristics and viral propagation properties of a new human diploid cell line, walvax-2, and its suitability as
a candidate cell substrate for vaccine production, Human Vaccines & Immunotherapeutics, 11:4, 998-1009
To link to this article: http://dx.doi.org/10.1080/21645515.2015.1009811
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Characteristics and viral propagation properties
of a new human diploid cell line, walvax-2, and its
suitability as a candidate cell substrate
for vaccine production
Bo Ma
1,2
, Li-Fang He
2
, Yi-Li Zhang
2
, Min Chen
2
, Li-Li Wang
2
, Hong-Wei Yang
2
, Ting Yan
2
,
Meng-Xiang Sun
1
, and Cong-Yi Zheng
1,
*
1
College of Life Sciences; WuHan University; Wuhan, Hubei, PR China;
2
Yunnan Walvax Biotechnology Co. Ltd.; Kunming, Yunnan, PR China
Keywords: biological characteristics, cell substrate, human diploid cell strain (HDCSs), human diploid cell vaccines (HDCVs), viral
sensitivities
Abbreviations: ATCC, American Type Culture Collection; CCID50, 50% cell culture infectious dose; CCTCC, China Center for
Type Culture Collection; CPE, cytopathogenic effect; ELISA, enzyme-linked immuno sorbent Assay; FFU, fluorescent focus units;
G6PD, glucose 6 phosphate dehydrogenase; GM, growth medium; HAV, hepatitis A virus; HDCSs, human diploid cell strains;
HDCV, human diploid cell vaccine; LD, lactate dehydrogenase; MCB, master cell bank; MDCK, Madin–Darby canine kidney;
MOI, multiplicity of infection; NIFDC, National Institute for Food and Drug Control; PAGE, polyacrylamide gelelectrophoresis;
PCB, primary cell bank; PFU, plaque forming units; PPLO; pleuropneumonia-Like organisms; STR, Short tandem repeats; VZV,
varicella zoster virus; WCB, Working cell bank
Human diploid cell strains (HDCSs), possessing identical chromosome sets known to be free of all known
adventitious agents, are of great use in developing human vaccines. However it is extremely difcult to obtain qualied
HDCSs that can satisfy the requirements for the mass production of vaccines. We have developed a new HDCS, Walvax-
2, which we derived from the lung tissue of a 3-month-old fetus. We established primary, master and working cell
banks successfully from reconstituted frozen cells. Observations during the concurrent propagation of Walvax-2 and
MRC-5 cells revealed differences in terms of growth rate, cell viability and viral sensitivities. Specically, Walvax-2 cells
replicated more rapidly than MRC-5 cells, with Walvax-2 cells attaining the same degree of conuence in 48 hours as
was reached by MRC-5 cells in 72 hours. Moreover, Walvax-2 cells attained 58 passages of cell doublings whereas MRC-
5 reached 48 passages during this period. We also assessed the susceptibility of these cells to rabies, hepatitis A, and
Varicella viruses. Analysis of virus titers showed the Walvax-2 cells to be equal or superior to MRC-5 cells for cultivating
these viruses. Furthermore, in order to characterize the Walvax-2 cell banks, a series of tests including cell identication,
chromosomal characterization, tumorigenicity, as well as tests for the presence of microbial agents, exogenous viruses,
and retroviruses, were conducted according to standard international protocols. In conclusion, results from this study
show that Walvax-2 cell banks are a promising cell substrate and could potentially be used for the manufacturing of
HDCVs.
Introduction
The replication of viruses occurs only when the virus enters
into host cells, often resulting in diseases that are difficult to treat.
Currently, there are no widely accepted therapeutics available to
treat such diseases, therefore prophylactic vaccines play an imper-
ative role in the fight against viral diseases. Antibodies produced
for most kinds of viral diseases when the immune system is stim-
ulated by intact viral particles,.
1,2
Owing to this property, the
vast majority of viral vaccines still adopt the traditional cell sub-
strate culture method. Three cell substrates, human diploid cells,
continuous cell lines and primary cell lines, are always used for
developing vaccines.
3
However, continuous and primary cell
lines used for vaccine production suffer from the limitation of
© Bo Ma, Li-Fang He, Yi-Li Zhang, Min Chen, Li-Li Wang, Hong-Wei Yang, Ting Yan, Meng-Xiang Sun, and Cong-Yi Zheng
*Correspondence to: Cong-Yi Zheng; Email: Cctcc202@whu.edu.cn
Submitted: 11/03/2014; Revised: 12/11/2014; Accepted: 12/12/2014
http://dx.doi.org/10.1080/21645515.2015.1009811
This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The
moral rights of the named author(s) have been asserted.
998 Volume 11 Issue 4Human Vaccines & Immunotherapeutics
Human Vaccines & Immunotherapeutics 11:4, 998--1009; April 2015; Published with license by Taylor & Francis Group, LLC
RESEARCH PAPER
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being potentially strongly tumorigenic. Four Additionally the
primary cell lines, which are obtained from animals, introduce
potentially risky exogenous agents.
4
In contrast, human diploid
cell strains (HDCSs), acquired from embryos or other tissue cells
of human origin, possess identical chromosome sets that are free
of all known adventitious agents.
5
These unique properties
explain the value of such materials and the current interest in
their use in the development of human viral vaccines.
6,7,8
Human
diploid cell vaccines (HDCVs) have been licensed all over the
world. Many studies have demonstrated superior immunogenic-
ity and safety of HDCVs relative to those using any other tissue
culture, such as hamster kidney cells or vero cell vaccines.
9
The
WHO recommends HDCS as the safest cell culture substrate for
the production of viral vaccines
10
and consequently they have
become the preferred cell substrate for vaccine production
worldwide.
Hayflick in 1961
8
and Jacobs in 1967
7
developed the 2 most
well known HDCSs, Wistar Institute (WI)-38 and Medical
Research Council (MRC)-5, respectively, that currently serve as
international standardized cell strains. Since then, there has
been continuing interest in the development of HDCSs.
Eleven,
12
However, it is extremely hard to obtain human fetal
tissue from which to derive qualified human diploid cell strains.
This is due to issues that include the requirement for strict ethi-
cal review, the possibility of environmental degradation, and
food safety hazards, all of which may lead to chromosomal aber-
rations such as the presence of aneuploidy and polyploidy for
the karyotype.
13
Most importantly, strict requirements regard-
ing the methods for obtaining suitable tissues from which to
derive HDCS via abortion render the acquisition of appropriate
material difficulty. Even if a new HDCS is derived successfully,
it might not satisfy requirements for industrial production due
to its inability to sustain multiple passages, the IMR-9 cell line
being an example.
14,15
Due to the diminishing supply of WI-
38
10
cells, the MRC-5 line has become the most widely used
cell strain in the production of HDCS-derived human vaccines.
