Mechanisms of Inactivation of HSV-2 during Storage in Frozen and Lyophilized Forms
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Biotechnol. Prog. 2005, 21, 911−917 911
Mechanisms of Inactivation of HSV-2 during Storage in Frozen and
Lyophilized Forms
Raino K. Hansen,†,‡ Suling Zhai,† Jeremy N. Skepper,§ Mike D. Johnston,⊥
H. Oya Alpar,| and Nigel K. H. Slater*,†
Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, United
Kingdom, Multi-imaging Centre, Department of Anatomy, University of Cambridge, Downing Site, Cambridge
CB2 3DY, United Kingdom, Xenova Limited, 310 Cambridge Science Park, Milton Road, Cambridge CB4 OWG,
United Kingdom, and The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N
1AX, United Kingdom
The structural integrity of herpes simplex virus 2 (HSV-2) during freezing, thawing,
and lyophilization has been studied using scanning and transmission electron
microscopy. Viral particles should be thawed quickly from -80 to 37 °C to avoid
artifacts of thawing. To avoid freezing damage, the virus should be rapidly frozen
(>20 K s-1) rather than slowly frozen as occurs on the shelf of a lyophilizer (912 Biotechnol. Prog., 2005, Vol. 21, No. 3
1964) since the closely related varicella zoster virus The experiments were conducted in duplicate, and virus
uncoats during freezing and lyophilization in the absence was diluted with Dulbecco’s medium and plated for
of cryoprotectants (Grose et al. 1981). It is therefore TCID50 determination within 3 h exactly as in (Zhai et
important to understand how HSV is inactivated under al. 2004). The control stored at 22 °C was used as 100%
these storage conditions in order to design better storage viral titer.
strategies with higher retention of viral titer. In this Negative Staining and Sample Preparation for
study, TCID50, electron microscopy, immunogold labeling Electron Microscopy. The negative staining procedure
techniques, and real-time PCR were used to analyze the used poly- or monoclonal antibodies against HSV epitopes,
inactivation mechanisms of HSV under various condi- which were visualized with 10 nm gold particles conju-
tions. This included liquid storage inactivation, which gated to secondary antibodies (British Biocell Interna-
may occur before filling vials with viral doses in a GMP tional, Cardiff, UK). The secondary antibodies were anti-
environment. Furthermore, the effect of the in-freezing mouse for targets of monoclonal antibodies (Virusys,
rates on a small viral sample was tested by repeated North Berwick, ME) or anti-rabbit for the polyclonal
freeze-thaw, where the freezing surface was either antibodies (Dako, Ely, UK) for HSV-2. The blocking
-40 °C (simulating an industrial lyophilizer) or -196 °C buffer used for blocking and dilution of primary antibod-
(simulating fast methods in the laboratory). Finally, ies consisted of 0.22 µm-filtered 5% v/v fetal calf serum
lyophilized viral samples were compared where the (Invitrogen) and 5% w/v serum bovine albumin (Sigma)
variable was the in-freezing rate (-40 or -196 °C in PBS buffer (Sigma). The dilution buffer used for
surfaces). The study also sought to examine whether washing and dilution of gold-conjugated antibodies con-
there is a difference in quality between virus from slow sisted of 0.22 µm-filtered 1% v/v fetal calf serum and 0.5%
(4 °C) and fast (37 °C, physiological) thawing from the w/v bovine serum albumin in PBS buffer. All prepara-
frozen state. tions and solutions were held at 22 °C throughout the
experiment, and after each step the electron microscopy
Materials and Methods (EM) grid was drained against filter paper.
Virus Propagation. The disabled infectious single- The negatively stained HSV electron microscopy (EM)
cycle HSV virus had a deletion of the DNA coding for preparations generally originated from the same batch
the essential membrane gH protein (Boursnell et al. 1997; used while 4-8 months old or from batches of similar
Farrell et al. 1994; Forrester et al. 1992). The virus is viral titer and quality as specified above. To prepare the
produced in host cells that express gH, resulting in the frozen virus for TEM, the virus was thawed on a water
viruses having a normal morphology (Lampert et al. bath (either 37 or 4 °C). The lyophilized samples (at
1969; Morgan et al. 1954). Vero CR2 cells were used to ∆K s-1 < 1 (Zhai et al. 2004)) were rehydrated at room
grow the genetically modified HSV-2, dH2A, as previ- temperature (rt, 22 °C) with distilled water. Furthermore,
ously described (O’Keeffe et al. 1998). The virus was the virus was stored under aqueous conditions in the
provided by Xenova Research, Ltd., as liquid crude, storage buffer at 4 °C.
