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Neutralization of the Plasmodium-encoded MIF ortholog confers protective immunity against malaria infection

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Plasmodium species produce an ortholog of the cytokine macrophage migration inhibitory factor, PMIF, which modulates the host inflammatory response to malaria. Using a novel RNA replicon-based vaccine, we show the impact of PMIF immunoneutralization on the host response and observed improved control of liver and blood-stage Plasmodium infection, and complete protection from re-infection. Vaccination against PMIF delayed blood-stage patency after sporozoite infection, reduced the expression of the Th1-associated inflammatory markers TNF-α, IL-12, and IFN-γ during blood-stage infection, augmented Tfh cell and germinal center responses, increased anti-Plasmodium antibody titers, and enhanced the differentiation of antigen-experienced memory CD4 T cells and liver-resident CD8 T cells. Protection from re-infection was recapitulated by the adoptive transfer of CD8 or CD4 T cells from PMIF RNA immunized hosts. Parasite MIF inhibition may be a useful approach to promote immunity to Plasmodium and potentially other parasite genera that produce MIF orthologous proteins.
PMIF neutralization confers complete protection to re-infection by wild type P. berghei ANKA. a Parasitemia after infection of BALB/cJ mice (10⁶PbAWT iRBCs) previously immunized with RNA replicons encoding PMIF (black circle) or a control (Con) RNA (white circle); *p < 0.05, **p < 0.01, by two-way ANOVA and error bars denote ±SD. b Kaplan–Meier survival plots for immunized mice following infection with PbAWT (black circle, PMIF and white circle, Con). Data are from two independent experiments with 10–15 animals per group; **p = 0.0016 by log-rank (Mantel Cox) test. c Percentage of iRBCs in BALB/cJ mice previously immunized with RNA encoding PMIF (black circle) or Con RNA (white circle), treated with chloroquine, and re-infected with 10⁶PbAWT iRBCs; *p < 0.05, #p < 0.0001 by two-way ANOVA and error bars denote ±SD. d Splenic parasite load 6 days after reinfection with iRBCs was measured by quantitative PCR of PbAWT 18S rRNA relative to host β-actin. Results are from two separate experiments. Bars represent the mean of 6 mice ± SD; **p < 0.01 by Mann–Whitney test. ePbAluc liver load and absolute luminescence values in PMIF (black circle) or Con (white circle) RNA replicon immunized mice 48 h after the first infection with 2000 PbAluc sporozoites. f Percentage of iRBCs after the first infection of BALB/cJ mice with 2000 PbAluc sporozoites. Data are from two independent experiments. Bars represent the mean of 10 mice ± SD; **p < 0.01, #p < 0.0001 by Mann–Whitney and two-way ANOVA. gPbA liver load and absolute luminescence values in PMIF (black circle) or Con (white circle) RNA replicon immunized hosts 48 h after the second infection with 2000 PbAluc sporozoites. h Percentage of iRBCs after the second infection of BALB/cJ mice. Data are from two independent experiments. Bars represent the mean of 10 mice ± SD; *p < 0.05, **p < 0.01 by Mann–Whitney test and two-way ANOVA
… 
PMIF neutralization decreases inflammatory cytokine production and enhances the development of CD4 T cells into effector memory and memory precursors during blood-stage infection. a, b Serum levels of the indicated cytokines were detected by specific ELISA 7 days after injection of 10⁶PbAWT iRBCs in mice immunized with RNA replicons encoding Con RNA or PMIF RNA. Data are representative of two independent experiments. Bars represent the mean of 6 mice ± SD; *p < 0.05,**p < 0.01 by Mann–Whitney test. c On day 7, 10, and 15 after infection, splenocytes were isolated and stimulated ex vivo with iRBC lysates in the presence of Brefeldin A. Representative dot plots and frequencies of PbAWT responsive CD4 T cells (Ki67⁺CD4⁺) expressing IFN-γ in spleens was detected by intracellular staining and analyzed by flow cytometry. Data are representative of two independent experiments. Bars represent the mean of 10 mice ± SD; *p < 0.05 by Mann–Whitney test. d, e Numbers of PbAWT responsive CD4 T cell (Ki67⁺CD4⁺) subsets, including T effector (Teff): CD62L⁻IL7Rα⁻, T effector memory (Tem): CD62L⁻IL7Rα⁺, and T memory (Tmem): CD62L⁺IL7Rα⁺ at day 7 and 15 after infection. The contribution of each memory CD4 T cell subset is expressed relative to the total number of PbAWT responsive CD4 T cells. Data are representative of two independent experiments. Bars represent the mean of 10 mice ± SD; n.s.: non-significant, *p < 0.05, **p < 0.001, #p < 0.0001 by Mann–Whitney test
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ARTICLE
Neutralization of the Plasmodium-encoded MIF
ortholog confers protective immunity against
malaria infection
Alvaro Baeza Garcia1,2,3, Edwin Siu1,2,3, Tiffany Sun1,2,3, Valerie Exler1,2,3,4, Luis Brito5, Armin Hekele5,
Gib Otten5, Kevin Augustijn6, Chris J. Janse6, Jeffrey B. Ulmer5,9, Jürgen Bernhagen4, Erol Fikrig 7,8,
Andrew Geall5& Richard Bucala1,2,3
Plasmodium species produce an ortholog of the cytokine macrophage migration inhibitory
factor, PMIF, which modulates the host inammatory response to malaria. Using a novel RNA
replicon-based vaccine, we show the impact of PMIF immunoneutralization on the host
response and observed improved control of liver and blood-stage Plasmodium infection, and
complete protection from re-infection. Vaccination against PMIF delayed blood-stage
patency after sporozoite infection, reduced the expression of the Th1-associated inamma-
tory markers TNF-α, IL-12, and IFN-γduring blood-stage infection, augmented Tfh cell and
germinal center responses, increased anti-Plasmodium antibody titers, and enhanced the
differentiation of antigen-experienced memory CD4 T cells and liver-resident CD8 T cells.
Protection from re-infection was recapitulated by the adoptive transfer of CD8 or CD4 T cells
from PMIF RNA immunized hosts. Parasite MIF inhibition may be a useful approach to
promote immunity to Plasmodium and potentially other parasite genera that produce
MIF orthologous proteins.
DOI: 10.1038/s41467-018-05041-7 OPEN
1Department of Internal Medicine, Yale School of Medicine, New Haven, CT 06520, USA. 2Department of Pathology, Yale School of Medicine, New Haven,
CT 06520, USA. 3Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06520, USA. 4Institut für Schlaganfall-
und Demenzforschung, Klinikum der Universität München Ludwig-Maximilians-Universität München, D-81377 München, Germany. 5Novartis Vaccines, Inc.,
350 Massachusetts Avenue, Cambridge, MA 02139, USA. 6Leiden Malaria Research Group, Department of Parasitology, Leiden University Medical Centre,
2300 RC Leiden, The Netherlands. 7Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven 06520
CT, USA. 8Howard Hughes Medical Institute, Chevy Chase 20815 MD, USA.