China consequently confronts 2 key challenges for the produc-
tion of viral vaccines from MRC-5 cells (which are mainly
obtained from abroad): concerns about influences of limited
passages, and the policies of the countries from which the cells
are imported. More specifically, the numbers of passages of the
imported MRC-5 cells are generally higher, generally later than
the 20
th
passage, resulting in restricted mass production due to
decreased growth vitality. Additionally, according to the stan-
dard for the Pharmacopoeia of the People’s Republic of China
(2010), Volume III, the use of the HDCSs is limited to genera-
tions within 2-thirds of the primary cell lifespan for the manu-
facture of vaccines. Due to the scarce HDCSs resources, the
research and production of viral HDCVs in China are substan-
tially restricted. For example, human diploid cell rabies vaccine,
which is considered to be the gold standard for rabies vaccines,
is not currently available in China.
16
Furthermore, the produc-
tive cell generations for the OKA-HDC on the Chinese market
from 3 manufactures are MRC-5 cells in the 32
nd
and 33
rd
pas-
sages,
17
which have therefore already reached the limit required
as described above in Chinese Pharmacopeia (the 33
rd
passage is
the last cell doubling that could be used in the production for
the MRC-5). Relying on imported HDCSs, may lead to unsta-
ble supply as well as unpredictable costs. Therefore the inten-
tion of this study is to develop a completely new HDCS of
Chinese origin that could be used in manufacturing viral
vaccines.
This study, therefore, aims to (i) establish and characterize
Walvax-2, a totally new HDCS; and (ii) evaluate the susceptibil-
ity to 3 kinds of viral vaccine strains, namely the CTN-V/PV
strain of rabies, the YN-5 strain of hepatitis A, and the Oka strain
of Varicella virus in Walvax-2, thereby preparing for the indus-
trial development of HDCVs in China.
Results
Source tissue material
We obtained 9 fetuses through rigorous screening based on
carefully specified inclusion criteria (see Methods section). The
Walvax-2 strain of cells met all of these criteria and proved to be
the best cell line following careful evaluation. Therefore it was
used for establishing a human diploid cell strain. Walvax-2 was
derived from a fetal lung tissue, similar to WI-38 and MRC-5,
and was obtained from a 3-month old female fetus aborted
because of the presence of a uterine scar from a previous caesar-
ean birth by a 27-year old healthy woman.
Primary cell stock and cell bank system
After incubation for 48 h, a confluent cell monolayer formed
and then increased in density over the following 48 hours. After
a series of successful cultures, these cells were specified as the pri-
mary cell seeds of the Walvax-2 human diploid cell line. Thereaf-
ter, a 3-tiered cell bank consisting of pre-master cell bank
(PCBP6), master cell bank (MCB, P14), and working cell
bank (WCB, P20) was established.
Figure 1 shows a gradual growing procedure for cells after
propagation. Initially, round cells with clear and relatively dark
edges were observed; as time went by, the cells elongated to
become spindle shaped and translucent fibroblasts (Fig. 1A).
Over a period of 24 hours during which the cells divided and
proliferated, the cells grew into flame shaped, typical plump dip-
loid fibroblasts with good refractive properties, and rearranged
into highly polarized areas with curling patterns (Fig. 1B).
Finally, the cells formed a dense confluent monolayer after
48 hours (Fig. 1C).
Walvax-2 cells maintained excellent capabilities for growth
and proliferation until the 50
th
passage, after which these abilities
degraded. At passage 58, cells exhibited blurred edges and could
not form a confluent monolayer after being cultured for 72 h.
Also noted were increasing black spots in cells, as well as dead
cells in the media. (Fig. 1D). Cell death was eventually observed
after 20 d
Cryopreservation stability and recovery viability
The Walvax-2 3-tiered cell bank, composed of PCB (P6),
MCB (P14), and WCB (P20), exhibited a homogeneous growth
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pattern and attained identical
population doublings (58) when
compared with the primary cell
seed. All the cells restored from
frozen stock reached adherent
growth in 2–8 h and formed a
confluent monolayer in 24 h,
with the percentage of viable
cells in the range of 80–90%
(Fig. 2). Each of the curves in
Figure 2 demonstrates the
growth features for the Walvax-2
primary cell seed as well as the
cell banks. Generally speaking,
the typical diploid cell with lim-
ited replicative lifespan follows a
“slow-logarithmic-slow” model.
However, Walvax-2 cells show
strong cell proliferation, with the
missing “slow” pattern at the
beginning for each curve, until
the 50th passage, after which the
viability of the cells decreases
dramatically. Furthermore, com-
parative cell doubling times
are summarized in Table 1.
The results confirm that the
Walvax-2 cells reconstituted
from the frozen state do not alter
their stability and viability, and could potentially be used as a cell
substrate due to these crucial properties.
Cell identification
As shown in Figure 3, the isozyme patterns of the Walvax-2
cells, using LD and G6PD as indicators, are identical to those of
human diploid cells (MRC-5) and the human cervical cancer cell
line (Hela) preserved in China Center for Type Culture Collection
(CCTCC), whereas the mouse fibroblast cell line (L929) exhibits
entirely different results. These findings confirm the fact that the
Walvax-2 cell banks behave in a manner similar to other human-
derived cell lines.
STR profiles of 16 DNA fragments of gene locus for Walvax-
2 cells are shown in Figure 4, from which we see that they match
the targeted alleles as expected. In Table 2 the data from this
study are compared with those of STR databases in ATCC of
USA and DSMZ of Germany. Three qualified laboratories,
CCTCC, NIFDC (National Institutes for Food and Drug Con-
trol) and Law School of Kunming Medical University, draw the
Figure 1. Morphology of the Walvax-2 cells. The cells were cultured and incubated at 37 C. The photos were
taken at 4 h (A), 24 h (B) and 48 h (C) and at 72 h post-subculture for the 58th passage (D).
Figure 2. The growth patterns of Walvax-2 cell banks. Primary cells were isolated from fetal lung tissue, frozen at the 6th, 14th and 20th passages, and
then recovered and subcultured continuously until cell senescence occurred.
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same conclusions that the Walvax-2 cell line displays its own spe-
cific DNA profile of human individual origin distinct from the
MRC-5 and the Hela cell lines.
Chromosomal characterization
The chromosomal characterization for PCB (P6), MCB
(P14), WCB (P20) from the 38th passage, which is the last pas-
sage that could be used for producing viral vaccines according to
the requirements of Chinese Pharmacopeia, are illustrated in
Figure 5. They show clearly that the Walvax-2 cells are 46/XX,
typical diploid type of human origin. The chromosomal analysis
of the Walvax-2 cells as summarized in Table 3 demonstrate that
the karyological properties of Walvax-2 cells satisfy the require-
ments of diploid cells of human origin to be used for producing
viral vaccines, with the frequencies of abnormalities being consid-
erably lower than the corresponding national standards.
