Robust Intrapulmonary CD8 T Cell Responses and
Protection with an Attenuated N1L Deleted Vaccinia
Anuja Mathew1*, Joel O’Bryan1, William Marshall2, Girish J. Kotwal2, Masanori Terajima1, Sharone
Green1, Alan L. Rothman1, Francis A. Ennis1
1Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America, 2Department of
Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America
Background: Vaccinia viruses have been used as a model for viral disease and as a protective live vaccine.
Methodology and Principal Findings: We investigated the immunogenicity of an attenuated strain of vaccinia virus
engineered to inactivate the N1L gene (vGK5). Using the intranasal route, this recombinant virus was 2 logs less virulent
compared to the wildtype VACV-WR. Infection by the intranasal, intraperitoneal, and tail scarification routes resulted in the
robust induction of cytolytic virus-specific CD8 T cells in the spleens and the lungs. VACV-specific antibodies were also
detected in the sera of mice infected 3–5 months prior with the attenuated vGK5 virus. Finally, mice immunized with vGK5
were significantly protected when challenged with a lethal dose of VACV-WR.
Conclusions: These results indicate that the attenuated vGK5 virus protects against subsequent infection and suggest that
the N1L protein limits the strength of the early antiviral CD8 T cell response following respiratory infection.
Citation: Mathew A, O’Bryan J, Marshall W, Kotwal GJ, Terajima M, et al. (2008) Robust Intrapulmonary CD8 T Cell Responses and Protection with an Attenuated
N1L Deleted Vaccinia Virus. PLoS ONE 3(10): e3323. doi:10.1371/journal.pone.0003323
Editor: Linqi Zhang, AIDS Research Center, Chinese Academy of Medical Sciences and Peking Union Medical College, China
Received June 23, 2008; Accepted September 11, 2008; Published October 2, 2008
Copyright: ? 2008 Mathew et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project has been funded in whole or part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of
Health, under Contract No. N01-AI-25490 and grant U19 AI057319 to FAE and R01 AI070940 and R21AI069167 to WLM.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Vaccinia virus (VACV) is a member of the Poxviridae family
and is well established as a model for the study of poxvirus biology
and disease, with the route of administration playing a major role
in outcome. VACV has also been studied as recombinant vector to
express foreign genes for human immunotherapy and as a vaccine
vector against a variety of diseases. Strains of vaccinia virus which
are in the licensed smallpox vaccines have played a central role in
the eradication of smallpox. Smallpox was eradicated in 1977, but
after September 11, 2001 there has been renewed interest in
variola virus (the etiologic agent for smallpox) and its potential use
as a bioweapon. The relatively high incidence of adverse events in
immunocompetent individuals following immunization with the
currently licensed vaccine (NYCBH virus/DryVax) , such as
myocarditis and eczema vaccinatum, has been a recent deterrent
to immunizing the general population .
Although mice are not a natural host for VACV, murine models
of VACV have been used to assess the efficacy of candidate
vaccines and define strategies for immune protection. The
Western Reserve strain of VACV (VACV-WR) is relatively more
pathogenic for mice than other strains of VACV, due to its
previous adaptation to grow in mouse brain . I.p. immunization
of mice with VACV-WR induces robust T cell responses .
Intranasal (i.n.) infection with VACV-WR simulates the spread of
smallpox virus throughout the respiratory tract with subsequent
spread to other visceral organs such as the ovaries, spleen, lungs,
and liver [5–7], but mice develop a low primary immune response.
Approaches that promote effective pulmonary T cell mediated
immunity are therefore important to evaluate T cell response in
mucosal sites where they serve as an important line of defense
following respiratory challenge with smallpox virus.
VACV encode for many proteins that modulate aspects of host
immunity [8–12]. For example the A44L protein of VACV is
immunosuppressive and affects local and systemic steroid levels
during VACV infection . The N1L protein is a known virulence
determinant of VACV [13,14] that functions by targeting
components of the IKK complex to inhibit NF-kB and IRF3
activation . The identification of N1L as an inhibitor of both
viruses deficient in the N1L gene would enhance vaccinia virus-
specific adaptive T cell responses. Recently a novel function for the
N1L proteinasanantiapoptotic molecule hasalsobeen suggested by
two groups [17,18]. Preliminary studies of the adaptive immune
response to the N1L-deficient virus were performed in balb/c mice
immunized by tail scarification and by footpad inoculation  and
prior to the identification of VACV-specific T cell epitopes.
Intracranial injection of the N1L deficient virus, vGK5 provided
protection against a subsequent challenge with VACV-WR .
While previous studies defined the attenuated VACV as significantly
PLoS ONE | www.plosone.org1October 2008 | Volume 3 | Issue 10 | e3323
less pathogenic than the mouse adapted neurovirulent VACV-WR,
the accompanying immune responses were not fully assessed .
Therefore it was not possible to determine the relationship of
pathogenicity to immunogenicity and viral replication.