9
Present address: Slaoui Center for Vaccines Research, GSK Vaccines, 14200
Shady Grove Rd., Rockville MD 20850, USA. Correspondence and requests for materials should be addressed to R.B. (email: richard.bucala@yale.edu)
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In 2013, there were approximately 200 million clinical cases
and 584,000 deaths from malaria caused by parasites of the
genus Plasmodium1.Plasmodium sporozoites enter the skin
through the bite of infected Anopheles mosquitoes, transit to the
liver, and replicate over several days to produce merozoites,
which then initiate an erythrocytic cycle of infection that pro-
duces the clinical manifestations of malaria2. Immunologically
naïve hosts are at the highest risk of lethal malaria but survivors
may develop partial immunity and tolerance to disease manifes-
tations. Such partial protection does not prevent re-infection and
declines in the absence of re-exposure to parasites2,3. One
mechanism for failure to develop sterilize immunity may be the
inability of the infected host to achieve immunologic memory and
maintain an effective anti-parasite immune response4,5.
The cellular processes responsible for ineffective immunity to
malaria are unclear, although studies support an impaired
development of the adaptive response with poor establishment of
germinal centers (GC) and a disruption of their architecture in
the spleen68. Effective GC formation requires CD4 T follicular
helper (Tfh) cells, which may be downregulated by an unresolved
pro-inammatory response and the expression of TNF-α, IL-12,
IFN-γ, and T-bet9,10. How Plasmodium infection negatively
impacts GCs is not understood, although parasite factors are
likely to play a central role2,4.
Many parasitic pathogens, including all studied Plasmodium
species, express an ortholog of the mammalian cytokine macro-
phage migration inhibitory factor (MIF)11,12. In studies of the
erythrocytic stage of Plasmodium berghei ANKA (PbA) malaria,
Plasmodium MIF (PMIF) was observed to be secreted into
infected erythrocytes and released upon schizont rupture13. PMIF
elicits a MIF receptor-dependent inammatory response that
interferes with the differentiation of Plasmodium-specic CD4 T
effector cells into long-lived memory precursors by increasing the
expression of TNF-α, IL-12, IFN-γ, and T-bet14. The role of
PMIF also has been examined in the Plasmodium liver-stage of
infection. Genetically-targeted Plasmodium strains that lack
PMIF do not show defects in virulence or in life cycle, however
infection with PMIF-decient Plasmodium yoelii may be asso-
ciated with retardation of parasite growth in liver and a delay in
blood-stage patency15.
Given the potential role of PMIF in modulating the immune
response and in liver-stage parasite development, we investigated
herein the impact of genetic deletion or immunoneutralization of
PMIF in the PbA experimental model of severe malaria. BALB/c
mice infected with PbAmifblood-stage parasites showed a
robust induction of antibody-secreting plasma cells and improved
differentiation of germinal Tfh cells when compared to wild type
parasites. PMIF immunization in turn recapitulated the pheno-
type observed with the PbAmifparasites, with improved
development of CD4 T effector cells into long-lived memory
precursors and enhanced differentiation of Tfh cells and
antibody-secreting B cells. PMIF-immunized mice showed
improved control of liver-stage infection that was associated with
an increase in the number Plasmodium-specic liver-resident
memory CD8 T cells. PMIF immunization also enhanced host
control of the rst infection and conferred complete protection to
re-infection.
Results
PMIF impairs germinal center formation during malaria.
Human and experimental mouse studies suggest that strong pro-
inammatory responses generated during blood-stage infection
can inhibit productive GC and Tfh cell responses7,8, and recent
data suggest a role for PMIF in the suppression of CD4 T cell
differentiation14. To assess whether PMIF pro-inammatory
activity affects the GC responses, we infected BALB/cJ mice
with PbAWT or PbAmifparasites. Infection with both strains
results in equivalent parasitemia and splenic parasite burden, and
comparable levels of circulating host MIF14. The frequency and
total numbers of GC B cells (CD19+CD38loGL7+) in the spleens
of PbAmifinfected mice was signicantly increased when
compared to PbAWT-infected mice (Fig. 1a and Supplementary
Figure 1a). The frequency and number of memory B cells
(CD19+IgDCD138CD38hi) also was signicantly lower in the
PbAWT-infected mice than in the mice infected with PbAmif
parasites (Fig. 1b), and this was associated with a 5-fold increase
in the parasite-specic antibody response (Fig. 1c). Immunohis-
tochemical staining at 15 days after infection of spleen sections
from PbAWT mice showed a signicant loss of the T cell zone
and a disorganized follicular architecture when compared with
PbAmifinfected mice. Taken together, these data suggest that
PMIF impairs GC reactions and antibody responses during
experimental malaria infection.
PMIF decreases Tfh cell responses during malaria infection.
Tfh cells are essential for the formation and maintenance of
GCs and enable proper B cell development into antibody-
producing plasma cells and memory B cells16. We investigated
if the impairment in GC formation associated with PMIF
was a consequence of defective Tfh differentiation. We
examined the frequency and number of activated Tfh cells
(CD4+CD62LCXCR5hiPD-1hi) in the spleens of mice at days 6
and 15 after infection with PbAWT or PbAmifparasites. Mice
infected with PbAmifparasites showed a signicant increase in
the number of Tfh activated cells at day 6 when compared to
PbAWT-infected mice (Fig. 2a, b and Supplementary Figure 2a).
This difference was maintained at day 15 of infection, and
without a change in the number of measured Tfh cells in the
PbAWT-infected mice. We also investigated if the difference in
the number of Tfh cells between the two groups was due to a
defect in their maturation7, despite a similar percentage of pre-
Tfh cells in mice infected with PbAWT or PbAmif. The number
of pre-Tfh cells (CD4+CD62LCXCR5intPD-1int) was sig-
nicantly elevated after 6 days of infection with PbAWT (Fig. 2c).
We measured the expression of the transcription factor Bcl-6, a
regulator of Tfh cell differentiation17. Bcl-6 expression was higher
in the PbAmif- infected mice, suggesting a defect in the
maturation of these cells in the presence of PMIF (Fig. 2d).
In the setting of malaria infection, the balance between Th1
and Tfh responses is determined by the expression of T-bet and
Bcl-6. Excessive expression of T-bet represses Bcl-6 expression
and interferes with Tfh cell expansion and GC formation7,9.In
the presence of PMIF, there is an evidence of increased
expression of the transcription factor T-bet by Plasmodium-
responsive CD4 T cells as a consequence of elevated host
production of IL-12 and IFN-γ14. We investigated the effect of
PMIF in driving Tfh responses toward Th1 development by
measuring T-bet expression in the Tfh lineage cells. T-bet was
signicantly higher in the CD4+CD62LBcl6hi splenic cells of
mice infected with PbAWT than PbAmifparasites (Fig. 2e).