Microbial agent tests
No cultivable bacteria or fungi were found in broth and agar
cultures. Mycoplasma tests using both the culture method and
DNA staining technique, also met the corresponding
requirements.
Exogenous virus agent tests
Results for the testing of general (non-specific) as well as spe-
cific adventitious viral agents were negative for all tested viruses
as described in detail in the “materials and methods” section.
Table 1. Population doubling times of the Walvax-2 cells with and without being subjected to freezing
Passage number Without being subjected to freezing Reconstituted from the frozen state
Population doubling time(h) Cell origin Population doubling time(h)
P10 1820 PCB,P6 1820
P20 2931 MCB, P14 3032
P25 3032 WCB, P20 3032
P32 3840 The 28th passage from the WCB 3941
P43 3941 The 38th passage from the WCB 4042
P55 5560 The 48th passage from the WCB 5762
Figure 3. Isoenzyme tests for the Walvax-2 cells. Firstly, LD and G6PD, 2 isoenzymes used as indicators, were isolated from HeLa, L929, MRC-5 and Wal-
vax-2 cells, and then subjected to PAGE and stained. The numbers of 20090327, 20090514 and 20090724 illustrated in the pictures represent Walvax-2
cells for the 18th, 30th and 50th passages.
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Retrovirus test
The results in Figure 6 make it clear that no retroviruses were
found in the Walvax-2 cells, as well as the system control cells of
MRC-5. However obvious retroviruses were found in the Sp2/0-
Ag14 cells of the positive control group, seen as tiny black dots
in the figure.
Tumorigenicity test
Tumorigenicity tests were conducted at 2 points following the
inoculation of cells into the nude mice, 21 and 84 d All mice sur-
vived in all study groups. During the animal tests, no pathologi-
cal abnormalities of nodule growths were found in the
experimental as well as the parallel negative control group
(MRC-5), and for both groups there was no pathological hetero-
geneous cell growth observed at the inoculating site or other sites
including heart, liver, spleen, lung, kidney, brain and mesenteric
lymph nodes after autopsy. In contrast, nodule and heteroge-
neous cell growth were easily found in the inoculating site in the
positive control group (Hela). These results show that the Wal-
vax-2 cells can be used for the production of vaccines with little
risk of potential carcinogenesis.
Susceptibility to viruse tests
Infectivity titers of the CTN-1V strain for the rabies virus are
presented in Table 4. CTN-1V virus was well adapted in Wal-
vax-2 relative to MRC-5. Maximum infectivity titers of CTN-
1V virus for Walvax-2 and MRC-5 were 8.14 and 7.41 FFU/ml,
respectively. During the period for virus propagation, the titers
in Walvax-2 cells were consistently higher than those of MRC-5
cells, although the differences were not always statistically signifi-
cant. However, analysis of the overall situation of the adaptation
of the CTN-1V in Walvax-2 cells relative to MRC-5 cells yielded
a significant difference (P0.001) by a 2-tailed t-test. Similarly,
the results for the PV strain adaptation in both human diploid
cells demonstrated a consistent trend, which exhibited distinct
differences for the titers.
Table 2. The STR mapping of the Walvax-2 cells
gene locus Walvax-2 MRC-5* HeLa* gene locus Walvax-2 MRC-5* HeLa*
Amelogenin X X,Y X D16S539 09,12 9,11 9,10
vWA 18 15 16,18 FGA 21,24 --
D21S11 29,30 --D3S1358 15,16 --
D18S51 15,18 --THO1 06,09 8 7
PentaE 05,18 --D8S1179 13,15 --
D5S818 10,11 11,12 11,12 TPOX 08,11 8 8,12
D13S317 11,12 11,14 12.13.3 CSF1PO 10,12 11,12 9,10
D7S820 08,12 10,11 8,12 PentaD 10,11 --
*Data from ATCC and DSMZ
Figure 4. The Short Tandem Repeat STRmap of Walvax-2 cells for the 18th passage. According to the instructions supplied with the Goldeneye 16A
identication kit (people spot), the DNA of Walvax-2 cells at the 18th passage were isolated and amplied by multiplex PCR with primers of 16 STR sites.
Then the STR map was obtained by analyzing the samples of PCR by capillary electrophoresis (CE). The STR maps of Walvax-2 cells at the 30th and 50th
passages (not shown) were the same as shown.
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Data for the VZV virus propagating in Walvax-2 and MRC-5
cells is given in Table 5. In Walvax-2 cells the virus titer grew
rapidly to 6.28 log PFU/ml, and reached a peak of 6.59 log
PFU/ml at passage 41. In contrast, the virus titers in MRC-5
were much lower, with the overall numbers less than 6.0 log
PFU/ml. All comparisons except that of the earliest generation
were all statistically significant, indicating strong adaptation of
the VZV strain for Walvax-2 cells relative to MRC-5 cells.
The comparative results for YN5 adaptability are listed in
Table 6. The titer after one generation in Walvax-2 reached 7.32
log CCID50/ml, even higher than the value in the original cells
(passage 23). During the course of 8 passages propagated contin-
uously in the Walvax-2 cells, the infectious virus titers increased
from 7.32 to 7.65 log CCID50/ml, which was marginally higher
than those of MRC-5 cells (7.0 to 7.36 log CCID50/ml).
Discussion
HDCS, deemed as the safest
cell substrate, play a vital role in
the production of viral human
vaccines. However, it is extremely
hard to obtain qualified HDCSs
that meet the requirements for
mass production. It took us 4 y
to successfully establish Walvax-2
cell lines and a 3-tiered cell bank,
namely PCB, MCB and WCB.
Complete records for the cell
bank establishment, cell culture
conditions, and tests are avail-
able. The criteria used for charac-
terizing the Walvax-2 cell banks
are those recommended interna-
tionally
18,19
and concurrent titra-
tions were set up using MRC-5
cells (the most widely used
human diploid cell substrate as a
parallel control. Walvax-2 cells
have received qualification test
reports from the NIFDC and
CCTCC, an important step in
their use for the production of
human viral vaccines in China.
Given that the availability of
HDCSs, and therefore the pro-
duction of HDCVs, is currently
subject to external forces, the development of an HDCS of Chi-
nese origin has great implications for improving the stability of
the supply of HDCVs in China.