We hypothesized that the attenuated N1L-deficient VACV
(vGK5) would be capable of initiating a robust T cell immune
response following intranasal infection. To understand the dynamics
of virus-specific CTL in detail, we used a tetramer against the
immunodominant B8R20–27epitope described in C57/BL6 mice to
both track and phenotype antigen-specific T cells in the lungs and
spleens of mice after infection . Our results indicate that the
recombinant attenuated vGK5 induced robust CD8+ T cell
responses when administered by the intranasal, tail scarification,
and systemic routes. Mice that were immunized with the attenuated
virus by the respiratory route were also significantly protected from a
induced a better balance between immunogenicity and virulence
than respiratory infection with the parent virus VACV-WR.
Recombinant VACV engineered to lack the N1L gene are
attenuated in C57/BL6 mice following intranasal
We first assessed the pathogenicity of the attenuated N1L
deficient vGK5 virus and the wildtype VACV-WR after intranasal
infection as this route of infection with VACV simulates smallpox
infection in humans. In agreement with published data [6,21–23],
the LD50 for VACV-WR in C57/BL6 mice was 4.26104PFU.
Mice were next monitored for weight loss and survival after
infection with varying doses of the recombinant attenuated virus,
vGK5 virus (Fig. 1A). Lines represent weight curves of individual
mice. The LD50 in age matched C57/BL6 mice was 4.26106
PFU based on the Reed and Muench method . We
determined the absolute numbers of lymphocytes recruited into
the spleens of mice infected with 106PFU VACV-WR and vGK5
by the intranasal route. We detected a significant decrease in the
total number of splenocytes in mice that were administered
VACV-WR. By day 7, these mice were moribund (Fig. 1B). In
contrast, when mice were administered 106PFU of the attenuated
vGK5 virus by the i.n. route, there was an increase in the total
number of lymphocytes in the spleens of infected mice with greater
than 50% of the CD8 T cells expressing high levels of the
activation marker CD11a (data not shown).
Increased expansion and activation of B8R20–27specific
CD8 T cells following respiratory infection with the
attenuated vGK virus
In order to more carefully dissect out the immunogenicity of the
attenuated N1L deleted VACV, we infected mice with varying
sublethal doses of vGK5 (106, 104.5, 103.5and 102.5) by the i.n.
route. To measure frequencies of antigen-specific T cells in target
Figure 1. Weight loss curves of individual mice and splenocyte counts following i.n. VACV infection of C57/BL6 mice. (A) Groups of
female C57/BL6 mice (n=5–8) were infected with 105, 106and 107PFU of vGK5 by the i.n. route. The percentage of weight relative to the initial body
weight (100%) was plotted and the data are presented as percent change in body weight following infection. ? depicts days that individual mice
were last alive. (B) Average spleen counts6standard deviation of mice were assessed by trypan blue exclusion at days 3, 5 and 7 post infection with
106VACV-WR and vGK5 by the i.n. route. Data shown are representative of 2 experiments performed and demonstrate that high dose VACV-WR i.n.
infections result in significantly (p,0.05) lower lymphocytes in the spleens during acute infection.
Immune Response to Vaccinia
PLoS ONE | www.plosone.org2October 2008 | Volume 3 | Issue 10 | e3323
organs, we obtained a tetramer directed against the immunodo-
minant epitope B8R20–27described in H-2b mice . Using the
gating strategy shown in Fig. 2A, the highest frequencies of
tetramer+ T cells were detected in spleens of mice that were infected
specific tetramer+T cells were lower in mice infected with 103.5PFU
of vGK5 and near background levels with 102.5of vGK5. Lungs of
mice infected with 106PFU of vGK5 also had the highest
frequencies (Fig. 2C) as well as total number of tetramer+ T cells
(Fig. 2D) compared to lungs of mice infected with lower doses of
virus. All of the tetramer positive cells expressed high levels of the
activation marker CD44 (data not shown). The data suggest that
administration of the attenuated vGK5 by the i.n. route induced
robust activation of CD8 T cells in mucosal and systemic sites.
Robust VACV-specific T and B cell responses to intranasal
We assessed the ability of splenocytes from mice infected
intranasally with different doses of vGK5 to secrete IFN-c in
response to the B8R20–27peptide as well as two additional VACV-
specific peptides K3L6–15and A47L138–146. Frequencies of
IFN-c secreting cells to all 3 peptides were significantly higher in
mice infected with 106PFU than lower doses of vGK5 (Fig. 3A).