In accordance with this increase in T-bet expression, there also
wasanelevationintheexpressionnumberofbothpre-Tfhand
Tfh cells in mice infected with PbAWT when compared to
those infected with PbAmif(Supplementary Figure 2b, c).
Finally, Tfh cells from PbAWT-infected mice expressed higher
levels of the cytokine IFN-γ(Supplementary Figure 2d). These
data suggest that a Th1 pro-inammatory response driven by
PMIF during acute infection has a detrimental effect on the
development of responsive Tfh cells, leading to inadequate
GC formation.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05041-7
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PMIF inuences PbA development in liver. Blood-stage infec-
tion with PbAmifparasites results in an augmented memory
CD4 T cell response when compared to infection with PbAWT,
although the survival of infected hosts is unchanged14.To
examine the impact of PMIF on liver-stage parasite development,
which may be impaired in the P. yoelii model15, BALB/cJ mice
were infected with 2000 freshly isolated PbAWT or PbAmif
sporozoites and blood-stage patency assessed. All mice infected
with PbAWT sporozoites developed patent infection at 3 days; by
contrast, fewer than 10% of mice infected with PbAmifparasites
showed blood-stage patency at 5 days and 25% of mice remained
free of parasitemia at 21 days (Supplementary Figure 3a). The
livers and spleens of mice infected with PbAWT or PbAmif
sporozoites were harvested 7 days after infection and the
CSP (Circumsporozoite protein) epitope-specic CD8 T cell
responses assessed by ow cytometry. CSP-specic CD8 T cells
(CD8+CD11ahiTetrCSPhi) were identied in both groups of mice
but increased numbers were evident in the livers of mice
infected with PbAmif(Supplementary Figure 3b). The pheno-
type of CSP-specic CD8 T cells was further characterized by
the expression of CD44, CD69, and KLGR1 to better
differentiate between the main subsets of CSP-specic cells. We
found two distinct populations of CD8 T cells in the livers of
PbAWT and PbAmifmice: resident memory cells (Trm:
CD44hiKLGR1CD69+) and effector memory cells (Tem:
CD44hiKLGR1hiCD69), which are two populations described
recently to persist long term and to be essential for protection
against re-infection11. Consistent with the CSP-specic cell
results, the number of liver Trm and Tem cells was signicantly
lower in the PbAWT than the PbAmifinfected mice (Supple-
mentary Figure 3c), with Trm cells representing ~64% of the to-
tal intrahepatic CSP-specic cells. These data support
the notion that PMIF deciency impairs liver-stage parasite
development, as previously suggested15, and this impairment
is associated with an augmentation in the liver-resident CD8 T
cell response.
Days after infection
Days 6
3.37 11.5 12.6
5.21 14 17.3
CD138
14.1
lgDCD19+
Days 9 Days 15 20
105
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100
105
104
103
102
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105
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100101102103104105
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105
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0 50K 100K 150K 200K 250K
0 50K 100K 150K 200K 250K
100101102103104105100101102103104105
15
**
** **
**
**
10
5
0
20
15
c
10
5
06915
Days after infection
15
n.s.
10
5
0
9
Days after infection
CD19+ CD138lgDCD38hi cells ×106
Anti-plasmodium lgG titer ×103
CD19+ CD38lo GL7+ cells ×106
15
6915
Days after infection
GC B cells
Memory B cells
PbAmif
PbA mif–
lgD PbA mif–
PbAWT
PbAmif–
PbAWT
PbAmif–
PbAWT
PbA WT
PbA WT
**
*
CD19
CD38
FSC
CD38
GL7
14.9
17.7
13.8
b
a
Fig. 1 PMIF impairs germinal center formation. BALB/cJ mice were infected with 106PbAWT or PbAmifiRBCs. On day 6, 9, and 15, splenocytes were
isolated and the total number of agerminal center (CD19+CD38loGL7+) and b(CD19+CD138IgDCD38hi) memory B cells were determined. Results are
from three separate experiments. Bars represent the mean of 12 mice ± SD. **p< 0.05 by MannWhitney test. cAnti-Plasmodium antibodies titers from
BALB/cJ mice that were infected with PbAWT or PbAmifiRBCs. On day 6, 9, and 15, sera of infected mice were collected, and the Plasmodium-specic IgG
responses measured by ELISA. Results are from three separate experiments. Bars represent the mean of 12 mice ± SD; n.s.: p> 0.05; *p< 0.05; **p< 0.01
by two-way ANOVA
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An RNA replicon encoding PMIF elicits humoral and cellular
immunity. The present and previous14 observations suggest
immunoregulatory actions for PMIF in the development of anti-
Plasmodium CD4 central memory T cells, GC Tfh responses, and
B cell maturation. We hypothesized that inhibition of PMIF
activity could improve host immunity against Plasmodium
infection and potentially confer protection against re-infection.
For immunization of naïve mice, we subcloned pmif into a self-
amplifying mRNA replicon, which is an antigen delivery
methodology that elicits cellular and humoral responses without
generating a limiting anti-vector response18,19. We studied the
impact of pmif or control RNA immunization on liver- and
blood-stage PbAWT infection in BALB/cJ mice, which under
normal circumstances results in a progressive parasitemia and
death from anemia at 23 weeks. If cured by chloroquine treat-
ment, the initially infected mice remain susceptible to the second
infection and develop a patent parasitemia that persists for at least
10 days20,21.
We assessed the immunogenicity of PMIF in mice given two
sequential pmif RNA replicon immunizations followed by
challenge infection with PbAWT-infected red blood cells (iRBCs)
(Supplementary Figure 4a). Single immunization resulted in a
primary anti-PMIF antibody response that increased in titer by 4-
fold after the second immunization (Supplementary Figure 4a).
Anti-PMIF antibody development was associated with the
elicitation of PMIF specic CD4 T cells (Supplementary
Figure 4c), and the elicited anti-PMIF IgG neutralized the
stimulatory action of PbAWT iRBCs or recombinant PMIF on
inammatory cytokine production by bone marrow-derived
macrophages (Supplementary Figure 4d, e). As host MIF
deciency may alter the course of Plasmodium infection22,we
also tested the specicity of the antibody response in the PMIF-
immunized mice and found that anti-PMIF IgG from immune
serum neutralized PMIF upregulation of host TLR4 expression
but failed to detect mouse MIF (Supplementary Figure 4fh). To
exclude a potential contribution for elicited anti-PMIF in the
clearance of free parasites after schizont release, we also
conrmed that PMIF is expressed only in the cytosolic fraction
and not on membranes (Supplementary Figure 4i). As a
specicity control, we studied the impact of anti-PMIF IgG in
mice infected with PbAmifiRBCs, which show similar
parasitemia and lethality in this model as mice infected with
PbAWT14. Anti-PMIF IgG administration to mice infected with
PbAmifdid not inuence parasitemia, and disease course
resembled that of PbAWT-infected mice treated with a non-
immune (Con) IgG (Supplementary Figure 4j). These results
demonstrate that pmif RNA replicon immunization elicits both a
cellular and humoral immune response against PMIF, and that
anti-PMIF IgG blocks the pro-inammatory action of PMIF
without inhibiting the action of host MIF.