Walvax-2 cells displayed a fibroblastic morphology similar to
that of MRC-5 cells. However, observations during the concur-
rent propagation of Walvax-2 and MRC-5 cells revealed differen-
ces in terms of growth rates and cell viability. The Walvax-2 cells
replicated more rapidly than MRC-5 –they attained the same
degree of confluence in 48 hours as was reached by MRC-5 in
72 hours, and the results are in line with measured cell doubling
times as listed in Table 1. After freezing and recovering, the
growth characteristics and patterns of the 3 life lines (PCB,
MCB, and WCB) are similar to those of the primary life line,
and attained 58 passages of cell doublings whereas MRC-5
reached 48 passages, with the difference decreasing gradually
Figure 5. Chromosomes from Walvax-2 cell banks. Walvax-2 cells were incubated 1 day post-subculture, after
which the colcemid and then Giemsa banded karyotype analyses were carried out. Pictures were the karyo-
type of Walvax-2 cells at the 6th (A), 14th (B), 20th (C) and 38th (D) passages
Table 3. The accumulated results of chromosomal analysis of Walvax-2 cells
Passage Structural abnormalities Aneuploidy Polyploidy Hyperdiploidy Breaks or gaps
Standard* 2% 18 % 4% 2% 8%
1019 0/3500 265/3500 (7.57%) 1/3500 (0.03%) 22/3500 (0.63%) 0/3500
2029 0/6000 538/6000 (8.97%) 3/6000 (0.05%) 49/6000 (0.82%) 1/6000 (0.17%)
3039 0/4500 423/4500 (9.4%) 1/4500 (0.02%) 40/4500 (0.89%) 1/4500 (0.02%)
4050 0/9000 945/9000 (10.5%) 7/9000 (0.08%) 113/9000 (1.26%) 7/9000 (0.08%)
*Chinese pharmacopeia, volume III, 2010 edition
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with increasing hours of freez-
ing.
8
In conclusion, these results
may indicate that Walvax-2 is a
cell line with superior characteris-
tic of high growth ability, as well
as strong viability compared to
MRC-5. It could be used as a
host for the cultivation and inoc-
ulation of viruses, although dif-
ferent schedules for inoculation
and propagation should be fur-
ther studied based on the growth
characteristics of particular
viruses. Furthermore, the stabil-
ity of the karyotype is another
crucial issue when using the
HDCS in the manufacture of
vaccines. The results for karyo-
logical data on Walvax-2 cells, as
summarized in Table 3, demon-
strate increases of aneuploidy and
hyperdiploidy with age. How-
ever, this is not a concern on the
grounds that the 2 “middle
groups," which are directly
related to those to be used in the
manufacture of vaccines accord-
ing to the requirements of Chi-
nese Pharmacopeia, have
frequencies of aneuploidy and
hyperdiploidy of 9.4% and
0.89% respectively, which are
substantially lower than the
national standards of 18% and
2%, respectively.
The susceptibility of the
human fetal cell strain MRC-5 to
viruses infectious in man has
Figure 6. Retrovirus tests of Walvax-2. The results were observed by mirror electron microscopy(200Kv 5000x/
160Kv 7800x). The arrows point to virus particles detected as shown in the picture. (A) and (A-1) were repre-
sented positive controls (Sp2/0-Ag14), (A-1) was partial enlarged detail of (A). (B) was represented negative
control (MRC-5). (C) was represented the cells of Walvax-2 of the 24th passage.
Table 4. Propagation of CTN-1V or PM virus in the Walvax-2 or MRC-5 cells
Virus Passage NO. CTN-1V virus ( log FFU/ml)
a
PVvirus ( log FFU/ml)
a
Walvax-2 cells MRC-5 cells P
b
Walvax-2 cells MRC-5 cells P
b
original 7.50 7.50 / 7.50 7.50 /
1 4.84 §0.62 4.58 §0.40 >0.05 4.40 §0.27 3.62 §0.23 >0.05
2 5.40 §0.21 5.02 §0.34 <0.05 5.04 §0.18 4.75 §0.24 <0.05
3 6.10 §0.37 5.41 §0.24 <0.05 5.30 §0.33 4.83 §0.25 <0.05
4 6.530.31 6.09 §0.17 <0.05 5.86 §0.10 5.02 §0.13 <0.05
5 6.78 §0.40 6.14 §0.16 <0.05 6.21 §0.21 5.63 §0.05 <0.05
6 7.08 §0.15 6.57 §0.42 >0.05 6.57 §0.53 6.02 §0.18 >0.05
7 7.34 §0.22 6.89 §0.21 <0.05 7.01 §0.70 6.00 §0.23 >0.05
8 7.51 §0.21 7.16 §0.08 >0.05 6.93 §0.19 6.28 §0.25 <0.05
9 7.67 §0.18 7.09 §0.10 <0.05 7.23 §0.23 6.59 §0.26 <0.05
10 8.14 §0.31 7.41 §0.35 <0.05 8.02 §0.19 7.11 §0.38 <0.05
Passages the 1th to 4th, subculture; Passages the 5th to 8th, cell-mixing; Passages the 9th to 10th, cell-free medium;
a
§SD.
b
Signicance of difference (P value) determined by 2-tailed t-test
1004 Volume 11 Issue 4Human Vaccines & Immunotherapeutics
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been well demonstrated over the past 10 years, indicating the
value of such material for the isolation of viruses and the develop-
ment of vaccines. In this study, the Walvax-2 cell line served as a
host for the cultivation of the CTN-V/PV strain of rabies, the
YN-5 strain of hepatitis A, the Oka strain of Varicella virus, with
results that demonstrate good sensitivity to these viruses. Com-
pared to the MRC-5 cells, titers for viruses in the Walvax-2 cells
are higher, with the overall numbers achieving statistical signifi-
cance. These discrepancies elucidate that Walvax-2, as a new
human diploid cell line, is equal or superior to MRC-5 for the
propagation of viruses. Generally speaking, as the cell passage
number increases the viral titers will experience an initial
decrease, and then increase gradually as the cell substrate adapts
to the virus. This trend is observed for the propagation of rabies
virus in our study. However, the results are not the same for the
propagation of VZV and HAV strains in HDCSs, which exhibit
increased titers after only one generation. To the best of our
knowledge, this may be attributed to the fact that these 2 virus
strains are quite sensitive to HDCSs, and particularly to the
Walvax-2 cells. Alternatively, the higher titers for Walvax-2 may
relate to the characteristics of high growth ability as well as strong
viability compared with MRC-5, as described in the “Results”
section. Nevertheless, more research needs to be done to investi-
gate the susceptibility of Walvax-2 cells to a greater variety of
viruses, and to develop fully the potential of Walvax-2 cells as a
cell substrate platform for producing viral vaccines for human
use in China.
The sensitivity to rabies virus of Walvax-2 has important
implications for China. Human diploid cell rabies vaccine,
which is free of complications but is highly immunogenic,
20
is
considered to be the gold standard for rabies vaccine.
16
Accord-
ing to the report by the WHO, there are roughly 55000 human
deaths caused by rabies annually.
