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Http://www.abbs.info e-mail:[email protected] ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2001, 33(5): 497-503 CN 31-1300/Q |
High
Frequency of Homologous Recombination in the Genome of Modified Vaccinia Virus
Ankara Strain (MVA)
( Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences,
MVA
was generated from vaccinia Ankara strain CVA by hundreds of passages in CEF
cells. During the process, multiple deletions of more than 30 kb DNA including
at least two viral host range genes occurred[6], which turned this VV
vector extremely safe for research and clinical use[6,7]. In
addition, the comparatively smaller genome may enhance the capacity of MVA to
incorporate large foreign sequences.
Live
vaccines with MVA as the vector were able to efficiently induce humoral[8–10]
and cellular immune responses[11–13] against antigens expressed by
the recombinant MVAs. To ensure further safety and more efficient selection of
recombinant MVA, Staib et al[14] recently developed a
transient selection marker system which introduced a selection marker flanked
by two identical sequences into MVA genome. In this system, the k1l gene
that has been reported to play a critical role in VV host range selection[15]
was used as the selection marker. The exact mechanism of its function remained
unclear. Therefore, it is necessary to limit the use of k1l gene to
selection of rMVA only and discard this marker from the final recombinant MVA.
In this transient selection system, k1l gene will automatically
disappear through homologous recombination if selection pressure is released.
The homologous recombination between these identical sequences can be easily
monitored by PCR. In this study, four recombinant MVAs based on this system
were generated and the homologous recombination of recombinant MVAs was
studied. We report here the detailed analysis and calculation of the frequency
of the homologous recombination. Our data will improve the current
understanding on the safety of the recombinant MVA system using transient
selection method, on the stability of MVA genome, and guide future generation
of recombinant MVAs.
1.1 Plasmids,
viruses and cells
Vector
pIIIdHR-P7.5 which contains the selection marker k1l and wild type
MVA(wtMVA) used in this study (provided by Dr. Sutter G, National research
center for environment and health, Germany) were described previously[14].
Baby hamster kidney BHK-21 (ATCC CCL-10) and rabbit kidney RK-13 (ATCC CCL-37)
cells were grown in RPMI1640 supplemented with 10% FCS. Cells were maintained
in a humidified incubator at 37 ℃
and 5% CO2.
1.2
Generation of recombinant MVAs
Structures
of MVA and recombination events in generation of recombinant MVAs were depicted
in Fig.1. Recombinant viruses were constructed mainly according to the
procedure described previously[14]. Briefly, foreign gene fragments
encoding a polyprotein of different lengths (2.60 kb, 2.22 kb, 1.98 kb and 1.53
kb, which will be described in detail elsewhere) were inserted into the MCS of
pIIIdHR-P7.5. Respectively, the resulting plasmids pIIIdHR-P7.5A,
pIIIdHR-P7.5B, pIIIdHR-P7.5C and pIIIdHR-P7.5D were then used to transfect
BHK-21 cells that were pre-infected with wtMVA. Cells were harvested two days
post-transfection and passaged in RK-13 cells that did not support the
replication of wtMVA. Only recombinant MVAs derived from homologous
recombination between wtMVA and transfected plasmid containing the selection
marker k1l (MVA-k1lFS) were recovered. The second homologous
recombination aiming to delete the k1l gene from the MVA genome was
allowed to happen in BHK-21 cells, where growth pressure was released. To
facilitate the generation of MVA-FS, we introduced blind passages in the
procedure. After several blind passages in BHK-21 cells, each recombinant MVA
was plaque purified and MVA-FS was isolated after one plaque-picking passage.
During each passage, viral genomic DNA from several plaques was extracted for
PCR analysis and cell lysates tested by western blot for the expression of
foreign gene products.