An RNA replicon encoding PMIF confers protection to re-
infection. Mice immunized with pmif or control RNA replicons
were injected with 106PbAWT-infected iRBCs and the progress
of infection followed over time. There was a more rapid increase
in parasitemia after day 5 in the control group, which became
moribund on day 21 (Fig. 3a, b). By contrast, the pmif RNA
replicon immunized mice showed better control of parasitemia
during the rst 15 days of infection and a 37% prolongation in
104
103
103104105
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Days after infection
66
200
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50
0
4
2
0
Tfh Pre-Tfh
CD4+ CD62L
CXCR5hi PD-1hi ×106 cells
CD4+ CD62L
CXCR5int PD-1int ×106 cells
T-bet MFI
CD4+ CD62LBcl-6hi ×106 cells
4
2
0
8
6
4
2
0
615
Days after infection
615
Days after infection
n.s.
Days 6
PbA WT
d e
PbA mif–
PbAWT
PbAmif–
PbAWT
PbAmif–
PbAWT PbAmif–PbAWT PbAmif–
PbA WTPbA mif–
CXCR5
Days 15
**
**
**
**
*
7.56
43.8
36.9
15.5
PD-1
Bcl6
CD4
37.9 51.3
ab c
Fig. 2 PMIF inhibits Tfh cell development. BALB/cJ mice were infected with 106PbAWT or PbAmifiRBCs. On days 6 and 15 after infection, splenocytes
were isolated and Tfh cells assessed. acRepresentative plots and absolute numbers of Tfh and pre-Tfh cells in the two groups of mice. Results are from
three separate experiments. Bars represent the mean of 12 mice ± SD (*p< 0.05, **p< 0.001 by MannWhitney test). dRepresentative plots and absolute
number of CD4+CD62LBcl-6hi cells at day 6 after infection, and eexpression of transcription factor T-bet in CD4+Bcl-6hi cells. Results are from three
separate experiments. Bars represent the mean of 12 mice ± SD; *p< 0.05, **p< 0.001 by MannWhitney test
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05041-7
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mean survival time. To test for the development of a protective
memory response, a cohort of pmif RNA replicon immunized and
PbAWT-infected mice was cured by treatment with chloroquine
and re-infected 4 weeks later (see scheme Supplementary Fig-
ure 4a). Mice that received the control RNA replicon developed a
rapidly increasing parasitemia that was resolved at day 30. By
contrast, patent parasitemia did not develop in the pmif RNA
replicon immunized mice, nor were parasites detected in organs
(Fig. 3c, d). Challenge infection was associated with a further
increase in anti-PMIF titer, indicating that PMIF immunization
produces a humoral response that persists after blood-stage
infection and is rapidly activated after the second challenge (see
Supplementary Figure 4b, third titer).
We studied the effect of PMIF on pre-erythrocytic stage
Plasmodium by immunizing mice with pmif or control RNA
replicons followed by i.v. injection of 2000 PbA expressing
luciferase (PbAluc) sporozoites. There was a 65% decrease
in the liver burden in the pmif RNA immunized mice at 48 h
after infection (Fig. 3e). While both groups of mice developed
blood-stage infection, the control mice showed a rapid
increase in parasitemia and became moribund on day 19 after
infection. The parasitemia in the pmif RNA immunized
mice, by contrast, never exceeded 2% and was eliminated in
all mice at day 25 (Fig. 3f). Cohorts of pmif or control RNA
replicon immunized mice also were cured of blood-stage
infection by treatment with chloroquine and re-infected 4 weeks
later with PbAluc sporozoites. While both groups of
mice showed a reduction parasite liver burden relative to
the rst infection (Fig. 3e), the pmif RNA immunized mice
showed a 70% reduction in liver parasites when compared with
the control mice and did not develop blood-stage infection
(Fig. 3g, h).
These data support the conclusion that PMIF blockade by
vaccination enhances the control of rst infection and prevents
100
a
e
g
bcd
f
h
100 81.5
1.0
PbA18s rRNA/host β-actin
0.5
0.0
6#
##
#
#
**
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0
50
% Survival
% Parasitemia
**
*
Con
PMIF Con
PMIF
Con
PMIF
Con
PMIF
Con
PMIF
80
60
% Parasitemia
40
20
0
0 5 10 15
Con PMIF 20
820
% Parasitemia
15
10
5
0
0 5 10 15 20
Days after infection
6
4
2
0Con PMIF
**
Radiance (p/s/cm2/sr)×105
Radiance (p/s/cm2/sr)×105
30
25
20
% Parasitemia
15
10
5
0
15
10
5
0
Con PMIF 0 5 10 15
Days after infection
20 25 30
20 25 30 0 10 20 30 40 0
2.5
2.0
×10
6
p/s/cm
2
/sr ×10
6
p/s/cm
2
/sr
1.5
1.0
1.0
0.8
0.6
0.4
0.2
0.5
5 1015202530
Days after infection
Con PMIF
**
**
**
**
*
p= 0.0016
Days post infection
Con PMIF
Days after infection
Fig. 3 PMIF neutralization confers complete protection to re-infection by wild type P. berghei ANKA. aParasitemia after infection of BALB/cJ mice (106
PbAWT iRBCs) previously immunized with RNA replicons encoding PMIF (black circle) or a control (Con) RNA (white circle); *p< 0.05, **p< 0.01, by two-
way ANOVA and error bars denote ±SD. bKaplanMeier survival plots for immunized mice following infection with PbAWT (black circle, PMIF and white
circle, Con). Data are from two independent experiments with 1015 animals per group; **p=0.0016 by log-rank (Mantel Cox) test. cPercentage of iRBCs
in BALB/cJ mice previously immunized with RNA encoding PMIF (black circle) or Con RNA (white circle), treated with chloroquine, and re-infected with
106PbAWT iRBCs; *p< 0.05, #p< 0.0001 by two-way ANOVA and error bars denote ±SD. dSplenic parasite load 6 days after reinfection with iRBCs was
measured by quantitative PCR of PbAWT 18S rRNA relative to host β-actin. Results are from two separate experiments. Bars represent the mean of 6
mice ± SD; **p< 0.01 by MannWhitney test. ePbAluc liver load and absolute luminescence values in PMIF (black circle) or Con (white circle) RNA
replicon immunized mice 48 h after the rst infection with 2000 PbAluc sporozoites. fPercentage of iRBCs after the rst infection of BALB/cJ mice with
2000 PbAluc sporozoites. Data are from two independent experiments. Bars represent the mean of 10 mice ± SD; **p< 0.01, #p< 0.0001 by
MannWhitney and two-way ANOVA. gPbA liver load and absolute luminescence values in PMIF (black circle) or Con (white circle) RNA replicon
immunized hosts 48 h after the second infection with 2000 PbAluc sporozoites. hPercentage of iRBCs after the second infection of BALB/cJ mice. Data
are from two independent experiments. Bars represent the mean of 10 mice ± SD; *p< 0.05, **p< 0.01 by MannWhitney test and two-way ANOVA
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05041-7 ARTICLE
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Content courtesy of Springer Nature, terms of use apply. Rights reserved
re-infection. Notably, protection was more pronounced in mice
infected with PbA sporozoites as these mice cleared blood-stage
parasites after challenge infection and failed to develop detectable
blood-stage infection after re-infection.