purified frozen or lyophilized (Loudon and Varley 2001), Samples for immunogold labeling and negative stain-
stored at -80 °C until use with batches of several ages ing were prepared by floating a carbon-coated 400 mesh
was possible. The benefit was the access to data on Formvar EM grid on top of one drop of undiluted virus
purity. The host cell line containing the complementing for 5 min. Alternatively (Figure 1E), the grid was drained
gene for the viral glycoprotein gH (Boursnell et al. 1997) and quench frozen (∆K s-1 > 500) (Echlin 1992) in
was seeded at 2 × 107 cells mL-1 and grown to confluence melting propane cooled in liquid nitrogen. The frozen grid
in Dulbecco’s modified Eagle’s medium over 4 days at (Figure 1E) was placed on a chilled (-196 °C) brass block
37 °C. The confluent cells were washed to remove bovine and lyophilized under high vacuum (10-6 mBar) over-
serum, refed with serum-free medium, and infected with night and later rehydrated with distilled water at rt. The
dH2A virus at a multiplicity of infection at 0.001 pfu grid was transferred to float on blocking buffer for 15 min
cell-1. During the three-day virus growing period, the and then transferred onto one drop of primary antibody
temperature was controlled at 34 °C. The virus was diluted with one volume of blocking buffer for 30 min.
released from the cells over a period of 2 h by addition of The grid was washed three times with dilution buffer
150 µg mL-1 of dextran sulfate to the supernatant before being transferred for 30 min to a drop of secondary
medium. Dextran sulfate was removed using diafiltration gold-conjugated antibody diluted in the ratio of 1:100 in
through a 300 kDa membrane. Contaminating cellular dilution buffer. Finally, the grid was floated for 30 s on
DNA was digested using 50 units mL-1 of benzonase. The 0.5% w/v potassium phosphotungstate (PTA) at pH 6.8
recovered virus was purified by chromatography through and drained, ready for electron microscopy using a
a Fractogel EMD SO3 column matrix from which it was Philips CM100 operating at 60 or 80 keV. The resolutions
eluted using 2 M NaCl. Then, the virus was transferred recorded were at 46 000 X for immunogold label density
into the storage buffer (200mM Tris-HCl at pH 7.4, 2.5% and at 10 500 X for viral morphology statistics.
w/v sucrose, and 0.5% w/v sodium glutamate) by dia- Scanning electron microscopy was performed on HSV
filtration using a 300 kDa membrane. to visualize the three-dimensional morphology. The
Effect of Multiple Freeze-Thaw Cycles on Hy- Formvar-coated, 400 mesh, TEM grid was floated on a
drated HSV Virus. The HSV virus was thawed from sample of virus for 3 min. The grid was then washed
-80 to 37 °C and divided into aliquots that were stored three times in ice-cold distilled water and immediately
at 4 °C until used on the same day. Virus (15 µL) was frozen in liquid propane, chilled by liquid nitrogen. The
spread on an aluminum pan and subjected to freeze- grid was lyophilized overnight and then sputtered with
thaw cycles using the following thermal treatments: gold-palladium to an average thickness of 10 nm and
22 °C for the control (no heat cycles), 37 °C alumina block imaged with a Philips XL30 FEG SEM operated at 5 keV.
for rapid thaw, slow freezing in the -40 °C shelf cabinet Real-Time PCR. Real-time PCR experiments on an
(a laboratory lyophilizer), and fast freezing on the surface ABI 7700 apparatus (Applied Biosystems) were set up
of liquid nitrogen at -196 °C (Zhai et al. 2004). A cycle to amplify a well conserved 157 base pair fragment using
of 30 s freezing (-40 or -196 °C) and 30 s for a complete the primers acgcgagagc ctgctga and ggtgaacccg tacaccga
thaw allowed for an experiment of 6 cycles to last 6 min. (Department of Biochemistry, University of Cambridge,Biotechnol. Prog., 2005, Vol. 21, No. 3 913
Figure 1. TEM micrographs of representative particles of HSV-2 prepared with polyclonal antibody for HSV-2 and then 10 nm
conjugated gold. (A) Fresh virus, 19 ( 4 virus-1. (B) Thawed from wet -80 °C, 17 ( 3 virus-1. (C) Bulk-lyophilized, stored at
-80 °C and rehydrated at rt, 4 ( 4. (D) Same as B, but stored for 7 days in the dark at 4 °C, 8 ( 4 virus-1. (E) Flash frozen on TEM
grid, lyophilized and rehydrated, approximately 95% of virus, 12 ( 5 virus-1. (F) Same as E, but minor representative (5%) with less
than 4 particles. Length of bar: 100 nm. A and C were prepared without 0.5% PTA; the remaining were prepared with 0.5% PTA.