21
Following India, China
ranks in second place for the highest number of human cases in
the world.
22
However, there is no such gold standard rabies vac-
cine on the Chinese market, where the disease burden is
remarkably high. Possible reasons are as follows: the vaccine,
regarded as liquid gold by the general public, represents a finan-
cial burden and hence has lower usage in developing countries.
To minimize costs as well as make it affordable for Indians, the
Serum Institute of India indigenously developed Ravivax ( Pit-
man-Moore strain, MRC-5), decreasing the cost for the vaccine
dramatically (from US $40 dropped to $7).
20
This is also one
of the motivations for this study, to develop a totally new
HDCS that could be used as a culture medium in manufactur-
ing viral vaccines in China. Recently, a document from the
Chinese pharmacopeia commission indicates that the current 2
kinds of cell substrate rabies vaccine presently on the interna-
tional market, PVRV and PHK, may not be included in the
updated Chinese pharmacopeia (2015).
23
The explanations for
the removed vaccines are that they will no longer be manufac-
tured or will be replaced by others. Human diploid cell rabies
vaccine is gaining increased national attention in China. We
tested the susceptibility of 2 rabies strains concurrently in our
study, CTN-V and PV. We found that the titers of CTN-1V
strain are higher than those of PV strain, independent of the
effects of adaptation by the cell substrates. Both strains have
been used for production in China over the years, and the safety
and immunogenicity of the vaccines have been verified.
24
Con-
sequently, considering the impact on future production, CTN-
1V will be the preferred rabies strain for research and produc-
tion in the future. Although we have reported results of the sus-
ceptibility of 3 viruses in this study, we prepared rabies vaccines
using the preferred CTN-1V-HDC (Walvax-2) viral strain
(15
th
passage) and determined the potency to be higher than
6.0IU/dose, which was significantly greater than the WHO-rec-
ommended standard of 2.5 IU/ dose
16
(described in detail in
another study
25
). The efficacies of the diploid rabies vaccines on
animal tests would further confirm the use of the Walvax-2 cells
in human viral vaccine production.
There are several limitations to this study. More work is
required regarding the adaptation of a greater variety of viruses
on the Walvax-2 cells and the possibility for the industrial
development of appropriate vaccines. In recent years, a large
number of research papers have reported the application of
Gene chip technology and high-throughput sequencing PCR
technology for detecting potential contaminations of viruses.
Thus, further screening of human-derived viruses needs to be
conducted, especially for tumorigenic DNA viruses, retroviruses
et al. Currently, we have been conducting tests for the human
Table 5. Propagation of VZV strain in the Walvax-2 or MRC-5 cells
Virus
Passage
No.
Virus Titer in
Walvax-2 cell
(log PFU/ml)
a
Virus Titer in
MRC-5 cell(log
PFU/ml)
a
P
b
31(original) 5.0 5.0
33 6.28 §0.28 5.42 §0.19 >0.05
35 6.13 §0.12 5.56 §0.11 <0.05
37 6.31 §0.28 5.52 §0.08 <0.05
39 6.27 §0.14 5.58 §0.12 <0.05
41 6.59 §0.06 5.74 §0.13 <0.05
a
§SD.
b
Signicance of difference (P value) determined by 2-tailed t-test
Table 6. The titers of HAV (YN5) adapted in human diploid cells
Virus
Passage
NO.
Infectivity titer
in Walvax-2 cells
(log CCID
50
/ml)
a
Infectivity titer
in MRC-5 cells
(log CCID
50
/ml)
a
P
b
23(original) 7.0 7.0
24 7.32 §0.28 6.27 §0.27 <0.05
25 7.47 §0.09 7.01 §0.23 >0.05
26 7.50 §0.17 7.35 §0.14 >0.05
27 7.62 §0.06 7.18 §0.38 >0.05
28 7.97 §0.09 7.50 §0.23 >0.05
29 8.21 §0.29 7.54 §0.24 <0.05
30 7.81 §0.17 7.35 §0.14 <0.05
31 7.65 §0.14 7.36 §0.34 >0.05
a
§SD.
b
Signicance of difference (P value) determined by 2-tailed t-test
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herpes simplex virus 6 and 7, and further screening will be car-
ried out soon.
In conclusion, we have successfully established and character-
ized a new human diploid cell line designated Walvax-2, and
evaluated its susceptibility to 3 kinds of viral vaccine strains. The
Walvax-2 cells are equally susceptible, and in some cases superior
to, the MRC-5 line for the cultivation of viruses. Results from
this study suggest that the Walvax-2 cell banks are a promising
cell substrate and could potentially be used for the manufacturing
of HDCVs.
Materials and Methods
Cells and viruses
HeLa, MRC-5, L929, MDCK, VeroandSp2/0-Ag14 cells
were obtained from the America Center for Type Culture Collec-
tion (CCL
-
2, CCL
-
171, CCL
-
1, CCL
-
34, CCL
-
81, andCRL-
158). Rabies fixed virus CTN-1Vstrain
26,27
and Pasteur strain
were provided by the National Institute for Food and Drug Con-
trol (NIFDC, P.R. China) and Jiangsu Simcere Vaxtec Bio-Phar-
maceutical Co., Ltd, respectively. The Varicella zoster virus Oka
strain
28
was provided by American Type Culture Collection
(ATCC). The hepatitis A virus (HAV) YN5 strain was isolated in
2003 from a hepatitis A patient in Kunming, China.
29
Laboratory animals
Kunming mice, guinea pigs and rabbits were supplied by
Guangdong Medical Laboratory Animal Center (Guangdong
Province, P.R. China). Specific-pathogen-free (SPF) eggs were
purchased from Beijing Merial Vital Laboratory Animal Tech-
nology Co., Ltd. Nude mice were purchased from Beijing Vital
River Laboratory Animal Technology Co., Ltd.
The care and use of laboratory animals were approved by the
Animal Care and Use Committee of Yunnan Walvax Biotechnol-
ogy Co., Ltd. All animals were treated humanely and euthanized
by cervical dislocation at the end of the experimental period.
Culture medium and other reagents
The growth medium (GM) for all cells was Eagle’s minimum
essential medium (M0769; Sigma) supplemented with 10 per-
cent calf serum, 2 percent 2 M Glutamine (G8540; Sigma) and
2 percent 0.83 M NaHCO
3
. The cryopreservation solution was
GM added with 10 percent DMSO (D8418, sigma). Inorganic
salts were purchased from Sinopharm Chemical Reagent Co. Ltd
(Shanghai, P.R. China).
