Memory CD8 T cell responses exceeding a large
but definable threshold provide long-term
immunity to malaria
Nathan W. Schmidt*, Rebecca L. Podyminogin*, Noah S. Butler*, Vladimir P. Badovinac*, Brad J. Tucker†,
Keith S. Bahjat‡§, Peter Lauer‡, Arturo Reyes-Sandoval¶, Claire L. Hutchings¶, Anne C. Moore¶, Sarah C. Gilbert¶,
Adrian V. Hill¶, Lyric C. Bartholomay†, and John T. Harty*?**
*Department of Microbiology and?Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, IA 52242;†Department of
Entomology, Iowa State University, Ames, IA 50011;‡ANZA Therapeutics, Inc., Concord, CA 94520; and¶The Jenner Institute, University of Oxford,
Oxford OX3 7DQ, United Kingdom
Edited by Michael J. Bevan, University of Washington, Seattle, WA, and approved July 14, 2008 (received for review June 6, 2008)
Infection of mice with sporozoites of Plasmodium berghei or Plasmo-
dium yoelii has been used extensively to evaluate liver-stage protec-
tion by candidate preerythrocytic malaria vaccines. Unfortunately,
effective malaria vaccines in humans. Thus, mice may be used better
as models to dissect basic parameters required for immunity to
Plasmodium-infection than as preclinical vaccine models. In turn, this
basic information may aid in the rational design of malaria vaccines.
Here, we describe a model of circumsporozoite-specific memory CD8
T cell generation that protects mice against multiple P. berghei
sporozoite challenges for at least 19 months. Using this model we
predicts long-term sterilizing immunity against liver-stage infection.
Importantly, the number of Plasmodium-specific memory CD8 T cells
required for immunity greatly exceeds the number required for
resistance to other pathogens. In addition, this model allowed us to
identify readily individual immunized mice that exceed or fall below
the protective threshold before infection, information that should
greatly facilitate studies to dissect basic mechanisms of protective
CD8 T cell memory against liver-stage Plasmodium infection. Further-
required for long-term protection in mice may have important impli-
cations for development of effective malaria vaccines.
the developing world (1), effects that have stimulated intense
efforts to develop efficacious vaccines. Protective CD8 T cell
immunity against liver-stage Plasmodium infection has been
demonstrated after vaccination of rodents with irradiated or
genetically attenuated parasites and after subunit vaccination
against liver-stage antigens (2–12). Immunity in rodents can last
for 6–12 months (3, 4, 7, 13), but in several studies also seems to
wane with time (7, 14–16). Although irradiated sporozoite
vaccines also protect humans (17–19), current subunit vaccina-
tions limit liver-stage infection but rarely prevent blood-stage
parasitemia (20). Importantly, it remains unknown whether
sterilizing long-term immunity to Plasmodium infection can be
achieved through subunit vaccines that predominantly evoke
memory CD8 T cell responses and, if so, precisely what memory
T cell parameters will be required.
A single mosquito bite delivers a few hundred infectious Plas-
modium sporozoites into dermal tissues (21), a fraction of which
traffic to the liver and establish hepatocyte infection leading to
release of blood stage parasites 2 days (P. berghei infection of mice)
(22) or 6–8 days (P. falciparum infection of humans) (23) later. As
such, infected cells may represent as few as 1 in 109hepatocytes in
humans and 1 in 106hepatocytes in mice. Thus, both temporal and
spatial challenges (analogous to rapidly finding a few needles in a
T cells to deal with all infected hepatocytes and prevent the
nfection of humans with Plasmodium species, the causative
agents of malaria, results in severe morbidity and mortality in
symptomatic blood stage of infection. The use of mouse models of
Plasmodium infection to determine how the immune system can be
manipulated by immunization to overcome these challenges may
have important implications for rational design of malaria vaccines.
Filling this knowledge gap will require immunization models to
reliably generate memory CD8 T cells that confer long-term
protection can be defined. Here, we describe an immunization
strategy that generates P. berghei circumsporozoite (CS)-specific
memory CD8 T cells capable of protecting mice from multiple
revealed that the threshold in memory CD8 T cell numbers
potentially important implications for development of effective
vaccines to protect against human malaria.