Fig.1 Recombination in generation of
recombinant virus MVA-k1lFS and MVA-FS
Schematic maps of the MVA genome (HindIII
restriction map) is shown at the top of the figure. MVA-k1lFS is the recombinant
MVA containing selection marker k1l gene. Flank1 and Flank2 refer to the
MVA-DNA sequences that are essential to target insertion of foreign sequences
(FS) to the site of deletion III within the MVA genome. rec2 indicates the
position of a 283 bp repetitive MVA-DNA fragment homologous to the right end of
Flank1, which allows deletion of the k1l expression cassette by
homologous recombination. MVA-FS is the recombinant MVA in which the k1l
gene has been deleted through further recombination. The annealing sites for
the primers (MVA-III-5' and MVA-III-3') used in PCR analysis are indicated on
the genome of MVA-k1lFS.
BHK-21
cells were infected with recombinant MVA or wtMVA with an MOI of 10 IU per
cell. Infected cells were harvested 48 hours post infection, washed twice with
PBS and directly lysed with SDS-PAGE loading buffer. After electrophoresis, the
separated proteins were transferred onto nitrocellulose membrane (Schleicher
& Schuell). The membrane was then blocked in 5% fat-free milk powder
prepared in PBST (PBS with 0.5% Tween 20) before probing with rabbit anti-serum
raised against the C region of the polyprotein. After that, the membrane was
incubated with HRP-conjugated goad-anti-rabbit IgG(Dianova, Germany) and developed
with Lumi-light Western blotting substrate(Roche).
1.4 Monitoring
recombination by PCR
The
recovered MVA-k1lFS (MVA-k1lA, MVA-k1lB, MVA-k1lC
and MVA-k1lD) were blindly passaged in BHK-21 cells for 3 or 4 times. To
reduce intermolecular recombination, MOI lower than 0.01 IU per cell was used.
For each passage, cells were harvested in 1 ml of PBS three days
post-infection, thoroughly homogenized, diluted, and used to re-infect BHK-21
cells for the next passage.
To
determine the recombination frequency of rMVA, cells were thoroughly
homogenized by the end of the final blind passage. Cell lysates were diluted
and plated on BHK-21 cell monolayers at 10–5 MOI per cell. Two days
later, several plaques were picked and amplified. Recombinant MVA DNA were prepared[16]
and analyzed by PCR.
The
primer pair used in this study (MVA-III-5' and MVA-III-3', see Fig.1) was
described previously[14]. They anneal to the flanking regions of
deletion III in MVA genome. With wtMVA, k1l+ rMVA and k1l-
rMVA as template, PCR with this primer pair generates DNA fragments of 0.76 kb,
2.28 kb＋FS
(with k1l) and 0.98 kb＋FS
(without k1l), respectively. PCR reactions were performed with PCR
Master kit (Roche) with additional DMSO added to each tube (final
concentration: 4%). After denaturation at 92 ℃
for 2 min, 30 cycles of amplification were carried out as following: 92 ℃,
30 s; 55 ℃,
40 s; 68 ℃,
4 min. The procedure was ended with an additional elongation of 8 min at 68 ℃.
1.5 Calculation
of recombinant frequency
Assuming
that the recombination frequency during each passage was a constant, we could
calculate the recombination frequency as following.
Let
r denote the recombination frequency during each passage. After one
passage in BHK-21 cells, we have:
Ratio
of recombinants: r. Ratio of non-recombinants: 1–r.
During
the second passage in BHK-21 cells, recombination continued with those
non-recombinants. Therefore, after two passages, we have:
Ratio
of recombinants: r+(1–r)r = 2r–r2 =
1–(1–r)2.
Ratio
of non-recombinants: (1–r)2.
Similarly,
after three passages in BHK-21 cells, we have:
Ratio
of recombinants: 2r–r2+(1-r)2r =
2r–r2+r3–2r2+r
= r3–3r2＋3r
= 1–(1–r)3.
Ratio
of non-recombinants: (1–r)3.
In
general, after n blind passages in BHK-21 cells, we have:
Ratio
of recombinants: 1–(1–r)n.