Similar responses were detected in mice infected with 103.5or
104.5PFU of vGK5 to all peptides. A cardinal property of
activated CD8 T cells during acute viral infections is their ability to
lyse and eliminate virus infected cells. Splenocytes and lung
lymphocytes from mice infected by the i.n. route were therefore
tested for their ability to lyse virus infected target cells in ex vivo
CTL assays. Splenocytes from mice infected with 103.5, 104.5and
106PFU of vGK5 all effectively lysed virus infected target cells
(Fig. 3B). No CTL activity was detected in the spleens of mice
infected with 102.5PFU of vGK5 (data not shown). CTL activity
was only detected in lung lymphocytes of mice infected with 106
and 104.5PFU of vGK5 by the i.n. route (Fig. 3C). Our data thus
far indicated that T lymphocytes in the spleens and lungs of mice
immunized with vGK5 were activated, elicited cytokine responses
to VACV-specific peptides and had lytic activity against virus
We next assessed VACV-specific antibody responses in mice
immunized with varying doses of vGK5 by the i.n. route. Sera
from mice immunized five months prior with 106, 104.5and 103.5
vGK5 had 50% PRNT neutralization titers which ranged from
160–640 (Fig. 3D) which were not significantly different from
PRNT50titers in the sera of mice immunized with 103.5VACV-
WR. Our data indicate that the attenuated vGK5 elicited robust T
cell as well as antibody responses in mice immunized by the i.n.
Figure 2. Tetramer frequencies following i.n. infection with vGK5. Mice were infected with varying doses of vGK5 by the i.n. route. (A) We
used a gating strategy to identify live CD3+CD8+T lymphocytes (live dead aqua negative and forward scatter positive; CD3+CD8+). Frequencies of
B8R20–27tetramer+ T cells were assessed in the (B) splenocytes and (C) lung lymphocytes 7 days after i.n. infection. (D) The absolute numbers of
B8R20–27tetramer+ T cells in the lung following i.n. infection. Each symbol represents the frequency of tetramer+ T cells obtained in target organs of
individual mice; median values are denoted by horizontal lines.
Immune Response to Vaccinia
PLoS ONE | www.plosone.org3October 2008 | Volume 3 | Issue 10 | e3323
Systemic infection and dermal scarification with vGK5
also results in robust adaptive immunity
Overall our studies thus far showed that mice could tolerate
high doses of vGK5 by the i.n. route and these doses elicited
robust CD8 T cell responses in the lungs and spleens of acutely
infected mice. To determine whether immune responses to the
attenuated vGK5 were comparable to wildtype VACV-WR, we
administered equivalent doses of both viruses by the i.p. and tail
scarification routes (106PFU) and lower doses by the i.n. route
(103.5PFU) since mice were unable to tolerate 104or greater doses
of wildtype VACV-WR intranasally. Mice that were administered
VACV-WR or vGK5 by the i.p. or tail scarification routes did not
lose any weight and remained healthy. Seven days post infection,
10% of the CD8+ T cells in the spleens and 13–16% of CD8+ T
cells in the lungs of mice infected systemically with VACV-WR or
vGK5 were tetramer positive with similar frequencies of B8R20–27
TET+ T cells detected in mice infected by the tail scarification
route (Fig. 4A). Splenocytes from mice infected with vGK5
systemically efficiently lysed VACV-infected target cells although
VACV-WR elicited slightly higher responses at all E/T ratios
tested (Fig. 4B). To compare antibody titers in mice immunized
with wildtype or the attenuated vGK5, we collected sera from
mice immunized 3 months prior with 106PFU VACV-WR or
vGK5 by the i.p. route. Sera from mice immunized with vGK5
had vaccinia-specific antibody titers ranging from 80–1280
(Geometric Mean Titer=380) while sera from mice immunized
with VACV-WR had PRNT50titers of 640 (Fig. 4C). There were
no statistical differences between the two groups.
At day 7 post infection, 5.4 (61.7)% of CD8 T cells in the
spleens of mice infected with 103.5VACV-WR i.n. were B8R20–27
specific which was similar to the frequencies in mice given 103.5
vGK5 by the i.n. route (Fig. 2B). Frequencies of lung lymphocytes
and elispot responses were also similar in mice administered 103.5
PFU of both viruses (data not shown). Our data indicate that the
attenuated vGK5 virus is immunogenic and elicits robust immune
responses that are comparable to the wildtype VACV-WR when
administered by multiple routes.
Distribution and titers of VACV in target organs after
intranasal and systemic infection
Since the timing of antigen exposure has been shown to
influence the magnitude and quality of the CD8+ T cell response,
Figure 3. Cytokine responses and cytolytic activity in target organs. (A) IFN-c responses of splenocytes from intranasally infected mice were
measured in response to 3 VACV-specific CD8 T cell peptides (1 mg/ml) in an Elispot assay. Assays were performed using triplicate wells for each
condition and individual mice/group. *=no responses detected. Seven days post infection, (B) splenocytes and (C) lung lymphocytes were isolated
and CTL assays were carried out using RMA cells infected with VACV-WR (moi=5), vGK5 (moi=5) at different effector to target (E/T) ratios. Data
shown are 1of 2 experiments performed. (D) PRNT50 antibody titers were measured in the sera of mice immunized 5 months prior with varying doses
of vGK5 or 103.5PFU of VACV-WR (n=5/group).