Generation of CS-Specific Memory CD8 T Cells. Protective immunity
to infection may be influenced by both the functional attributes
and numbers of memory CD8 T cells (24–26). We reasoned that
the extremely low frequencies of infected hepatocytes might
dictate that a large number of memory CD8 T cells would be
required to ensure all infected liver cells are located and dealt
with to prevent blood stage infection. To test this hypothesis, we
made use of an accelerated ‘‘prime-boost’’ immunization strat-
egy, developed in our laboratory, that rapidly generates large
numbers of memory CD8 T cells (27). BALB/c mice initially
were immunized with mature dendritic cells (DC) coated with a
P. berghei epitope (CS252–260, also known as ‘‘Pb9,’’ DC-CS
immunization) that is a target of protective CD8 T cells (8). As
we reported for other epitopes (27), DC-CS immunization
resulted in accelerated acquisition of memory characteristics
(CD127hi, KLRG-1lo, IL-2?) by the responding CD8 T cells (Fig.
1A), including the ability to respond to booster immunization
with recombinant attenuated (actA-, inlB-deficient) (28) L.
Author contributions,: N.W.S., A.C.M., S.C.G., A.V.H., L.C.B., and J.T.H. designed research;
P.L. contributed new reagents/analytic tools; N.W.S. and J.T.H. analyzed data; and N.W.S.,
L.C.B., and J.T.H. wrote the paper.
Conflict of interest statement: P.L. is an employee of ANZA Therapeutics, Inc, which owns
intellectual property covering the compositions and methods described in this manuscript.
In addition, ANZA employees hold stock and/or stock options in the company. The remain-
ing authors disclose no known financial conflicts.
This article is a PNAS Direct Submission.
§Present address: Medarex, Inc., Milpitas, CA 95035.
**To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
September 16, 2008 ?
vol. 105 ?
no. 37 ?
monocytogenes expressing the CS252–260 epitope (here on re-
ferred to as ‘‘LM-CS252’’) that is embedded within a secreted
ovalbumin fusion protein and does not contain known antibody
or CD4 T cell epitopes. This DC-CS ? LM-CS immunization
generated large frequencies (Fig. 1B) and total numbers (Fig.
1C) of effector and memory CS252-specific CD8 T cells that were
?10-fold greater than generated by DC-CS or LM-CS252immu-
nization alone. Splenic memory CD8 T cell frequencies and total
numbers (Fig. 1B and C) in all groups were maintained stably
between day 41 and day 96 and, of critical importance for
resistance to liver-stage Plasmodium infection, numbers of
CS252-specific CD8 T cells in the liver and spleen were propor-
tional at day 54 (Fig. 1D and E) and at day 72 (data not shown).
Additionally, the frequency of CS252-specific CD8 T cells of all
peripheral blood lymphocytes (PBLs) was proportional to the
Thus, DC-CS ? LM-CS immunization rapidly generated large
and stable populations of P. berghei-specific memory CD8 T cells
in the spleen, PBL, and liver.
It should be noted that this immunizations strategy, based on
a short peptide epitope prime and epitope-fusion protein boost,
does not induce a detectable CD8 negative (i.e., CD4 T cell)
IFN-? response (data not shown). Also, antibodies induced by
this vaccination would be directed either to the CS252-MHC class
I complex (after DC-CS252immunization) or to the short CS
epitope embedded in the ovalbumin carrier protein (LM-CS252
boost). If such antibodies are induced, they are unlikely to react
in a meaningful way with the conformationally intact CS protein
expressed by P. berghei sporozoites. Thus, our immunization
strategy permits a focus on the ability of CS252-specific CD8 T
cells to provide immunity against sporozoite challenge.
DC-CS ? LM-CS CD8 T Cells Prevent Blood-Stage Parasitemia. To
evaluate individual-to-individual variability and to mimic sam-
pling of humans, frequencies of memory CS252-specific CD8 T
cells of all PBL were determined at day 98 in individual mice.
DC-CS- and LM-CS252-immunized mice had ?0.2% CS252-
specific PBL, whereas DC-CS ? LM-CS mice exhibited ?2%
CS252-specific PBL (Fig. 2A). This frequency represented a
substantial fraction (?21%) of circulating CD8 T cells in the
DC-CS ? LM-CS mice (data not shown). All naı ¨ve and LM-
CS252-immune and 11 of 12 DC-CS-immune mice developed
blood stage parasitemia after sporozoite challenge (Fig. 2B). In
contrast, 10/11 DC-CS ? LM-CS mice were protected, with
blood-stage parasitemia observed only in the mouse that had the
lowest frequency of CS252-specific PBL (?0.5%). These data
suggest that immunity to liver-stage parasites may depend on the
numbers of antigen-specific memory CD8 T cells.