Ratio
of non-recombinants: (1–r)n.
After
blind passages, the final recombination frequency (R) could be
determined by PCR analysis.
So
from R＝1–(1–r)n,
we can calculate the recombination frequency during each passage.
2.1 Construction
of rMVA carrying k1l selection marker flanked by two identical sequences
Recombinant
MVA carrying two identical sequences (Fig.1) is a convenient system for study
of homologous recombination. For this purpose, four MVA-k1lFS were
constructed by homologous recombination between wtMVA and plasmids that
contained foreign sequence (FS) and k1l selection marker flanked by two
identical rec2 sequences (see materials and methods). Only recombinant MVA
carrying thek1l selection marker could replicate and propagate in RK-13
cells while k1l- MVA could not. Recombinant MVAs from
positive plaques were picked, diluted and used to re-infect RK-13 cells at a
low MOI per cell. After several passages in RK-13 cells, pure recombinant MVAs
carrying k1l selection marker flanked by two identical sequences of 283
bp were recovered.
Recovered
four MVA-k1lFS were subsequently characterized by PCR and western blot
analysis. PCR with primers specific for FS demonstrated that all rMVAs carried
foreign sequences. No wtMVA signal (0.76 kb) was observed in PCR with
MVA-III-5'/MVA-III-3' primer pair (data not shown). The result indicated that
recovered viruses were recombinant and free of wild type MVA. Western blot
analysis with antibodies specific for FS product confirmed that all rMVAs were
able to express foreign inserts (Fig.2). These rMVAs are then subjected to the
recombination study.
Fig.2 Western blot analysis of the
expression products of all MVA-k1lFS
The
specific protein bands reacted with the anti-serum against the C region of
polyprotein were detected for each virus confirming the correct generation of
all MVA-k1lFS.
When
directly plating the above-mentioned rMVAs on BHK-21 cell monolayers which do
not exert any selection pressure, incubating for two days and analyzing
isolated viral plaques by PCR, we always observed two bands instead of only a k1l-
single band (Fig.3). The lack of a 0.76 kb band indicated that the isolates
were free of wtMVA. As to the PCR products from recombinant MVA templates, the
upper band was amplified from the k1l gene containing MVA genome, while
the lower band was from the k1l free recombinant MVA genome, which could
be generated during one passage of MVA-k1lFS on BHK-21 cells. As we and
others in our lab never observed pure k1l free MVA isolate from hundreds
of MVA-k1lFS isolates, we drew the conclusion that all original isolates
were k1l positive.
Fig.3 PCR analysis showing the
difficulty in isolation of k1l free recombinant MVA
Wild type free MVA-k1lD was directly
plated on BHK-21 cell monolayer. Six plaques were isolated and analyzed by PCR
method with primer pair MVA-III-5′/MVA-III-3′.
For all the samples of recombinant MVA, two bands were detected, the upper band
was amplified from the MVA genome containing k1l gene, while the lower
one from k1l free MVA genome. No pure k1l free virus represented
by a single lower band was detected. Analysis of other plaques gave similar
results. M, 1 kb ladder.
Fig.4 Analysis of the recombination
frequency after blind passages
(A) Four MVA-k1lFS were subjected
to 3 or 4 times of blind passages, when the k1l free recombinant MVAs
were accumulating. P1, P2, P3, P4 indicate the number of blind passages carried
on BHK-21 cells. (B) The recombinant MVA from final blind passages were then
subjected to plaque-picking passage on BHK-21 cell monolayer. S1-S8
indicate the isolated plaques (samples) analyzed by PCR. Pure k1l free
viruses represented by a single lower band were detected for each recombinant
virus. All PCR were performed with primer pair MVA-III-5'/MVA-III-3'. M, 1 kb
ladder.
From
Fig.4, recombinants were counted and the final recombination frequency was
calculated (Table 1).