Length of bar: 100 nm.
Table 1. Morphology Classes Given in % for Herpes
U.K.). The primer concentrations of 1200 nM/1200 nM Simplex Virus Stored at -80 °C and then Thawed at the
were found to be most effective for amplification. The dye Temperature Givena
system was SYBR Green with Polymerase (Applied
Biosystems). Intact HSV virus for standard curves was full viral multiple naked empty
particle capsids capsids envelopes
inactivated in the presence of 1% SDS, 95 °C for 10 min,
and then diluted 1000-fold or more. Freeze-thaw samples 37 °C thaw 53 1 6 40
were not treated with SDS but were treated in parallel 37 °C thaw + 7 days 24 1 12 63
4 °C thaw 43 5 7 45
to assay for free DNA. The control of SDS-dissolved virus 4 °C thaw + 7 days 25 2 8 65
gave a dilution curve by real-time PCR with a coefficient
a Viral preparation stored at 4 °C for 7 days is spontaneously
of 1.9, close to the optimal value of 2.
degraded from approximately 48% particles of morphology, as in
Figure 1, to approximately 23% of the particles. 859 particles were
Results and Discussion analyzed from a total of two batches and eight to nine electron
Viral Titer Measurements and Initial Assessment micrographs per preparation with an estimated standard deviation
of (6 for classification of empty and full viral particles. A total of
of Viral Morphology. Viral titer measurements (TCID50) 21 cases of multiple capsids in one envelope were observed, 16 in
(Reed and Muench 1938) were made to obtain an 4 °C preparations and 5 in 37 °C preparations (428 and 431
estimate of the number of infectious particles present in particles evaluated). The viral titer was 100 ( 5% for the period.
a sample after physical handling. The effect of thawing
and freezing rates was observed in two series of experi-
ments. storage at 4 °C, both samples appear to be identical with
The recoveries of infectious virus after thawing of a a decrease to 25% intact particles. The samples thawed
frozen sample at either 37 or 4 °C were not significantly at 4 °C (slowly) have a marginally lower (43% ( 6)
different (p > 0.05); however, there were a number of proportion of intact viral particles (as Figure 1AB) than
morphological differences between the two samples. The samples thawed at 37 °C (53% ( 6). As thawing at 37 °C
results are summarized in Table 1. After 1 week of (physiological) presented fewer artifacts than thawing at914 Biotechnol. Prog., 2005, Vol. 21, No. 3 Figure 2. Sequence of events leading to the uncoating of HSV-2 as shown with particles that are not intact. (A, B) Labeled with polyclonal antibody and 10 nm conjugated gold antibody. (C) Labeled with antibody against ICP-8 (capsid) and 10 nm conjugated gold antibody. (D) Labeled with antibody against VP16 (tegument) and 10 nm conjugated gold antibody. (E, F) Nuclei that are almost naked; no colloid gold was used. Length of bar: 100 nm 4 °C (Figure 3), the rapid 37 °C thaw was chosen for using liquid nitrogen is the more reliable of the two further work. This thaw is equivalent to that reported freezing methods. Furthermore, they indicate that it is in a recent paper on plasmid vectors (Armstrong and detrimental to viral quality to remove water from shelf- Anchordoquy 2004) and liposomes (Hansen et al. 2002). frozen material. However, it is clear that complete viral Aliquots of viral particles were frozen, by either liquid recovery can be achieved by liquid nitrogen freezing even nitrogen or placement onto a prechilled shelf, followed after several cycles of thawing. by rapid 37 °C thawing for 6 cycles. The viral morphology Immunogold Labeling. Uncoating mechanisms were and viral titer were analyzed, and the results are studied quantitatively using immunogold labeling with summarized in Table 2. A viral titer recovery of 100 ( polyclonal and monoclonal antibodies raised against HSV 5% was achieved after 6 cycles of freeze-thaw experi- envelope proteins (Stannard et al. 