Source tissue material
The fetal material was provided by the Department of Obstet-
rics and Gynecology of Yunnan Hospital, with legal and ethical
agreements from the donator. Before the study, we made strict
and comprehensive inclusion criteria in order to guarantee a high
quality cell strain: 1) gestational age 2 to 4 months; 2) induction
of labor with the water bag method; 3) the parents career should
not involve contact with chemicals and radiation; 4) both parents
are in good health without neoplastic and genetic diseases, and
with no history of human tissue or organ transplantation in the
families traced for 3 generations; and 5) no infectious diseases.
The tissues from the freshly aborted fetuses were immediately
sent to the laboratory for the preparation of the cells.
Preparation of primary cell stock and cell banks
The preparations of the primary cell stock and serial propaga-
tion of cells were carried out according to the methods of Jacobs
in 1970
8
and Hayflick in 1961.
30
The selected primary Walvax-2
cell seed was used for passaging, with the inoculation concentra-
tion of 5£10
5
cells /ml. Subsequent subculture was conducted at
a 1:2 split ratio immediately subsequent to the formation of a
dense cell monolayer. When the cells reached the 6
th
,14
th
, and
29
th
cell doublings, cultures were harvested and frozen to form a
pre-master cell bank (PCB), master cell bank (MCB) and work-
ing cell bank (WCB).
Cryopreservation stability and recovery viability
Cryopreservation was performed 6 times for Walvax-2 cells
with the cell lines designated as P6, P14, P20, P28, P38 and
P48. These particular cell lines were chosen on the grounds that
they represented the corresponding cell banks of PCB, MCB,
WCB, the major working passages, and the entire lifecycle of the
Walvax-2 cells. The cells were centrifuged and re-suspended in
cryopreservation solution, and the cell concentration was
adjusted to 6»10£10
6
cells /ml. The suspension was dispensed
in 1.0 ml to 2.0 ml Cryogenic Vials (#430659Corning). Fol-
lowing the manufacturer’s instructions for the use of pro-
grammed cooling boxes (Nalgene Mr. FrostyThermo Fisher),
the cryogenic vials were then sealed and put into the boxes at
¡70C overnight. Then cryogenic vials were placed directly into
liquid nitrogen for long term cryopreservation. The frozen cells
were recovered according to the procedures given by Jacobs in
1970
8
and Hayflick in 1961.
30
Cells reconstituted from the frozen state were taken immedi-
ately for the calculation of population doubling times by cell
counting. The three-tiered banks were propagated serially for
doubling time assessments. The experiments were repeated
8 times, and the doubling times were compared with that of cells
that had not been frozen.
Cell identification
The cell identification was evaluated by a 2-step procedure:
Firstly isoenzyme analysis was performed using lactate dehydro-
genase (LD) and Glucose 6 phosphate dehydrogenase (G6PD) as
indicators to confirm Walvax-2 cell banks were human-derived
cells. Then, Short Tandem Repeat (STR) analysis was conducted
using MRC-5 as a parallel control by 3 qualified laboratories:
China Center for Type Culture Collection (CCTCC), National
Institutes for Food and Drug Control (NIFDC), and Law School
of Kunming Medical University to assure that the cells were
derived from the tissue of a specific human individual and differ-
ent from any other established human diploid cell lines.
1006 Volume 11 Issue 4Human Vaccines & Immunotherapeutics
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Chromosomal characterization
Chromosome examinations were conducted for every 10 pas-
sages by counting percentages for 5 types of chromosomal aberra-
tions, including structural abnormalities, aneuploidy, polyploidy,
hyperploidy and breaks or gaps. Chromosome specimen slides
were obtained using the method of Coburn and Leykauf,
31
and
then stained with Giemsa. Giemsa-banded karyotypes were
recorded by Applied Imaging SoftwareKaryotyping 3.0
(England).
Microbial agents tests
The presence of bacterium, fungus and mycoplasmas for
Walvax-2 cells were tested according to the requirements of
ATCC and WHO.
18,19
Bacillus subtilis (CMCC(B)63501),
Clostridium sporogenes (CMCC(B)64941) and Candida albi-
cans (CMCC(F)98001) were used as positive controls for the
tests of bacteria and fungi. A total of 19 cell passages were tested
for sterility. The cell samples were tested under different tempera-
tures for 2 weeks to confirm that no bacterial and fungal contam-
ination was present. The mycoplasma test was conducted as per
requirement in Volume III of Chinese Pharmacopeia, using the
culture method and DNA staining technique, and B6yh4 cells
were used as a positive control. All positive controls were pro-
vided by the National Institute for Food and Drug Control
(NIFDC, P.R. China).
Exogenous virus agents tests
Tests for adventitious viral agents of Walvax-2 cells were con-
ducted as per requirements for Preparation and Control of Ani-
mal Cell Substrates Used for Production and Testing of
Biologics in Volume III of Chinese Pharmacopeia, including test-
ing for general adventitious viral agents (non-specific virus) and
specific adventitious viral agents.
General adventitious agents included embryonated egg inocu-
lation by the yolk sac, allantoic cavity; i.c. and i.p. inoculation of
adult and suckling mice, i.p. inoculation of guinea pigs; mono-
layer cell culture using MRC-5, and vero cells for detection of
various human viruses.
Tests for specific adventitious agents consisted of human
derived virus, bovine derived virus and porcine virus. For the
human derived virus test, 6 viruses including HBV, HCV, HIV,
Human cytomegalovirus, human nasopharyngeal virus and
human parvovirus B19, were carried out based on per testing kit,
using ELISA and PCR methods, respectively. For the bovine
derived virus test, 3 methods were used: (i) the microscopic CPE
observation method; (ii) different cell culture conditions for
hemadsorption activity, and (iii) fluorescence quantitative RT-
PCR method (bovine adenovirus, bovine parvovirus, bovine diar-
rhea virus, bovine influenza virus, bovine parainfluenza virus,
rabies virus and retrovirus). The possible swine viral contamina-
tion was examined using RT-PCR and PCR methods for classical
swine fever virus, Japanese encephalitis virus and Pseudorabies
virus.
Retrovirus test
The retrovirus test was performed according to procedures
described in “ Reverse transcriptase activity assay in attenuated
live vaccine”(Yan Kong et al)
32
and “Development of an
improved product enhanced reverse transcriptase assay” (Audrey
Chang, et al).
33
More specifically, the testing methods included
product-enhanced reverse transcriptase (PERT) assay, infection
test and direct observation by transmission electron microscopy.
The mouse bone marrow cell line Sp2/0-Ag14 served as a posi-
tive control while MRC-5 cells were used for the system control.
Tumorigenicity test
To ascertain whether the cells had any neoplastic properties,
P10, P20, P28, P38 and P48 Walvax-2 cells were implanted into
10 nude mice aged 4–6 weeks, in the thigh of the right hind leg
of each mouse according to the requirements of Chinese Pharma-
copeia. MRC-5 cells served as the negative control, and Hela cells
served as the positive control. All animals were examined after 21
and 84 d following the inoculation of the cells. Animals not sur-
viving the full period were examined post mortem, and observa-
tions for neoplastic growth were conducted for all tested animals.