A PMIF vaccine enhances liver-resident memory CD8 T cells.
As infection with PbAmifsporozoites is associated with an
increased number of liver-resident memory CD8 T cells (Trm)
(Supplementary Figure 3), we examined if immunization with
pmif RNA also leads to enhanced Trm numbers after sporozoite
infection. We hypothesized that vaccination with PMIF could
have an impact in the host immunity against Plasmodium liver-
stage by increasing the number of liver-resident memory CD8
T cells. We immunized mice with pmif or control RNA replicons
followed by i.v. injection of 2000 PbA sporozoites 1 month later
and characterized the phenotype of liver CD8 T cells 7 days after
infection. There was a 48% increase in the number of CSP-
specic CD8 T cells in the liver (Fig. 4a) but not the spleen
(Supplementary Figure 5a) in the pmif RNA versus the control
RNA immunized group, and examination of CD8 T cell subsets
revealed a corresponding increase in CSP-specic Trm cells
(Fig. 4b). Liver CD8 Trm cells directed against Plasmodium are
long-lived23. To examine their development and response to a
second infection, we cured immunized mice that had primary
blood-stage infection by chloroquine treatment and examined
CD8 Trm cell frequency 1 month later, both before and after re-
infection with PbAWT sporozoites. While the number of liver
CD8 Trm cells decreased after 1 month in both the pmif and the
control RNA immunized groups, there was a more than 3-fold
increase in the pmif RNA immunized mice when compared with
the control group (Supplementary Figure 5b). Seven days after re-
infection with sporozoites, there was an expansion of this liver
CD8 Trm population, with a 60% increase in CSP-specic CD8
Trm cells in the pmif RNA immunized cohort when second
infection is compared to the number of Trm 1 month after the
rst infection (Fig. 4c). Moreover, there was an increase in the
number of IFNγ-expressing CD8 Trm cells after the second
a
b
c
CD8+CD11ahiCSPtetrhi
×106 cells
Con
CD8loCD11ahi
7.01
CD8
CSPTetr
CD69
KLGR1
CD69
KLGR1
9.09
6.36
9.55
14.6 33
10.7
12.9
16.3 28.8
Con
CD8+CSPTet r
Con
CD8+CSPTet r
PMIF
CD8loCD11ahi
PMIF
CD8+CSPTet r
PMIF
CD8+CSPTet r
CSPTetraCD44hiKLGR1CD69+
×105 cells
CSPTetrCD44hiKLGR1CD69+
×104 cells
CSP-specific
Tr m
Tr m
**
**
**
4
105
104
103
102
101
100
100101102103104105100101102103104105
100101102103104100101102103104
100101102103104105
100101102103104105
3
2
1
0
3
2
1
0
5
4
3
2
1
0
Con
Con
Con
PMIF
PMIF
PMIF
105
104
103
102
101
100
105
104
103
102
101
100
105
104
103
102
101
100
105
104
103
102
101
100
105
104
103
102
101
100
Fig. 4 PMIF neutralization enhances the development of Plasmodium liver memory CD8 T cells. BALB/cJ mice immunized with replicons encoding Con RNA
or PMIF RNA were challenged with 2000 PbAWT sporozoites by i.v. injection. On day 7 after the rst or second infection, liver immune cells were isolated
and the percentage and total number of CD8 T cells assessed. aRepresentative plots and absolute numbers of CSP-specic CD8 T cells
(CD8+CD11ahiCSPTetrhi) in the livers of PMIF or Con RNA immunized mice. Results are from two separate experiments. Bars represent the mean of
6 mice ± SD; **p=0.0022 by MannWhitney test. Representative plots and absolute number of CSP-specic tissue resident memory CD8 T cells (Trm:
CSPTetrCD44hiKLGR1CD69+) at day 7 after the rst (b) and second (c) infection. Results are from two separate experiments. Bars represent the mean
of 6 mice ± SD; **p=0.0022 by MannWhitney test
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05041-7
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Content courtesy of Springer Nature, terms of use apply. Rights reserved
infection in the pmif versus control RNA group when tested by
ex vivo stimulation with sporozoites lysates (Supplementary
Figure 5c). Taken together, these ndings indicate that pmif RNA
vaccination promotes a liver CD8 Trm cell response that func-
tionally expands after re-infection.
PMIF neutralization increases the memory CD4 T cell
response. Infection with blood-stage PbAmifparasites, when
compared to PbAWT parasites, is associated with lower circu-
lating levels of IFN-γand increased numbers of Plasmodium-
responsive CD4 T cells that develop into memory precursor CD4
T cells14. We observed a 48% lower serum concentration of IL-12
in mice infected with PbAWT iRBCs that were immunized with
pmif versus control RNA, as well as a 30% and 45% reduction
respectively, in circulating IFN-γand TNF-αlevels (Fig. 5a).
Serum concentrations of PMIF and host MIF also were measured
by specic ELISA. PMIF levels were reduced by 89% in infected
mice previously immunized with pmif RNA and, as expected,
there was no alteration in the levels of circulating host MIF
(Fig. 5b). While there were similar numbers of Plasmodium-
responsive CD4 T cells in both groups of mice during acute
infection (day 7), there was a 50% reduction in the percentage of
CD4 T cells producing IFN-γin the pmif RNA immunized group
(Fig. 5c), which is consistent with reduced development of an
initial inammatory CD4 T effector population in the setting of
PMIF neutralization or genetic absence. This difference in IFN-γ
producing CD4 T cell population disappeared by day 10, and with
resolution of infection (day 15) there was a comparative increase
in the Plasmodium-responsive, IFN-γexpressing memory CD4 T
cell population in the pmif RNA immunized group. These data
suggest a time-dependent development and preservation of a
600
a
c
de
b
400
200
**
**
**
**
IL-12, pg/mL
0
Con
7 days
11 11.7 6.4
5.7
CD4
IFN-γ
CD62L
CD62L
Tem Tmem
Day 7
Day 15
Teff
Teff Tem Tmem
Teff
n.s.
n.s.