Immune Response to Vaccinia
PLoS ONE | www.plosone.org4October 2008 | Volume 3 | Issue 10 | e3323
we examined viral replication in multiple organs including the
spleens, lungs and ovaries of mice infected by the i.n. and i.p.
route. 7 days post infection, viral titers were low but detectible in
the spleens of mice infected with 106, 104.5and 103.5vGK5 by the
i.n. route (Fig. 5A) and titers were several logs higher in the lungs
of these mice at the same time point (Fig. 5B). Viral titers were 3–4
logs higher in the lungs of mice administered 103.5VACV-WR
compared to lungs of mice administered 103.5vGK5 (Fig. 5B).
Equivalent viral loads were achieved in the lungs of mice infected
with 104.5PFU of vGK5 and 103.5VACV-WR 7 days post
infection. On day 5, mice that were infected via the i.p. route with
both viruses had similar titers of virus in the ovaries, lungs and
spleens (Fig. 4D). By day 7, virus was cleared from both the lungs
and spleens of mice infected by the i.p. route while titers in the
ovaries were detectible but at similar levels (data not shown).
These data indicate that the attenuated vGK5 virus replicates to
high levels in target organs. The route of inoculation influences the
level of replication between the attenuated vGK5 virus and
wildtype VACV-WR when similar doses are administered.
Mice immunized with vGK5 are protected from a lethal
respiratory challenge with VACV-WR
Finally, to determine whether vGK5 infected mice were
protected from a lethal challenge with the neurovirulent VACV-
WR, mice that were immunized with 104.5and 106PFU of vGK5
intranasally 1 month earlier were challenged with 106PFU of
VACV-WR by the i.n. route. Mice were monitored for weight loss
for 5 days and organs were isolated to measure viral load in naı ¨ve
and immunized mice. While naı ¨ve mice rapidly lost weight and
appeared moribund on d5, mice immunized with both doses of
vGK5 did not lose significant weight (Fig. 6A). Viral titers at day 5
post challenge were significantly reduced in the lungs (Fig. 6B) and
ovaries (Fig. 6C) of mice immunized with 106and 104.5of vGK5
compared to unimmunized groups.
Using a murine model, we assessed the immunogenicity of a
recombinant VACV that was engineered to lack a known virulence
Figure 4. Immune responses following infection by the tail scarification and i.p. routes. Mice were infected with 16106PFU VACV-WR or
vGK5 by the i.p. and t.s. routes. (A) Lung lymphocytes and splenocytes obtained from mice (n=4 mice/group except for infection with VACV-WR by
the t.s. route where splenocytes and lung lymphocytes from 2 mice were pooled together) infected 7 days prior were stained with B8R20–27tetramer.
The data shown represent frequencies of cells that were tetramer positive within the CD3+CD8+ gate. Each symbol represents the frequency of
tetramer+ T cells obtained in target organs of individual mice; median values are denoted by horizontal lines. (B) Seven days post infection,
splenocytes were isolated and CTL assays were carried out using RMA cells infected with VACV-WR (moi=5), vGK5 (moi=5) at different (E/T) ratios.
Data shown are representative of 2–3 experiments performed for each condition for the i.p. route. (C) PRNT50 antibody titers were measured in sera
of mice immunized 3 months prior with 106PFU of VACV-WR (n=3) or vGK5 (n=4). (D) VACV titers were determined in organs 5 days post infection
by the i.p. route and expressed as log10PFU per gram of lung and spleen tissue and PFU/ovary. – represents median values of titers in respective
organs. N.S.=Not significant. P values were determined by Student’s t test.
Immune Response to Vaccinia
PLoS ONE | www.plosone.org5 October 2008 | Volume 3 | Issue 10 | e3323
determinant of VACV, the N1L gene. Mice infected with sublethal
doses of the attenuated vGK5 virus by the i.n., tail scarification, and
i.p. routes had high frequencies of activated antigen-specific CD8 T
cells in both mucosal and systemic sites. In addition, mice infected
with vGK5 had levelsof VACV-specific antibodies 3–5 months after
immunization which were similar to levels elicited in mice
immunized with VACV-WR. Our results indicate that the
attenuated vGK5 virus, though more attenuated in vivo, is still
immunogenic and able to elicit robust T as well as B cell responses.
The lung is the major portal of entry and transmission for
variola virus, the etiologic agent of smallpox. Therefore it is critical
to assess vaccination strategies that lead to robust cell mediated
immunity in pulmonary as well as extrapulmonary tissues. VACV
inoculation by the i.n. route has been used as a model that more
closely approximates the route of infection with natural variola
(smallpox) virus infections in humans; at high doses of VACV-WR
i.n., mice develop fatal lung disease with high viral titers in lung
tissue and the brain [6,21]. Our data suggest that the N1L protein
limits the strength and magnitude of the CD8 T cell immune
response during acute intranasal VACV infections since deletion
of the N1L gene allows mice infected with the attenuated virus to
respond with a robust CD8 T cell response.