CS-Specific Memory CD8 T Cells Afford Long-Term Sterile Immunity.
Although many vaccination strategies are successful in protect-
ing rodents from sporozoite challenge for several months (29),
the feasibility of long-term immunity based solely on memory
CD8 T cells remains unknown. A representative analysis of
DC-CS ? LM-CS mice revealed ?106CS252-specific CD8 T cells
per spleen on day 209 (Fig. 3A), a number similar to that
observed at day 41 (Fig. 1). One hundred percent of additional
mice from this group were protected from sporozoite challenge
on day 210, indicating that CD8 T cells in DC-CS ? LM-CS mice
can protect for at least 7 months (Fig. 3B). Immunity in
malaria-endemic areas must protect the host from repeated
sporozoite exposures. To address this issue and to evaluate
further the duration of memory and protection, CS252-specific
(A) Frequency and phenotype of splenic CD8 T cells that are CS252specific as determined by ICS 8 days after DC-CS prime. (B) Frequency of splenic CD8 T cells that
are CS252specific on days 14, 41, and 96 as determined by ICS. Profiles from representative mice are shown; numbers represent mean ? SD; n ? 3 per group. (C)
Total number (mean ? SD, n ? 3 per group) of CS252-specific CD8 T cells in the spleen on various days after initiation of immunization. Total number (mean ?
SD, n ? 3 per group) of CS252-specific CD8 T cells (D) in the spleen and (E) liver or (F) percent CS252-specific CD8 T cells of all PBL 54 days after initiation of
immunization for the indicated groups. Data are representative of three experiments.
Generation and maintenance of P. berghei CS252-specific CD8 T cells. BALB/c mice were primed with 3 ? 105bone marrow-derived dendritic cells coated
www.pnas.org?cgi?doi?10.1073?pnas.0805452105 Schmidt et al.
PBL were determined at day 406 in the 5 mice that had resisted
challenge at day 210 and in an additional group of unchallenged
DC-CS ? LM-CS mice (Fig. 3C). Both groups exhibited ?2%
CS252-specific PBL and were protected from sporozoite chal-
lenge at day 422 (Fig. 3C). Both groups of mice maintained high
and all resisted additional sporozoite challenges at day 455, day
485, and day 565 (19 months after immunization, data not
shown). Interestingly, we did not observe ‘‘boosting’’ in memory
cell frequencies in these groups despite the repeated challenges
(data not shown). This may result from the very large memory
populations already present in the DC-CS ? LM-CS immune
mice or suggest that the amount of antigen present in the
challenges was insufficient to cause noticeable boosting in
memory numbers. However, it is possible that the repeated
challenge infections induced antibody and CD4 T cell responses
that contributed to resistance of multiply challenged mice. These
issues are under investigation. Over multiple experiments, 136/
141(?96%) of DC-CS ? LM-CS mice were protected from
initial sporozoite challenge from day 28 to day 422 after immu-
nization. Thus, CD8 T cell protection can last for ?14 months,
and immunity is maintained up to 19 months in the face of
multiple sporozoite challenges. Importantly, the ability to gen-
erate large numbers of CS252-specific memory CD8 T cells and
sterilizing immunity to sporozoite challenge was not limited to
DC-CS ? LM-CS immunization but also could be achieved by
boosting LM-CS252-immune mice at ?40 days with a higher dose
of LM-CS252 (LM-CS ? LM-CS data in Fig. 4). These data
demonstrate that single-epitope specific-memory CD8 T cells
can provide long-term sterilizing immunity against Plasmodium
Defining the Threshold Frequency of Memory CD8 T Cells Required for
Sterilizing Immunity. The reliable long-term protection afforded
by DC-CS ? LM-CS immunization suggests that this model may
be well suited to dissect the basic parameters of CD8 T cell
immunity to liver-stage Plasmodium infection. Initially, we
sought to determine whether a threshold frequency of protective
memory CD8 T cells could be defined with this immunization
model. BALB/c mice were primed with DC-CS and then boosted
with a range of LM-CS252(from 2 ? 104-2 ? 107) to stimulate
different magnitudes of CD8 T cell memory. Because DC-CS,
LM-CS252, and DC-CS ? LM-CS immunization stimulated
memory CD8 T cells that exhibited some differences in pheno-
type (for example, CD27 and CD62L expression and IL-2
production) [supporting information (SI) Fig. S1], we investi-
gated whether boosting the mice with different doses of LM-
CS252altered the phenotype of the CS252-specific CD8 T cells.