Susceptibility to viruses test
Particular cell generations that would potentially be used for
producing viral vaccines, were used to determine susceptibility to
viruses after 25 to 30 cell doublings. Three kinds of viral vaccine
strains (rabies, Varicella zoster and Hepatitis A) were used for the
assays. To determine the susceptibility of Walvax-2 cells relative
to MRC-5 cells, concurrent titrations were compared for the
same cell doublings.
Rabies Virus
Virus propagation
The CTN-1V and Pasteur strains were propagated in Walvax-
2 and MRC-5 cells by the method of Wiktor et al.
34
The virus
maintenance medium was consistent with GM with the addition
of 2% (v/v) fetal calf serum. A multiplicity of infection (MOI) of
0.01 was used. The viruses were incubated at 34–35C.
Virus titration
The rabies virus was titrated using a modified test as described
by Smith et al.
35
Virus titer was expressed in fluorescent focus
units (FFU)/ml. Briefly, a monolayer of BSR cells in 96-well
plates was incubated with serial fold5- virus dilutions at 37Cin
a5%CO
2
humidified incubator for 24 h. The cells were then
fixed with 80% cold acetone at -20C for 30 minutes, and then
stained with the Rabies DFA Reagent (5100; Millipore). The
plates were examined by fluorescence microscopy (Olympus
Corp., Tokyo, Japan), and the numbers of fluorescent foci pre-
sented in the wells were recorded. The highest dilutions with
fluorescent foci less than 30 were defined as endpoints, and virus
titers were calculated by the following formula: virus titer (FFU/
ml) D(the mean foci number in the endpoint wells £5Cthe
mean foci number in the wells with lower dilutions next to the
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endpoint well) ÷2£the dilution factor of the lower dilutions ÷
the volume of virus dilution inoculated into each well.
Varicella Zoster Virus (VZV)
Virus propagation
The Oka strain at passage 31 was inoculated into Walvax-2
and MRC-5 cells and grown into a confluent monolayer at an
MOI of 0.01.
36
Infected cells were incubated at 36 C for 48–52
hrs till the cytopathogenic effect (CPE) was estimated to be
approximately 75% to 100%. The cells were then trypsinized
and resuspended in cryopreservation solution and stored at
-196C. The virus was serially propagated 8 times as described
above for Walvax-2 and MRC-5 cells.
Virus titration
A plaque assay
37,38
was used and virus titer was expressed in
plaque forming units (PFU)/ml. When Walvax-2 and MRC-5
cells in 6-well plates grew to a near confluent monolayer, the old
medium was poured off and the monolayer was infected with
cell-associated virus in fresh medium (with 2% fetal calf serum
and 1% penicillin-streptomycin). Infections were allowed to pro-
ceed for 8–9 days, at which point the first signs of CPE was visi-
ble, the cells were stained and plaques counted.
Hepatitis A Virus
Virus propagation
According to the method of Wang et al,
39
the HAV YN5
strain was propagated in Walvax-2 cells and MRC-5. Briefly,
Walvax-2 cells were trypsinized and inoculated with HAV at a
MOI of 0.01 and stirred gently with a magnetic stirrer for 2 h at
37C. The cells were then seeded in T225 flasks filled with GM
at 37C for 3–4 d until a confluent monolayer was formed. The
GM was replaced by virus maintenance medium, consisting of
MEM supplemented with 2% (v/v) fetal calf serum, 0.35% (m/
v) NaHCO
3
, 2% (v/v) and 2 M Glutamine; Cells were incu-
bated at 35C for 25 d Afterwards the cells were harvested and
stored at ¡80C.
Virus titration
An enzyme-linked immunosorbent assay (ELISA) was used to
determine the virus infectivity titer of HAV.
40
The monolayers
of Walvax-2 and MRC-5 were inoculated with serial fold5- cell-
associated virus dilutions and incubated at 37C for 1 h. Each
dilution was assayed in quadruplicate. Then the inoculums were
removed and replaced with a 1 ml nutrient MEM overlay con-
taining 2 % fetal calf serum and incubated at 35C for 25 d. The
infected cells were harvested and sonicated. The presence of
HAV-Ag was tested by ELISA. The CCID
50
value was calculated
by a modified Reed-Muench’s method.
41
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We are grateful to Dr Shen Chao (College of Life Sciences at
Wuhan University) for generous technical assistance and advice.
Many thanks to all those who were involved in this work.
Funding
This work was financially supported by National High Tech-
nology Research and Development Program of China
(2014AA021002), the National Key Technology Support Pro-
gram (2008BAI54B04–09), National Science and Technology
Major Project (2013ZX10004003) and Major Project of Yunnan
Provence Programs for Fundamental Research (2013FC005).
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www.taylorandfrancis.com 1009Human Vaccines & Immunotherapeutics
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... In the recent past, vaccine manufacturing industry was found to be relying increasingly on the human diploid cells and continuous cell lines such as Vero cells as cell substrates of choice, because of their well characterized nature. 7,8 Human diploid cells such as MRC-5 cells and the continuous cell line of African green monkey kidney cell such as Vero cell line are the most studied and hence the safest known cell substrate for the manufacture of human vaccines. These cells can be preserved in-house, in liquid nitrogen as a cell bank with ease of revival at any time with a short notice, thus eliminating the efforts on advanced planning, logistics and dependency on external source for the supply of the starting material. ...
Chapter
Lyssaviruses continue to evolve and pose threats to humans, domestic animals, and wildlife. As a fatal disease of zoonotic importance, rabies is fortunately preventable, thanks to the advent of potent biologics. Pioneering works done during the last two centuries act as cornerstone of research making rabies diagnosis, prevention, control, and selective elimination possible. The field has benefited immensely from the tremendous scientific advancement during the last three decades in virology, molecular biology, vaccinology, and delivery systems. Vaccines have evolved from the first generation of crude nerve tissue-based products to recombinant vaccines. Cell culture-based inactivated rabies vaccines for intramuscular (IM) and intradermal (ID) use in humans continue to play a pivotal role when it comes to rabies prophylaxis. Parenteral and oral vaccinations prove time and again promising tools for rabies control in domestic animals and wildlife. Improvements likely to cause paradigm shift include products based on virus-like particles, obviating the need for a high containment facility; sparing usage of antigens using new adjuvants; delivery using novel systems; and direct inoculation of potent vaccines without the need of rabies immune globulin (RIG).