Recently two groups solved the structure of N1L which has
striking homology to the Bcl-2 family of antiapoptotic genes
[17,18]. In vitro, the N1L protein inhibits NF-kB signaling after
IL-1, TNF-a, LT-b, and TLR stimulation . Under normal
conditions, NF-kB is an antiapoptotic transcription factor and
therefore inhibition of NF-kB signaling under these conditions
could induce programmed cell death . Replication of the N1L-
deleted virus in cell culture has however been found to be
indistinguishable from a wildtype as well as a revertant virus .
We hypothesize that the Bcl-2-like structure of N1L reconciles the
observed lack of positive or negative effect on cell survival in vitro
following N1L expression, with its otherwise fatal NF-kB inhibitory
Graham et al recently confirmed that transfected N1L DNA
inhibited IL-1 and TRAF 6 signaling to NF-kB . Vaccinia
virus proteins A52 and B14 share a Bcl-2-like fold but have
evolved to inhibit NF-kappaB rather than apoptosis. N1L appears
to inhibit NF-kB dependent inflammatory cytokine production in
mice, based on the observation that N1L-deficient vaccinia virus
permits greater expression of NF-kB driven genes during in vivo
VACV infection . Furthermore, N1L also suppresses signaling
to IRF3, more robustly than it does NF-kB. IRF3 signaling was
Figure 5. Viral titers following i.n. infections. (A) Spleens and (B) lungs (n=4–8 per group) were harvested on day 7 from mice infected with
VACV-WR and vGK5 by the i.n. route. VACV titers were determined and expressed as log10PFU per gram of lung and spleen tissue. – represents
median values of titers in respective organs. N.S.=not significant. Each symbol represents the titer obtained in target organs of individual mice;
median values are denoted by horizontal lines. P values were determined by Student’s t test.
Immune Response to Vaccinia
PLoS ONE | www.plosone.org6 October 2008 | Volume 3 | Issue 10 | e3323
not investigated by Cooray et al, although Bcl-2 family members
also influence IRF3 signaling. Programmed cell death, IRF3
signaling and NF-kB signaling are three key pathways in the innate
immune response, and Bcl-2 proteins, like N1L, are capable of
inhibiting all three innate immune response pathways [27–32].
Since signaling via the innate immune system is thought to be
involved in the adaptive immune response [33,34], the N1L
protein may contribute to impaired adaptive immune responses by
inhibiting any combination of these innate signaling pathways.
In vivo, VACV-WR and vGK5 had different replication
kinetics. In our studies, when equal doses of virus were
administered after i.n. infection (103.5PFU), VACV-WR replicat-
ed to a 3–4 log higher titer compared to the vGK5 virus.
Interestingly, when equal doses were administered by the i.p. route
(106PFU), viral titers were not significantly different. Since the
N1L protein was hypothesized to have an antiapoptotic function,
increased survival of cells infected with VACV-WR which express
the N1L protein versus cells infected with vGK5 virus could
contribute to increased viral titers in the lungs after i.n. infection.
Intranasal infections with respiratory viruses result in the
recruitment of virus-specific CD8+ T cell effectors in the lung
during acute infection and persistence of these virus-specific T cells
in the respiratory tract months after the infection has resolved [35–
37]. Frequencies of antigen-specific T cells that are maintained in
memory following virus infections are likely influenced by several
factors including the amount of initial antigen available for T cell
priming, viral replication in target tissues, the route of inoculation
and the cytokine milieu. Virus titers in the lungs of mice infected
with the attenuated N1L deleted virus by the i.n. route were
several logs higher compared to lungs of mice infected with by the
i.p. route. While frequencies of antigen-specific cells in the lungs
during acute responses were not significantly impacted by these
differences in viral loads, whether frequencies of B8R20–27specific
T cells are differentially maintained in memory is still unknown.
Memory responses of these and other VACV-specific T cells
therefore need to be further evaluated in mice infected with
attenuated N1L deficient viruses.
Several factors including the initial antigen dose, the kinetics of
virus replication in mucosal and systemic sites, the innate immune
response, T cells as well as antibodies are likely to contribute to
protection. Our data show that mice immunized with attenuated
vGK5 virus by the intranasal route induced robust immunity and
subsequently was able to protect mice from a lethal challenge with
VACV-WR. The vGK5 virus is not currently a strain with
satisfactory attenuation or safety profile and further clinical
development would likely involve testing the effect of N1L
inactivation in an established vaccine strain. We propose that
the attenuated vaccinia virus lacking a major virulence gene N1L
is an alternative that balances immunogenicity and safety. Our
data have implications for the rational design of recombinant live
vaccines against foreign antigens.
Materials and Methods
RMA murine lymphoma line (H-2b) was provided by Dr.
Raymond M. Welsh, at the University of Massachusetts Medical
School, Worcester, MA.
Peptides of VACV were based on published reports .