Importantly, the surface expression of certain memory markers
(CD27, CD62L, and CD127) was similar in all groups, as was the
against a P. berghei sporozoite challenge. (A) Frequency of CS252-specific CD8
T cells in the PBL 98 days after priming. Data (mean ? SD) are from 10 to 12
mice per group. (B) Percentage total PBL that are CS252-specific CD8 T cells as
determined by ICS in individual mice from the indicated immunization group
before challenge. Filled circles indicate naı ¨ve or immune mice that developed
blood-stage malaria after a challenge with 1000 P. berghei sporozoites.
Numbers represent protected mice/total challenged for each group. n.d., not
CS252-specific memory CD8 T cells afford protection for ?3 months
DC-CS + LM-CS
with 2 ? 107LM-CS252. (A) Total number (mean ? SD, n ? 3 mice per day) of
CS252-specific CD8 T cells in the spleen on various days after the start of
immunization. (B) Naı ¨ve or DC-CS ? LM-CS mice were challenged with 1000 P.
berghei sporozoites on day 210. (C) The percent of total PBL that are CS252-
individual unchallenged or previously challenged DC-CS ? LM-CS immunized
mice. Naı ¨ve and immune mice were challenged with 1000 P. berghei sporo-
zoites at day 422. Filled circles indicate mice that developed blood-stage
malaria. Numbers represent protected mice per total challenged for each
group. n.d., not determined.
Long-term protection against multiple P. berghei sporozoite chal-
Schmidt et al.
September 16, 2008 ?
vol. 105 ?
no. 37 ?
fraction of CS252-specific memory cells that could produce IL-2
after in vitro stimulation (Fig. 5A and Fig. S2). Thus, differences
in the resistance of individual immunized mice probably would
be based on the number of memory CD8 T cells. The average
frequency of memory CD8 T cells in the PBL of each group
increased with increasing doses of LM-CS252boosting, as did the
frequency of protected mice in each group (Fig. 5B). Because
there was individual-to-individual variation within each group,
and the phenotypes of the memory CD8 T cells were similar,
we evaluated all immunized mice based on the frequency of
CS252-specific CD8 T cells and protection. Strikingly, 20 of 22
DC-CS ? LM-CS mice that had ? 1% CS252-specific PBL were
protected from sporozoite challenge at day 98 after immuniza-
tion, whereas only 1 of 16 similarly immunized mice with ?1%
CS252-specific PBL were protected (Fig. 5B). When we include
all DC-CS ? LM-CS mice in which memory CD8 T cells have
been evaluated in the blood at day 98 or later, our data
demonstrate a 97% chance of protection from sporozoite chal-
lenge in mice containing ? 1% CS252-specific PBL and a 6%
chance of protection in mice with ?1% CS252-specific PBL (Fig.
5C). Thus, long-term sterilizing immunity to P. berghei infection
in this model requires maintenance of CS252-specific memory
CD8 T cells that exceed a large (? 1%) but definable frequency
of PBL (Fig. 5C).