*
**
*
#
Tem Tmem
Naive
57.9 27
5.71 9.31
41.5 34.8
4.97 18.7
IL7R-α
IL7Rα
100
101
102
103
104
105
100
101
102
103
104
105
100101102103104105
100101102103104105
100
101
102
103
104
105
100101102103104105
100
101
102
103
104
105
100101102103104105
11 11.3
10 days 15 days
PMIF Con PMIF
Con PMIF
Con
PMIF
7
0
5
10
%CD4+Ki67+IFNγhi
15
0
3
2
1
0
5
10
CD4+Ki67+
CD62LIL7Rα×107 cells
CD4+Ki67+
CD62LIL7Rα+ ×107 cells
CD4+Ki67+
CD62LIL7Rα+ ×107 cells
CD4+Ki67+
CD62L+IL7Rα+ ×107 cells
CD4+Ki67+
CD62L+IL7Rα+ ×107 cells
CD4+Ki67+
CD62LIL7Rα×107 cells
15
*
*
*
10
Days after infection
15
Con PMIF Con PMIF
Con PMIF
Con PMIF Con PMIF Con PMIF
Con PMIF Con PMIF
Con PMIF
Con
PMIF
600
400
200
IFN-γ, pg/mL
0
400
200
300
100
TNF-α, pg/mL
0
10
5
PMIF, ng/mL
0
100
150
50
n.s.
MIF, ng/mL
0
2.0
1.5
1.0
0.5
0.0
2.0
1.5
1.0
0.5
0.0
10
8
6
4
2
0
4
3
2
1
0
Fig. 5 PMIF neutralization decreases inammatory cytokine production and enhances the development of CD4 T cells into effector memory and memory
precursors during blood-stage infection. a,bSerum levels of the indicated cytokines were detected by specic ELISA 7 days after injection of 106PbAWT
iRBCs in mice immunized with RNA replicons encoding Con RNA or PMIF RNA. Data are representative of two independent experiments. Bars represent
the mean of 6 mice ± SD; *p< 0.05,**p< 0.01 by MannWhitney test. cOn day 7, 10, and 15 after infection, splenocytes were isolated and stimulated
ex vivo with iRBC lysates in the presence of Brefeldin A. Representative dot plots and frequencies of PbAWT responsive CD4 T cells (Ki67+CD4+)
expressing IFN-γin spleens was detected by intracellular staining and analyzed by ow cytometry. Data are representative of two independent
experiments. Bars represent the mean of 10 mice ± SD; *p< 0.05 by MannWhitney test. d,eNumbers of PbAWT responsive CD4 T cell (Ki67+CD4+)
subsets, including T effector (Teff): CD62LIL7Rα
, T effector memory (Tem): CD62L
IL7Rα+, and T memory (Tmem): CD62L+IL7Rα+at day 7 and 15
after infection. The contribution of each memory CD4 T cell subset is expressed relative to the total number of PbAWT responsive CD4 T cells. Data are
representative of two independent experiments. Bars represent the mean of 10 mice ± SD; n.s.: non-signicant, *p< 0.05, **p< 0.001, #p< 0.0001 by
MannWhitney test
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memory CD4 T cell response during blood-stage infection after
pmif RNA immunization.
Plasmodium infection is associated with a down-regulation of
the T cell survival receptor IL7Rαand an upregulation of T-bet,
which are markers of the terminal differentiation of effector CD4
T cells14,24. Using the markers CD62L and IL7Rαto assess the
phenotype of Plasmodium-responsive memory CD4 T cells, we
observed at 7 days a 90% increase in CD4 T effector memory cells
(Tem: CD62LIL7Rα+), an 80% increase in CD4 T memory cells
(Tmem: CD62L+IL7Rα+) (Fig. 5d, e), as well as a 20% reduction
in the number of CD4 T cells expressing the exhaustion marker
PD-1 in the pmif RNA versus control RNA immunized mice
(Supplementary Figure 6). This observed phenotype of Plasmo-
dium-responsive effector memory and memory CD4 T cells was
further evidenced by measurements at 15 days of infection
(Fig. 5e). Taken together, these ndings support the relative
preservation of a memory CD4 T cell response by PMIF
neutralization during blood-stage Plasmodium infection.
We next examined the impact of re-infection in a cohort of
pmif RNA immunized mice that were cured of primary blood-
stage PbAWT infection by chloroquine treatment. Lower
circulating concentrations of IFN-γwere noted after challenge
infection in the pmif RNA immunized group when compared to
controls, and this was associated with a 94% reduction in serum
PMIF (Supplementary Figure 7a). There also was evidence of
preservation and expansion of the CD4 T effector memory and
memory cell population by 100%. (Supplementary Figure 7b).
After re-infection, the number of Plasmodium-responsive CD4
T cells was similar in both groups but there were 40% fewer
Plasmodium-responsive CD4 T cells producing IFN-γin the pmif
RNA immunized mice than in the control group (Supplementary
Figure 7c). Moreover, there was a> 25% decrease in the
proportion of memory CD4 T cells expressing PD-1, suggesting
that the neutralization of PMIF during blood-stage infection
reduces memory CD4 T cell exhaustion (Supplementary
Figure 7d).
PMIF neutralization promotes anti-PbA cellular and humoral
immunity. Immunohistochemical staining of spleens 15 days
after blood-stage infection with PbAWT revealed an expanded
and less disorganized B cell relative to T cell zone in the pmif
RNA versus control RNA immunized mice (Supplementary Fig-
ure 8a, b). We examined the development of Tfh cells and GC B
cells in spleens, rst by enumerating Tfh cells
(CD4+CD62LCXR5hiPD-1hi)7,25 in mice infected with PbAWT
parasites that had been immunized previously. There was a 2.5-
fold increase in the number of CD4 Tfh cells when compared to
infected mice immunized with a control RNA replicon. The
number of pre-Tfh cells also was higher in the controls than in
the pmif RNA immunized mice (Fig. 6a, b), supporting a
maturation defect in Tfh cells in the control group. Consistent
with this observation, we measured the expression of the Tfh
differentiation regulator Bcl-6 and conrmed that its expression
was signicantly higher in the Tfh cells from the pmif RNA versus
the control RNA immunized mice (Fig. 6a, c)17. Consistent with
this observation, we found a signicant increase in the number
of GC B cells (CD19+CD38loGL7+) and memory B cells
(CD138CD19+IgDCD38hi) during the rst infection, and the
difference was maintained after the second infection in pmif RNA
immunized mice when compared with the controls (Fig. 6d, e).
That pmif RNA immunization is associated with an improvement
in the host Tfh and B cell responses was conrmed by serum
antibody titers against Plasmodium blood- and liver-stage (CSP)
antigens, and a 6-8-fold higher titer of total IgG, was observed
against blood-stage and liver-stage antigen, respectively, in the
pmif RNA versus control RNA immunized groups (Fig. 6f, g).
Taken together, these results demonstrate that immunoneu-
tralization of PMIF reduces its detrimental effect on the devel-
opment of Plasmodium-responsive Tfh cells, restores GC
formation, and promotes a more effective cellular and humoral
response against pre- and erythrocytic Plasmodium infection.