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MRC-5 represents the most frequent human diploid cells (HDCs)-type cell substrate in the production of human viral vaccines. However, early-passage MRC-5 is diminishing and, due to both technical and ethical issues, it is extremely difficult to derive novel HDCs from fetal lung tissues, which are the common sources of HDCs. Our previous studies suggested that human umbilical cord may represent an alternative but convenient source of new HDCs. Here, we established a three-tiered cell banking system of a hUC-MSC line, designated previously as Cell Collection and Research Center-1 (CCRC-1). The full characterization indicated that the banked CCRC-1 cells were free from adventitious agents and remained non-tumorigenic. The CCRC-1 cells sustained its rapid proliferation even at passage 30 and were susceptible to the infection of a wide spectrum of viruses. Interestingly, the CCRC-1 cells showed much higher production of EV71 or Rubella viruses than MRC-5 and Vero cells when growing in serum-free medium. More importantly, the EV71 vaccine produced from CCRC-1 cells induced immunogenicity while eliciting no detectable toxicities in the tested mice. Collectively, these studies further supported that CCRC-1, and likely other hUC-MSCs as well, may serve as novel, safe and high-yielding HDCs for the production of human viral vaccines.
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Rabies is a neglected viral zoonosis with the highest case fatality of any infectious disease. Pasteur’s historical accomplishments during the late 19th century began the process of human vaccine development, continuing to evolve into the 21st century. Over the past 35 years, great improvements occurred in the production of potent tissue culture vaccines and the gradual removal from the market of unsafe nerve tissue products. Timely and appropriate administration of modern biologics virtually assures survivorship, even after severe exposures. Nevertheless, in the developing world, if not provided for free nationally, the cost of a single course of human prophylaxis exceeds the average monthly wage of the common worker. Beyond traditional approaches, recombinant, sub-unit and other novel methods are underway to improve the availability of safe, effective and more affordable rabies biologics.
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This study was conducted to investigate body mass index (BMI), levels of cholesterol and triglycerides in prison inmates at the Institution for Reform and Rehabilitation in Southern Libya to be considered as an indication about their health and the provided foods. The results of this study showed that 26.5% of BMI of the prison inmates were found to be higher than the normal levels. Generally, the average level of cholesterol and triglycerides concentrations were found to be within normal range 142.6 mg/dl and 135.4 mg/dl, respectively. The findings also established that there were a significant relationship and direct correlation between BMI levels and age and concentration of cholesterol and triglycerides levels. The results of this showed that the served foods for these prison inmates are well balanced as indicated by their cholesterol and triglycerides levels.
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The isolation and characterization of 25 strains of human diploid fibroblasts derived from fetuses are described. Routine tissue culture techniques were employed. Other than maintenance of the diploid karyotype, ten other criteria serve to distinguish these strains from heteroploid cell lines. These include retention of sex chromatin, histotypical differentiation, inadaptability to suspended culture, non-malignant characteristics in vivo, finite limit of cultivation, similar virus spectrum to primary tissue, similar cell morphology to primary tissue, increased acid production compared to cell lines, retention of Coxsackie A9 receptor substance, and ease with which strains can be developed. Survival of cell strains at - 70 °C with retention of all characteristics insures an almost unlimited supply of any strain regardless of the fact that they degenerate after about 50 subcultivations and one year in culture. A consideration of the cause of the eventual degeneration of these strains leads to the hypothesis that non-cumulative external factors are excluded and that the phenomenon is attributable to intrinsic factors which are expressed as senescence at the cellular level. With these characteristics and their extremely broad virus spectrum, the use of diploid human cell strains for human virus vaccine production is suggested. In view of these observations a number of terms used by cell culturists are redefined.
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The coxsackie A16 virus (CA16), along with enterovirus 71 (EV71), is a primary pathogen that causes hand, foot, and mouth disease (HFMD). To control HFMD, CA16, and EV71 vaccines are needed. In this study, an experimental inactivated CA16 vaccine was prepared using human diploid cells, and the vaccine's immunogenicity was analyzed in mice and rhesus monkeys. The results showed that the neutralizing antibody was developed in a dose-dependent manner, and was sustained for 70 days with an average GMT (geometric mean titer) level of 80 to 90 in immunized mouse and for 56 days with GMT of higher than 300 in monkeys. The neutralizing antibody had a cross-neutralizing activity against different viral strains (genotype A and B), and the specific IFN-γ-secreting cell response was activated by these virus strains in an ELISPOT assay. This study provides evidence for the potential use of inactivated CA16 as a candidate for use in vaccines.
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In 1999, Serum Institute of India indigenously developed an adsorbed human diploid cell rabies vaccine (Rabivax). During 2000 – 04, this new vaccine was subjected to a series of immunogenecity and safety studies. Initially, an experimental batch of Rabivax (adsorbed) was assessed on 10 healthy adult volunteers and its response was comparable with that of Merieux inactivated rabies vaccine (MIRV, lyophilized) which was used as a control. Subsequently, Rabivax (adsorbed) was assessed on forty-five suspect rabid dog bite cases with MIRV as control. The vaccine was found to be equally safe and immunogenic as MIRV and showed better rabies virus neutralizing antibody (RVNA) response on day 90 than MIRV. A post-licensing study conducted on 150 cases of suspect rabid animal bites showed it to be safe and immunogenic. To assess its long-term sero-efficacy some of these subjects tested after one year of follow up showed that 84% of them had adequate RVNA titers. In addition, a routine post-marketing surveillance done on 1608 animal bite cases demonstrated that Rabivax (adsorbed) was safe and efficacious. The adverse events to Rabivax (adsorbed) included pain at injection site (3.4%), swelling with induration (2.8%), fever and headache (1.4%). No serious adverse event was reported from the studies. In conclusion, Rabivax (adsorbed) is an immunogenic, safe, and efficacious vaccine for rabies prophylaxis in humans.
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The isolation and characterization of 25 strains of human diploid fibroblasts derived from fetuses are described. Routine tissue culture techniques were employed. Other than maintenance of the diploid karyotype, ten other criteria serve to distinguish these strains from heteroploid cell lines. These include retention of sex chromatin, histotypical differentiation, inadaptability to suspended culture, non-malignant characteristics in vivo, finite limit of cultivation, similar virus spectrum to primary tissue, similar cell morphology to primary tissue, increased acid production compared to cell lines, retention of Coxsackie A9 receptor substance, and ease with which strains can be developed.Survival of cell strains at − 70 °C with retention of all characteristics insures an almost unlimited supply of any strain regardless of the fact that they degenerate after about 50 subcultivations and one year in culture. A consideration of the cause of the eventual degeneration of these strains leads to the hypothesis that non-cumulative external factors are excluded and that the phenomenon is attributable to intrinsic factors which are expressed as senescence at the cellular level.With these characteristics and their extremely broad virus spectrum, the use of diploid human cell strains for human virus vaccine production is suggested. In view of these observations a number of terms used by cell culturists are redefined.