Peptides were synthesized at AnaSpec Inc. (San Jose, CA) and the
Protein Chemistry Core Facility at the University of Massachusetts
Medical School using an automated Rainin Symphony peptide
Immunization of mice and preparation of splenocytes
and lung lymphocytes
Female C57BL/6 (4–8 weeks old) mice were purchased from the
Jackson Laboratories (Bar Harbor, ME). Mice were infected with
16106PFU VACV-WR or vGK5 by the i.p. and tail scarification
routes. For dermal scarification, 16106PFU VACV-WR or vGK5
in 50 ml PBS was placed at the base of the tail and 10–20 scratches
were made in a crosshatch pattern using a 21 gauge needle. For the
i.n. infections, mice were anesthetized with isoflurane and 50 ml PBS
containing the indicated dose of virus was instilled into the nares.
This volume allows predominant but not exclusive installation
intranasally into infected mice. Splenocytes and lung lymphocytes
were collected at the indicated time points post-immunization. To
isolate lymphocytes from the lungs of infected mice, lungs were
minced and treated with 0.14 U/mlBlendzyme (Roche Diagnostics)
and DNase I (Sigma Biochemicals) for 45 mins at 37 C. Cells were
passed through a 40 micron cell strainer, lysed with RBC lysis buffer
Figure 6. Mice immunized with vGK5 are protected from a lethal
challenge with VACV-WR. Mice were infected with 104.5, 106PFU of
vGK5(n=3/group) bythe intranasalroute.1 month later immunized mice
and age matched naı ¨ve controls were challenged with a lethal dose of
VACV-WR (106PFU) by the i.n. route. (A) Weights of mice were monitored
over 5 days. Viral titers were measured in the (B) lungs and (C) ovaries and
expressed as viral titers/gm lung tissue or ovaries. Each symbol represents
the titer obtained in target organs of individual mice.
Immune Response to Vaccinia
PLoS ONE | www.plosone.org7 October 2008 | Volume 3 | Issue 10 | e3323
(Sigma) and resuspended in RPMI 1640 medium with 10% heat-
inactivated fetal bovine serum (FBS) and 561025M 2-mercapto-
ethanol (2-ME). All mice were maintained in the Animal Facility at
the University Of Massachusetts Medical School, which is regulated
by AWA-1995, PHS-1986, MA140-1985, and following the
Ovaries, lungs and spleens of infected mice were collected at the
indicated timepoints and frozen at 280uC for virus titration. Briefly,
organs were freeze- thawed 3 times in 0.5 ml MEM/2% FBS.
0.5 ml of a 0.25% trypsin solution was added to the tubes and they
were incubated at 37uC for 30 mins. Organs were homogenized and
plates.After48 hoursmediawasremoved andcrystalvioletaddedto
the platesand plaquesenumerated.Data shownindicatethenumber
of plaque forming units (PFU) of vaccinia virus/gram of tissue in the
lungs and spleen and PFU/ovary.
51Cr release assay
RMA cells were infected with VACV-WR or vGK5 (MOI=5)
for 18–20 hrs. Uninfected or virus-infected RMA target cells were
then labeled with 0.25 mCi of51Cr for 60 min at 37uC. Following
labeling, the cells were washed three times and then resuspended
in RPMI 1640 containing 10% FBS. Effector cells (ex vivo
splenocytes or lung lymphocytes) were then added to virus-infected
RMA cells in 96-well round-bottom plates at various effector:tar-
get cell (E:T) ratios. Plates were incubated for 4 hr at 37uC,
supernatants were harvested (Skatron Instruments, Sterling, VA),
and specific lysis was calculated as [(experimental release-
lease)]6100. All assays were performed in triplicate. All experi-
ments were performed at least twice. Spontaneous lysis was less
than 15% in all assays.
Tetramer and cell surface staining
The tetramer containing the immunodominant epitope of
VACV-B8R20–27was synthesized in the NIH Tetramer Facility.
Splenocytes, lung cells and whole blood from infected and
uninfected mice were initially stained with the viability marker
LiveDead Aqua (Molecular Probes) for 30 mins. at 4uC. Cell
suspensions were then washed in FACS buffer, blocked with
CD16/CD32 mAb (Fc block 24G.2) (BD Biosciences, San Diego
CA.) for 15 minutes at 4uC, stained with the PE conjugated
B8R20–27 tetramer at room temperature for 30 mins. mAb
directed at surface phenotypic markers CD3 (clone 145-2C11),
CD8 (clone 53-6.7), CD11a (clone 2D7) and CD44 (clone 1M7)
were added for 30 minutes at 4uC. Cells were washed and all cell
preparations were fixed with Cytofix (BD Biosciences). Samples
were analyzed on a FACSARIA flowcytometer. FlowJo (TreeStar
Inc. Ashland, OR.) version 7.1 was used to analyze all the data.