1987) in conjunction ment by in-freezing at -196 °C as well as after a single with TEM. Absence of immunogold labeling on intact in-freezing followed by lyophilization. Only 74 ( 5% viral viral particles was taken as indicative of the denaturation titer recovery was achieved after exposing a sample to 6 of the relevant envelope proteins. The reference for the freeze-thaw cycles of freezing a sample on a metal shelf experiments was fresh HSV stained with polyclonal at -40 °C followed by thawing at 37 °C on a metal block. antibodies and labeled with 19 ( 4 particles virus-1 When the control was compared with the -40 °C freeze- (Figure 1A). The other viral particles were stored at thaw sample, a change in morphology was observed with -80 °C for 4-8 months and then thawed (Figure 1B) or the quantity of intact viral morphology decreasing from lyophilized and then rehydrated (Figure 1C). The virus 33 to 11% and with the proportion of naked capsids thawed from -80 °C gave a gold label density identical increasing from 31 to 56%. In-freezing to -196 °C to fresh virus with 17 ( 3 particles virus-1. Once thawed, followed by lyophilization (at -40 °C) is reported in virus was stored at 4 °C for 1 week (Figure 1D), and the further detail in another paper (Zhai et al. 2004). number of bound secondary antibodies fell significantly The lowest viral titer recovery of only 40% was from 17 ( 3 to 8 ( 4 particles virus-1. Lyophilization achieved after freezing on a metal shelf at -40 °C reduced the number of bound immunogold particles to followed by lyophilization as reported by (Loudon and 4 ( 4 particles virus-1 (Figure 1C). When the virus was Varley 2001) (Figure 1C). These results clearly show that frozen in liquid propane and lyophilized as a monolayer freezing particles slowly on a shelf, a common laboratory on the EM grid, the quality of the virus was high (Figure practice, is detrimental to viral quality and that freezing 1E) with 12 ( 5 particles virus-1, although ca. 5% of
Biotechnol. Prog., 2005, Vol. 21, No. 3 915
Figure 3. Spontaneous uncoating of HSV virus and artifacts of HSV-2 thawed at 4 °C (Table 1). (A) Virus thawed at 4 °C; several
irregular envelopes (like Figure 2A) are indicated by arrows. (B) Preparation A stored for 7 days at 4 °C; severe loss of nuclei. Arrow
indicates an intact viral particle. (C-E) Fused viral particles. Arrow indicates an electron lucent capsid, which does not contain
DNA. Length of bar: 200 nm.
Table 2. Figures in % for Viral Morphology (1200 tons, and Figure 2F shows a rare capture of the tegument
Particles Smaller than 200 nm) and Viral Titer in a hanging off the capsid in only one point. From these
Freeze-Thaw Experimenta observations it can be concluded that pathways of un-
full viral multiple naked coating could be via the following three approaches that
particles capsids capsids empty titer could take place simultaneously in solution: (1) by the
control on bench 33 1 31 35 100 simultaneous release of tegument and membrane with
nitrogen (-196°C) 26 1 32 40 100 the sequence of events illustrated with Figure 2A-C to
shelf (-40°C) 11 1 56 32 74 release a naked nucleocapsid; (2) by the loss of membrane
a Six freeze-thaw cycles were conducted lasting a total of 6 min, structure, followed by the release of tegument, with the
with the thaw taking place on a 37 °C block of metal. The sequence being 2A, 2B, 2D, and 2F to release a naked
aluminum pan was chilled either on the shelf or on the surface of nucleocapsid; and (3) by the pathway known for un-
liquid nitrogen. The control was at room temperature for 6 min, coating of HSV in vivo (Sodeik et al. 1997) with a
and the nitrogen freeze-thaw had the same viral titer and the sequence of 2A, 2B, (possibly 2D) on to 2E, and finally a
same distribution of morphologies as the control. The shelf freezing
caused a reduction of viral particles by two-thirds (from 33 to 11%), naked nucleocapsid.