Immunity to P. berghei Requires More CD8 T Cells than Other Patho-
gens. Although it is clear that the magnitude of CD8 T cell
memory can influence resistance to infection (25, 26), there are
few data comparing the actual numbers of antigen-specific T
cells required for protection against diverse pathogens. To
memory CD8 T cells reduced infection by the liver pathogen
Listeria monocytogenes by ?100-fold (Fig. S3). Similarly, adop-
tive transfer of ?85,000 memory CD8 T cells dramatically
decreased virus titers after lymphocytic choriomeningitis virus
against a P. berghei sporozoite challenge. (A) In an initial experiment to
evaluate conventional prime-boost responses, BALB/c mice were primed with
7 ? 106LM-CS252 and boosted with 2 ? 107LM-CS252 53 days later (LM-
CS(d53)?LM-CS). On the day of the LM-CS boost, naive BALB/c mice were
group) of CS252-specific CD8 T cells in the spleen and liver and percentage of
PBL at day 61 after boost or first immunization was determined by ICS. (B) In
a second experiment BALB/c mice were primed with 7 ? 106LM-CS252and
boosted with 2 ? 107LM-CS25244 days later (LM-CS(d44)? LM-CS). On the day
of the LM-CS252boost, naive BALB/c mice were primed with 7 ? 106LM-CS252
(LM-CS). Percentage of total PBL that were CS252-specific CD8 T cells at day 75
after the last immunization was determined by ICS in individual mice. Filled
circles indicate naı ¨ve or immune mice that developed blood-stage malaria
after a challenge with 800 P. berghei sporozoites on day 83 after boost.
Numbers represent protected mice per total challenged for each group. n.d.,
LM-CS252-primed and LM-CS252-boosted BALB/c mice are protected
Challenged Protected % Protected
(A) Phenotype of PBL CS252-specific CD8 T cells as determined by ICS at day 98.
Data (mean ? SD) are from 10 mice per group except for 2 ? 107, in which n ?
7. (B) Before sporozoite challenge the percentage of total PBL CS252-specific
CD8 T cells was determined in individual mice by ICS. Naı ¨ve and immune mice
were challenged with 1000 P. berghei sporozoites. Filled circles indicate mice
that developed blood-stage malaria. n.d. ? not determined. (C) Cumulative
results from DC-CS ? LM-CS immunized mice in which the frequency of PBL
CS252-specific CD8 T cells was determined before challenge with sporozoites.
Numerical requirements for sterile immunity mediated by CS252-
www.pnas.org?cgi?doi?10.1073?pnas.0805452105Schmidt et al.
infection (30). Finally, the presence of ?50,000 memory CD8 T
cells in the spleen converted a lethal L. monocytogenes infection
into a sublethal infection that was cleared from all mice (26).
This last situation is analogous to the biological ‘‘bar’’ that must
be overcome for CD8 T cell protection against Plasmodium
infection, in which elimination of all infected hepatocytes is
required for survival of the mouse. Thus, the 1% of CS252-
specific PBL threshold (equivalent to ?106in spleen and ?2 ?
105in liver) required for long-term sterilizing immunity to
liver-stage Plasmodium infection is 100-1000-fold higher than
the numbers of memory CD8 T cells required for meaningful
immunity against a bacterial or viral pathogen.
In this study, we developed a model of epitope-specific immu-
nization to generate large memory CD8 T cell responses capable
of protecting mice from sporozoite challenges. Although several
studies have shown that protection from challenge at short
intervals after boosting correlates with large CD8 T cell re-
sponses (6, 8, 9, 31, 32), our results extend the field in at least 3
ways. First, we demonstrate that memory CD8 T cells specific for
Plasmodium liver-stage antigens are capable of providing long-
term sterilizing immunity, approaching the entire lifespan of the
laboratory mouse. Protection lasting ?6 months has been de-
scribed only for immunization with irradiated or genetically
attenuated sporozoite immunizations, suggesting that long-term
immunity after subunit vaccination may not be possible. Our
results clearly argue against this notion. Second, our results also
reveal that a large but definable threshold of memory CD8 T
cells is required for protection against sporozoite challenge.
Importantly, this threshold greatly exceeds the number of mem-
ory CD8 T cells required for protection against specific bacterial
and viral pathogens. These results suggest that the biology of the
pathogen will affect the number of memory CD8 T cells required
for resistance to infection. In the case of Plasmodium infection,
the low frequency of infected hepatocytes in combination with
the requirement of preventing even 1 infected cell from releasing
blood-stage parasites provides a challenge to the immune system
that requires commitment of a substantial fraction of the CD8 T
cell repertoire to achieve sterilizing immunity. Third, these data
describe a novel model system that should facilitate studies to
address how CD8 T cells provide immunity against liver-stage
Our results were generated with an epitope-specific immuni-
zation protocol in inbred mice, and this scenario is unlikely to
have immediate relevance as a vaccine strategy in humans.