PMIF vaccination elicits malaria-protective CD4 T cells. The
observation that immunoneutralization of PMIF promotes the
differentiation and maintenance of a memory CD4 T cell
response, improves anti-Plasmodium antibody responses, and
prevents re-infection to blood-stage malaria prompted us to
examine more closely the contribution of the adaptive and
humoral responses to protective immunity. We assessed the
functional signicance of an augmented CD4 T cell response by
adoptive transfer into naïve recipients of splenic CD4 T cells
isolated from PbAWT-infected mice that had been immunized
against pmif or control RNA. For this protocol, mice were
sacriced 7 days after the second infection and 2 × 107splenic
CD4 T cells (CD45.2) were CFSE-labeled and transferred into
congenic CD45.1 BALB/cJ mice. The recipient mice then were
infected with blood-stage PbAWT 3 days after adoptive cell
transfer (Fig. 7a). Infection was established in recipient mice that
received CD4 T cells from the control group, as evidenced by
increasing parasitemia and organ parasite content, but not in
mice that received CD4 T cells from the pmif RNA immunized
donors (Fig. 7b). The phenotype of the transferred CD4 T cells
also was characterized in mice euthanized at day 7 after infection.
The protection conferred by the adoptive transfer of CD4 T cells
from the pmif RNA immunized donors was associated with a
higher number of proliferating CD4 T cells (CFSElo) (Fig. 7c, d),
higher levels of IFN-γproduction (Fig. 7e), and reduced
expression of the exhaustion marker PD-1 when compared to
CD4 T cells adoptively transferred from the control group
(Fig. 7f). These data indicate that the augmented CD4 T cell
response that develops after pmif RNA immunization in infected
mice is sufcient to prevent the establishment of blood-stage
infection.
PMIF vaccination promotes a protective CD8 T cell response
against sporozoite infection. Immunization with pmif RNA
partially protects mice from sporozoite challenge and protects
completely from re-infection when the initially infected mice are
cured by chloroquine treatment (Fig. 3eh). As this protection is
associated with an expansion of liver CSP-specic CD8 T cells
(Fig. 6), we adoptively transferred 2 × 107liver CD8 T cells from
immunized CD45.2 mice after the second infection to evaluate
the functional signicance of this expanded CSP-specic T cell
population. Three days after adoptive transfer, we infected reci-
pient CD45.1 Balb/cJ mice with 2000 PbAWT sporozoites and
assessed the development of infection and the CD8 T cell
response (Fig. 8a). Hepatic parasite content was signicantly
reduced at 48 h in mice that received liver CD8 T cells from the
pmif RNA versus the control RNA replicon immunized hosts
(Fig. 8b). Blood patency was established in recipient mice that
received liver CD8 T cells from the control RNA immunized
hosts but not in mice that received CD8 T cells from the pmif
RNA immunized hosts (Fig. 8c). We euthanized the mice 7 days
after infection to assess the phenotype of the transferred CD8+
T cells. The protection conferred by the adoptive transfer of liver
CD8 T cells from the pmif RNA immunized hosts was associated
with a higher number of proliferating CSP-specic CD8 T cells
(CFSElo) producing IFNγ(Fig. 8d, e). These data indicate that the
augmented liver CD8 T cell response that develops in infected
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05041-7
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mice after pmif RNA immunization is sufcient to prevent the
establishment of infection by Plasmodium sporozoites.
Antibodies elicited by PMIF vaccination enhance malaria
control. We puried serum IgG from PbAWT-infected mice
previously immunized with RNA encoding PMIF or GFP and
tested its effect on malaria development in both the BALB/cJ and
the cerebral malaria-sensitive C57BL/6J mouse strains (Supple-
mentary Figure 9a). Administration of IgG from PMIF-
immunized mice into naïve mice that were infected with
PbAWT provided partial protection, with a delayed rise in
CD4
+
CD62L
CD4+CD62L
CXCR5hiPD-1hi ×106 cells
CD4+CD62L
CXCR5intPD-1int ×106 cells
Tfh Bcl6+ ×105 cells
Pre-Tfh Bcl6+ ×105 cells
CD19+CD38loGL7+ ×107 cells
Con
a
de f
g
bc
CXCR5
CD38
GL-7
CD38
FSC
PD-1
Con
5
PMIF
8
PMIF
29
Con
20
Con
32.8
PMIF
90
PMIF
62
Con
56
1st Infection
CD19
+
1st Infection 2nd Infection 1st Infection 2nd Infection
1st Infection
CD19
+
CD138
lgD
2nd Infection
CD19
+
CD138
lgD
2nd Infection
CD19
+
Bcl-6
100101102103104105
100101102103104105100101102103104105
100101102103104105100101102103104105
100101102103104105
100101102103104105
100101102103104105
10540
30
20
10
0
0
20
40
60
104
103
102
101
100
105
104
103
102
101
100
105
104
103
102
101
100
105
104
103
102
101
100
105
104
103
102
101
100
105
104
103
102
101
100
0 50K 100K 150K 200K 250K
0 50K 100K 150K 200K 250K
0 50K 100K 150K 200K
0 50K 100K 150K 200K
105
104
103
102
101
100
105
104
103
102
101
100
105
104
103
102
101
100
105
104
103
102
101
100
PMIF
Pre-Tfh
Pre-Tfh
Tfh
Tfh
Pre-Tfh
3
2
1
0
3
4
5
2
1
0
2
1
0
Con
*
**
**
PMIF Con PMIF
Con PMIF Con PMIF
Con
15
Anti-Plasmodium IgG titer ×103
Anti-CSP IgG titer ×103
10
5
0
31.0
0.8
0.6
0.4
0.2
0.0
0.0
Con
PMIF
Con PMIF
**
CD19+CD138IgDCD38hi ×107 cells
2.0
1.5
1.0
0.5
Con
PMIF
**
**
#
#
2
1
0
PMIF
#
Ψ
1565
1765
2298
1699
Tfh
10
8
6
4
2
0
Fig. 6 PMIF inhibition enhances the development of CD4 Tfh, plasma cells, and anti-Plasmodium antibody responses. BALB/cJ mice immunized
with replicons encoding Con RNA or PMIF RNA were infected with 2000 PbAWT sporozoites and splenocytes isolated on day 7 after infection.
a,bRepresentative plots of absolute numbers of Tfh cells (CD4+CD62LCXCR5hiPD-1hi) and pre-Tfh cells (CD4+CD62LCXCR5intPD-1int). cExpression
of transcription factor Bcl-6 in Tfh and pre-Tfh cells for both groups of mice during rst PbAWT infection. Results are from two separate experiments. Bars
represent the mean of 6 mice ± SD; *p< 0.05, **p< 0.001, Ѱp< 0.0001 by MannWhitney test. d,eRepresentative plots and absolute number of germinal
center (CD19+CD38loGL7+) and memory B cells (CD19+CD138IgDCD38hi) after the rst and second infection. Results are from two separate
experiments. Bars represent the mean of 6 mice ± SD; **p< 0.001, #p< 0.0001 by MannWhitney test. Serum titers of specic anti-Plasmodium blood-
stage (f) and anti-CSP liver-stage antigen (g) IgG from immunized mice analyzed 1 week after the second infection with PbAWT sporozoites. Data shown
are from three and two independent experiments, respectively. Bars represent the mean of 10 mice ± SD; #p< 0.0001 by MannWhitney test
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parasitemia, a 30% reduction in peak parasitemia, a 30% pro-
longation in survival time in BALB/c mice (Supplementary Fig-
ure 9b), and a 30% reduction in lethality in C57/BL/6J mice
(Supplementary Figure 9c). These data indicate that while
humoral IgG was not as protective as CD4 T cell transfer, which
resulted in complete protection to PbAWT infection in BALB/c
mice, antibody produced in the setting of PMIF vaccination was
more effective in ameliorating lethality than antibody from the
vaccine controls.