but this was not proportional to the loss of viral titer by one-fourth The degradation of virus following thaw and hydration
(from 100 to 74%). was assessed for 859 virus particles, subdivided into four
groups: group A, intact virus, as shown in Figure 1; group
viruses had anomalously low labeling of 0-3 particles B, fused viral particles, as shown in Figure 3; group C,
virus-1 (Figure 1F). naked nucleocapsid; and group D, empty viral coats (as
Figures 4A and 1A demonstrate that freshly shed and shown in Figure 4B). Viral counts indicated a reduction
purified virus have a uniform, spherical morphology. In in the number of full, intact virus (group A) from
all preparations with a low immunogold labeling density, approximately 50% of particles to 25% of particles after
there were several morphologies present (Figure 3B), and 1 week of wet storage at 4 °C. Group B of fused viral
intact particles had a tegument of an uneven thickness particles was small, and the differences between condi-
(Figure 3A). Figure 2 shows the putative stages of viral tions of thaw were insignificant when using a Student T
uncoating, and Table 1 shows the statistics for viral test. Group C of naked particles increased significantly
morphology. Viral particles thawed from -80 °C often during hydrated storage at 4 °C, as seen in Figures 3A
had irregular membranes (Figure 2A and 2B, polyclonal and 3B of the same viral preparation. The preparation
antibody), probably illustrating that tegument is of for Figure 3A was thawed from -80 at 4 °C and most
variable thickness (Zhou et al. 1999). In viral prepara- particles had electron dense nucleocapsids. Upon further
tions that had been stored wet at 4 °C or thawed on ice, storage for 1 week at 4 °C, the sample for Figure 3B was
it was common to see viral particles that were partially obtained, which had few viral particles with nucleo-
naked. Where the virus was labeled for ICP-8, the DNA capsids and a large proportion of empty coats. Evidence
binding capsid protein (Figure 2C), the capsid stayed of damage to membranes during freezing was found in
intact and there were no immunogold labels on the intact preparations thawed at 4 °C (Figure 3C-E). Figure
side. Figure 2D shows a viral particle labeled for the 3C-E indicates the importance of both preparation for
VP16 protein of the tegument. This image indicates that storage and the subsequent revival of virus. Fusion of
the tegument is stretched away from the nucleus, and it viral particles at low temperatures demonstrates ice
appears that the membrane has been lost (absence of damage to viral membranes, which occurred during bulk
white rim). Figure 2E shows a rare form with a partially freezing. To investigate further, virus was prepared for
naked nucleocapsid with tegument attached to the pen- scanning electron microscopy (Figure 4), a technique that916 Biotechnol. Prog., 2005, Vol. 21, No. 3
Conclusion
This paper describes an analysis of the process of the
inactivation of HSV virus during freezing and thawing
and demonstrates the efficiency of different viral han-
dling procedures. There appears to be three mechanisms
by which viral particles are degraded. First, the integrity
of membrane glycoprotein structure can be lost, as
evidenced by the immunogold labeling experiments.
Second, viral uncoating occurs, shown clearly in a series
of EM images and the well-known loss of viral titer
(Smith 1964), although the uncoating process does not
necessarily give a lower immunogold count (Figures 2A
and 2B). A third mechanism for viral inactivation could
be via breakdown of the viral capsid; however, no HSV
DNA was detected by real-time PCR, unlike in previous
studies of infected tissue (Nicoll et al. 2001). The capsids
were found to be robust, and only a heat denaturation
with SDS detergent released DNA. These results agree
with the observation that naked capsids of HSV can be
re-encapsulated into infectious particles (Fu and Zhang
2001) and that capsids form a homogeneous layer during
ultracentrifugation (Smith 1964).
Although the viral TCID50 values were virtually identi-
cal for fresh virus and virus stored at -80 °C and revived
under different conditions, the two viral samples were
still clearly distinguishable using EM techniques. Par-
ticles that were thawed from -80 to 4 °C had more fused
particles and irregularly shaped particles than rapidly
thawed samples. The observation that 6 cycles of rapid
freezing (>20 K s-1) and rapid thaw (Table 2) led to an
unchanged viral titer makes it possible to recommend a
better handling procedure. For quantitative virology of
the same experimental batch and for biotechnology, we
recommend rapid freezing and a physiologic temperature
thaw for the reduced growth of ice crystals and the best
quality of virus, even if this should be counterintuitive
in relation to previous stability studies (Taniguchi and
Yoshino 1964).
Figure 4. Scanning electron microscopy of HSV. (A) Fresh virus
flash frozen in propane and lyophilized. Arrows indicate regular, Acknowledgment
round spheres of viral particles. (B) Rapidly thawed from
-80 °C and processed like A. The particles (arrows) were flatter Funding: BBSRC for R.K.H. J.N.S. was funded by the
and not as spherical. (C) Bulk lyophilized virus, which was re- Wellcome Trust. The British Oxygen Company sponsored
hydrated. The particles here (arrows) had lost their three- S.Z. Mr. T. Burgess, Ms. J. Powell, and Ms. L. Carter
dimensional structure. Relative viral titers were 100, 100, and
>40%, respectively. Length of bar: 1 µm. are acknowledged for excellent technical support in
relation to electron microscopy. Dr. R. Falconer is thanked
for giving critical comments.
involves freeze-drying of the virus. A sample containing
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