Although the mouse in general and our approach specifically
may have limitations as a preclinical model, the results still may
have relevance for understanding why subunit vaccines that
evoke sterilizing immunity against human malaria have been
difficult to obtain. For example, accumulating data from human
clinical trials show that current prime-boost immunizations
generate Plasmodium-specific T cell responses in the range of
0.1% of PBL at the peak after boosting and ?0.01% at memory
stages (20, 33–36). These frequencies are 10-fold and 100-fold
lower than required to protect mice from Plasmodium infection
and consist mainly of CD4 T cells, which may explain why these
vaccines delay the onset but rarely prevent blood-stage para-
sitemia (20, 36). Clearly, delayed onset of blood-stage para-
sitemia indicates partial protection by these vaccines, and such
partial protection could have real benefits in malaria-endemic
areas. However, results from our model suggest that additional
or stronger booster immunizations may be required to generate
memory CD8 T cell frequencies that exceed the threshold for
optimal resistance to Plasmodium infection in humans. Further-
multiple liver-stage antigens will decrease the large frequency of
CS-specific memory CD8 T cells required for sterilizing immu-
nity. The model system we describe here is ideally suited to
address this question.
Alternatively, vaccination of humans to achieve the large
frequencies of memory CD8 T cells that are required for
sterilizing immunity to Plasmodium infection in mice may not be
feasible. In this regard, CD8 T cells are not the only effectors of
immunity to Plasmodium infection, and efforts are underway to
develop vaccines that also engage Plasmodium-specific CD4 T
cells and antibodies and that are capable of targeting multiple
stages of the parasite infection (23). Importantly, the relation-
ships between these various arms of the immune response
are unknown. The model system described here is well suited for
determining these relationships, because it permits quantitative
assessment of whether and how Plasmodium-specific antibodies
and CD4 T cells decrease the threshold frequencies of CS-
specific CD8 T cells required for protective immunity.
CD8 T cell immunity to liver-stage Plasmodium infection remain
to be defined. The DC-CS ? LM-CS immunization approach
used here provides an informative and reliable model in which
immune and susceptible mice can be identified before infection.
The ability to differentiate prospectively between resistant and
susceptible subjects provides a level of resolution that is partic-
ularly important for liver-stage studies because the host must be
killed for tissue sampling before the outcome of challenge is
known. This feature of the model will facilitate studies to address
in detail the molecular and cellular features of long-term CD8
T cell protection against liver-stage Plasmodium infection. In
turn, this basic information should be useful in devising the most
efficacious malaria vaccines.
Mice and Immunizations. BALB/c mice were housed at the University of Iowa
and Iowa State University animal care units. Mice were primed with DC (2.5 ?
105-5 ? 105) coated with CS252–260or with LM-CS252(7 ? 106) through i.v.
methods are available in SI Text.)
Quantification of Antigen-Specific T Cells. The total number of spleen CS252-
specific CD8 T cells was determined by ICS for IFN-? after 5 h of incubation in
brefeldin A in the presence or absence of CS252–260. Total liver CS252-specific
ICS for IFN-? after 5 h of incubation in brefeldin A in the presence or absence
of CS252–260-coated P815 cells.
ATCC) were reared in controlled environments (27°C ? 1°C and 80% ? 5%
relative humidity) and a 16:8-hour photoperiod. Mosquitoes were fed on
anesthetized mice ?3 days after subpassage or when parasitemia reached
5–20%. To confirm infection before sporozoite collection, oocyst prevalence
and intensity were monitored 7–14 days after exposure.
Sporozoite Challenge. P. berghei (ANKA strain clone 234) sporozoites were
isolated from the salivary glands of infected A. stephensi. Naı ¨ve and immu-
Identification of Protected Mice. Thin blood smears were performed 7 to 12
days after sporozoite challenge. Parasitized red blood cells were identified by
Giemsa stain. Protected mice were defined as those not having blood-stage
discussion and S. Perlman for critical comments on the manuscript. We thank
with Anopheles stephensi eggs (donated by William E. Collins) and Susan
Paskewitz for advice in culturing parasites in mosquitoes. Work in the J.T.H.
laboratory is supported by grants from the National Institutes of Health and
by support from the Department of Microbiology and Carver College of
Medicine, University of Iowa.
Schmidt et al.
September 16, 2008 ?
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no. 37 ?
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