Enzyme-linked immunospot (ELISPOT) assay for single-
cell IFN-c secretion
ELISPOT assays were performed according to the manufac-
turer’s protocol (Mabtech AB, Sweden) and as previously
described . Briefly, 96 well Multiscreen-IP plates (Millipore,
Bedford, MA) were coated with 15 mg/ml of rat anti-mouse IFN-c
monoclonal antibody (AN-18) over night at 4uC. Then, freshly
isolated splenocytes (2.56105/well) were incubated with the
indicated peptides (1 mg/ml), or concanavalin A (ConA) (5 mg/
ml) at 37uC for 18–20 hr in RPMI 1640 containing 10% FBS.
Biotinylated rat anti-mouse IFN-c monoclonal antibody (R4-6A2)
was added and incubated for 2 hr at room temperature, followed
by addition of streptavidin-horseradish peroxidase for 1–2 hr at
room temperature. Spots were stained with Vector NovaREDTM
Substrate kit for peroxidase (Vector Laboratories, Burlingame,
CA). The precursor frequency was calculated as [(number of spots
in experimental well - number of spots in medium control well)/
total number of cells per well]6106. Experiments were performed
in triplicate wells.
Plaque Reduction Neutralization titers
Serial two fold dilutions of heat inactivated sera from infected
mice were incubated with VACV-WR for 60 minutes at 37c.
Following the incubation, 0.25 mls of the virus/antibody mixture,
virus alone or media was added to appropriate wells of confluent
BSC-40 cells cultured in MEM. After 48 hours media was
removed and crystal violet added to the plates and plaques
enumerated. Titers were defined as the reciprocal serum dilution
that caused a 50% reduction in viral plaques (PRNT50).
Statistical significance of the data was determined by using
Student’s t test.
We thank Kim West, John Cruz and Anita Leporati for technical
assistance. We thank Pamela Pazoles and Marcia Woda for help with flow
cytometry analysis. We thank the NIH tetramer facility for providing us
with the vaccinia virus-specific B8R20–27Kb restricted MHC tetramer.
Conceived and designed the experiments: AM WSM GJK MT SG ALR
FAE. Performed the experiments: AM JO. Analyzed the data: AM JO
ALR. Contributed reagents/materials/analysis tools: WSM GJK MT SG.
Wrote the paper: AM WSM GJK ALR FAE.
1. Rosenthal SR, Merchlinsky M, Kleppinger C, Goldenthal KL (2001)
Developing new smallpox vaccines. Emerg Infect Dis 7: 920–926.
2. Fulginiti VA, Papier A, Lane JM, Neff JM, Henderson DA (2003) Smallpox
vaccination: a review, part II. Adverse events. Clin Infect Dis 37: 251–271.
3. Turner GS (1967) Respiratory infection of mice with vaccinia virus. J Gen Virol
4. Harrington LE, Most Rv R, Whitton JL, Ahmed R (2002) Recombinant
vaccinia virus-induced T-cell immunity: quantitation of the response to the virus
vector and the foreign epitope. J Virol 76: 3329–3337.
5. Smee DF, Bailey KW, Sidwell RW (2001) Treatment of lethal vaccinia virus
respiratory infections in mice with cidofovir. Antivir Chem Chemother 12: 71–76.
6. Williamson JD, Reith RW, Jeffrey LJ, Arrand JR, Mackett M (1990) Biological
characterization of recombinant vaccinia viruses in mice infected by the
respiratory route. J Gen Virol 71(Pt 11): 2761–2767.
7. Andrew ME, Coupar BE, Boyle DB (1989) Humoral and cell-mediated immune
responses to recombinant vaccinia viruses in mice. Immunol Cell Biol 67(Pt 5):
8. Haga IR, Bowie AG (2005) Evasion of innate immunity by vaccinia virus.
Parasitology 130 Suppl: S11–25.
9. Gurt I, Abdalrhman I, Katz E (2006) Pathogenicity and immunogenicity in mice
of vaccinia viruses mutated in the viral envelope proteins A33R and B5R.
Antiviral Res 69: 158–164.
10. Reading PC, Smith GL (2003) Vaccinia virus interleukin-18-binding protein
promotes virulence by reducing gamma interferon production and natural killer
and T-cell activity. J Virol 77: 9960–9968.
11. Reading PC, Moore JB, Smith GL (2003) Steroid hormone synthesis by vaccinia
virus suppresses the inflammatory response to infection. J Exp Med 197:
Immune Response to Vaccinia
PLoS ONE | www.plosone.org8 October 2008 | Volume 3 | Issue 10 | e3323
12. Clark RH, Kenyon JC, Bartlett NW, Tscharke DC, Smith GL (2006) Deletion
of gene A41L enhances vaccinia virus immunogenicity and vaccine efficacy.
J Gen Virol 87: 29–38.
13. Bartlett N, Symons JA, Tscharke DC, Smith GL (2002) The vaccinia virus N1L
protein is an intracellular homodimer that promotes virulence. J Gen Virol 83:
14. Kotwal GJ, Hugin AW, Moss B (1989) Mapping and insertional mutagenesis of
a vaccinia virus gene encoding a 13,800-Da secreted protein. Virology 171:
15. DiPerna G, Stack J, Bowie AG, Boyd A, Kotwal G, et al. (2004) Poxvirus protein
N1L targets the I-kappaB kinase complex, inhibits signaling to NF-kappaB by
the tumor necrosis factor superfamily of receptors, and inhibits NF-kappaB and
IRF3 signaling by toll-like receptors. J Biol Chem 279: 36570–36578.