The
recombination frequencies during one blind passage were also estimated as
described in Materials and Methods. An average of 0.136 with a standard
deviation of 0.056 was obtained. It indicated that MVA genomes carrying two identical
sequences underwent efficient homologous recombination when the growth pressure
was released.
Homologous
recombination of VV was initially observed in cells co-infected with two distinct
VV strains. The studies of recombination in VV system with Southern blot
demonstrated a high frequency of homologous recombination[17].
Furthermore, the recombination frequency correlated linearly to the length of
homologous sequence[18].
In
this study, we constructed four MVA-FS based on the transient selection marker
system developed recently and carried out a detailed analysis on the events of
homologous recombination during the generation of these recombinant MVAs. We
observed that the recombination of MVA genome is significant, though not as
frequent as that of VV studied previously which reach as high as 40%[17].
After several blind passages on BHK-21 cell monolayer without selection
pressure, we calculated the recombination frequency per passage to be within 8%-20%.
This suggests that rMVA without the k1l selection marker can be easily
selected after three or four passages in BHK-21 cells. Therefore, our data
presents important evidence in supporting the conclusion that recombinant MVA
with the transient selection marker system is both safe and convenient, which
may turn out to be a valuable live vaccine and gene therapy vector for human.
The standard deviation of the calculated recombination frequencies was
comparably high, which was understandable, as the quantity of analyzed samples
was limited. For a precise measurement of the recombination frequency, more
samples will be required.
The
homologous recombination of vaccinia virus can be both intramolecular and
intermolecular. Southern blot analysis showed that intermolecular recombination
was much less frequent than intramolecular recombination[17],
possibly because two crossovers are needed for intermolecular recombination,
while intramolecular recombination only involves one crossover. To identify
rMVAs generated from intermolecular recombination would give a more accurate
estimation of the recombination frequency, which should be close to the present
result.
To
avoid multiple infection of one cell in analyzing the recombination frequency, our
approach was to homogenize the virus preparation as much as possible and to use
an extremely low MOI of MVA to infect cell monolayers. Comparing with Southern
blot analysis, PCR method was much more convenient and sensitive in analyzing
the structure of MVA genome, which had also been demonstrated in the procedure
of generating rMVAs[14]. However, the data from PCR analysis on each
plaque (Fig.3) was not quantitative. We noticed that the lower band signal that
represented the k1l- rMVA in the last blind passages
[Fig.4(A)] was stronger than that of the upper band for the k1l+
rMVA. This did not mean that there were more k1l- rMVAs than k1l+
rMVAs, since the recombination frequency was estimated within 8%-20%
per passage. It was likely that k1l- signal was much
exaggerated by PCR due to smaller fragment (1.3 kb smaller than k1l+)
being preferentially amplified.
The
capability to induce protective immune responses and safety of MVA made it a
promising vector in vaccine development. Recently, recombinant MVA-HIV was used
as the main component of an HIV vaccine in a study on macaques model[19].
Animals inoculated with such a vaccine were reported to produce high T-cell
immune responses that contained viruses after SHIV challenge. This breakthrough
in HIV vaccine development made rMVA more attractive in fighting viral diseases
and cancers. Therefore, the improvement on the technique to generate
recombinant MVAs, namely, transient selection marker system, will be more
widely used. We demonstrated that 3 or 4 blind passages would facilitate the
selection of k1l- rMVAs. Since blind passage translates to
much less work and more cost effective means than plaque-picking passage, our
data reported here would help to guide future generation of rMVAs based on the
transient selection marker system.
Acknowledgements The authors would like to thank Dr.
Sutter G and Dr. Staib C for their kind help in MVA manipulation.
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Received: April 13, 2001 Accepted: May 29, 2001
This work was supported by a grant from
the State 863 High Technology R&D Project of China (No.863-102-07-02-02)
and the Project CHN 98/112 (WTZ-Internationales Büro
des BMBF, Germany)
*Corresponding authors: Tel,
86-21-64374430; Fax, 86-21-64338357; e-mail, [email protected]