16. Zhang Z, Abrahams MR, Hunt LA, Suttles J, Marshall W, et al. (2005) The
Vaccinia Virus N1L Protein Influences Cytokine Secretion in Vitro after
Infection. Ann N Y Acad Sci 1056: 69–86.
17. Aoyagi M, Zhai D, Jin C, Aleshin AE, Stec B, et al. (2007) Vaccinia virus N1L
protein resembles a B cell lymphoma-2 (Bcl-2) family protein. Protein Sci 16:
18. Cooray S, Bahar MW, Abrescia NG, McVey CE, Bartlett NW, et al. (2007)
Functional and structural studies of the vaccinia virus virulence factor N1 reveal
a Bcl-2-like anti-apoptotic protein. J Gen Virol 88: 1656–1666.
19. Billings B, Smith SA, Zhang Z, Lahiri DK, Kotwal GJ (2004) Lack of N1L gene
expression results in a significant decrease of vaccinia virus replication in mouse
brain. Ann N Y Acad Sci 1030: 297–302.
20. Tscharke DC, Karupiah G, Zhou J, Palmore T, Irvine KR, et al. (2005)
Identification of poxvirus CD8+ T cell determinants to enable rational design
and characterization of smallpox vaccines. J Exp Med 201: 95–104.
21. Smee DF, Bailey KW, Wong MH, Sidwell RW (2001) Effects of cidofovir on the
pathogenesis of a lethal vaccinia virus respiratory infection in mice. Antiviral Res
22. Phelps A, Gates AJ, Hillier M, Eastaugh L, Ulaeto DO (2005) Comparative
efficacy of replicating smallpox vaccine strains in a murine challenge model.
Vaccine 23: 3500–3507.
23. Hayasaka D, Ennis FA, Terajima M (2007) Pathogeneses of respiratory
infections with virulent and attenuated vaccinia viruses. Virol J 4: 22.
24. Reed LaM H (1938) A simple method of estimating 50% end points. American
Journal of Hygiene 27: 493–497.
25. Karin M, Lin A (2002) NF-kappaB at the crossroads of life and death. Nat
Immunol 3: 221–227.
26. Graham SC, Bahar MW, Cooray S, Chen RA, Whalen DM, et al. (2008)
Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to
inhibit NF-kappaB rather than apoptosis. PLoS Pathog 4: e1000128.
27. Adams JM, Cory S (1998) The Bcl-2 protein family: arbiters of cell survival.
Science 281: 1322–1326.
28. Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, et al. (1993)
bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic
cell death. Cell 74: 597–608.
29. de Moissac D, Zheng H, Kirshenbaum LA (1999) Linkage of the BH4 domain of
Bcl-2 and the nuclear factor kappaB signaling pathway for suppression of
apoptosis. J Biol Chem 274: 29505–29509.
30. Hiscott J (2007) Convergence of the NF-kappaB and IRF pathways in the
regulation of the innate antiviral response. Cytokine Growth Factor Rev 18:
31. Parker LC, Whyte MK, Dower SK, Sabroe I (2005) The expression and roles of
Toll-like receptors in the biology of the human neutrophil. J Leukoc Biol 77:
32. Reed JC, Tsujimoto Y, Alpers JD, Croce CM, Nowell PC (1987) Regulation of
bcl-2 proto-oncogene expression during normal human lymphocyte prolifera-
tion. Science 236: 1295–1299.
33. Hoffmann JA, Kafatos FC, Janeway CA, Ezekowitz RA (1999) Phylogenetic
perspectives in innate immunity. Science 284: 1313–1318.
34. Medzhitov R, Janeway CA Jr (1998) Innate immune recognition and control of
adaptive immune responses. Semin Immunol 10: 351–353.
35. Woodland DL, Hogan RJ, Zhong W (2001) Cellular immunity and memory to
respiratory virus infections. Immunol Res 24: 53–67.
36. Wiley JA, Hogan RJ, Woodland DL, Harmsen AG (2001) Antigen-specific
CD8(+) T cells persist in the upper respiratory tract following influenza virus
infection. J Immunol 167: 3293–3299.
37. Lawrence CW, Ream RM, Braciale TJ (2005) Frequency, specificity, and sites of
expansion of CD8+ T cells during primary pulmonary influenza virus infection.
J Immunol 174: 5332–5340.
38. Mathew A, Terajima M, West K, Green S, Rothman AL, et al. (2005)
Identification of murine poxvirus-specific CD8+ CTL epitopes with distinct
functional profiles. J Immunol 174: 2212–2219.
Immune Response to Vaccinia
PLoS ONE | www.plosone.org9 October 2008 | Volume 3 | Issue 10 | e3323