Discussion
Immunity to malaria is slow to develop and the tolerance that
may develop to clinical disease requires repeated infection over
many years. The cellular and humoral mechanisms responsible
for the failure by the host to develop sterile immunity are not well
understood but have been considered to be features of an
immunosuppressive response. Several lines of evidence indicate
that acute malaria can inhibit T cell development with signicant
impact on GC development and activity5,2629, and recent
studies have identied that many of the same inammatory fac-
tors that mediate severe malaria have a deleterious effect on Tfh
and B cell development7,8. Herein we provide evidence for a
mechanism by which Plasmodium parasites negatively regulate
the adaptive response via the expression of PMIF. PMIF upre-
gulates IL-12 and IFN-γexpression to increase the inammatory
environment of the CD4 T cell response and interfere with the
differentiation of Plasmodium-specic CD4 T effector cells14. The
pro-inammatory action of PMIF during Plasmodium infection
blocks Tfh cell development, leading to a higher frequency of Tfh
a
ef
b
d
c
PMIF or C on
mice
Cd45.2+
CD4+ cells
+CFSE
CD45.1+
BALB/c
Rest
3 days
1 week
PbA
infection
Parasitemia
CD4 proliferation
8
6
2
% Parasitemia
4
0
03456
Days after infection
Con
PMIF
#
*
150
100
Counts
50
0
10
0
10
1
10
2
CFSE
10
3
10
4
10
5
Con
PMIF
CD4+CD45.2+
recovered × 107 cells
CD4+CD45.2+CFSElo
cells/spleen ×107
% CD4+CD45.2+CFSElo IFNγ cells
Mean CD4+CD45.2+CFSElo PD-1+
** **
2.5
2.0
1.5
1.0
0.5
0.0
25
**
*
20
15
10
5
0
50
40
30
20
10
0
1.0
0.8
0.6
0.4
0.2
0.0
Con PMIF
Con PMIF Con PMIF
Con PMIF
Fig. 7 Adoptively transferred CD4 T cells from PMIF-immunized mice confer protection to challenge by iRBCs. aBALB/cJ mice immunized with replicons
encoding Con RNA or PMIF RNA were infected with 106PbAWT iRBCs and treated with chloroquine on days 712. Four weeks later, the mice were
reinfected with 106PbAWT iRBCs and splenocytes isolated 7 days after infection, incubated with chloroquine to eliminate blood-stage parasites, and
labeled with CFSE. Puried CD4+CD45.2+T cells (2 × 107) then were transferred into naïve congenic CD45.1 BALB/cJ hosts and the mice infected 3 days
later with 106PbAWT iRBCs. bFrequency of iRBCs in mice adoptively transferred with CD4 T cells from Con (white circle) or PMIF (black circle) RNA
immunized mice. Results are from two separate experiments. Bars represent the mean of 6 mice ± SD; *p< 0.05, #p< 0.001 by two-way ANOVA.
cRepresentative CFSE dilution histogram of adoptively transferred CD4+T cells (CD45.2) from donors immunized with Con or PMIF RNA and
enumeration of recovered CD45.2 CD4+T cells, and dproliferative response of transferred CD4 T cells into CD45.1 recipients 7 days after infection.
ePercentage of proliferating CD45.2+CD4+T cells (CFSElo) producing IFN-γafter stimulation ex vivo with iRBC lysates in the presence of Brefeldin A.
fMean uorescence intensity of PD-1 in PbAWT responsive CD45.2+CD4+T cells (CFSElo) from Con or PMIF RNA immunized donors. Results are from
two separate experiments. Bars represent the mean of 8 mice ± SD; *p< 0.05, **p< 0.01 by two-tailed MannWhitney test
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Content courtesy of Springer Nature, terms of use apply. Rights reserved
precursors expressing Th1-type molecules such as T-bet and
IFNγ, and a lower frequency of pre-Tfh cells expressing Bcl-6.
This interference in Tfh cell maturation by PMIF is associated
with a detrimental effect on the induction of GC and B cell
responses (Supplementary Figure 11).
These ndings prompted us to examine if immunization
against PMIF could reduce its deleterious action during pre-
erythrocytic or erythrocytic stage infection. While initial spor-
ozoite infection produced patent infection in immunized hosts,
parasitemia was signicantly attenuated. Moreover, mice that
were cured of infection but received a second sporozoite challenge
were fully protected against re-infection. This protection was
associated with enhanced development of liver CSP-specic CD8
T cells that were predominantly of the Trm phenotype. The
protective action of liver-resident CD8 T cells was conrmed by
adoptive transfer, which fully protected naïve hosts from devel-
oping detectable parasitemia after sporozoite infection. When
infection was initiated with Plasmodium blood-stage malaria,
prior immunization against PMIF reduced the expression of IL-
12 and IFN-γ, resulting in an increased number of Plasmodium-
responsive CD4 T cell memory precursor cells, and an expansion
of CD4 T cell effector memory and memory population. A dis-
organization of GC architecture has long been noted to occur in
human and experimental malaria, and is associated with an
impairment in an effective antibody response7,30,31. PMIF
immunization led to a preservation of splenic GC architecture
and B cell zonal expansion, an increase in the number of CD4 Tfh
cells and GC B cells, and a higher anti-Plasmodium antibody titer.
Adoptive transfer of the CD4 T cells that develop in PMIF-
immunized mice during Plasmodium blood-stage infection also
conferred full protection to blood-stage infection in naïve hosts.
Upon transfer, these CD4 T cells showed enhanced Plasmodium-
specic proliferation and IFN-γproduction, and reduced
exhaustion.
Taken together, these ndings demonstrate remarkable pro-
tection in a lethal murine model of malaria initiated by sporozoite
or blood-stage infection. These data also afrm the role of the
Plasmodium-encoded factor PMIF in actively interfering with the
adaptive immune response by a pro-inammatory mechanism
involving engagement of the host MIF receptor14 to suppress the
differentiation of memory CD4 and CD8 T cell subsets. We
suggest that the marked protection observed by PMIF immuni-
zation may prompt consideration of this antigen as a vaccine
candidate, either as a standalone immunogen or in combination
with other Plasmodium antigens, where it could act to ensure the
development and maintenance of adequate memory responses in
endemic settings. It is notable that closely homologous MIF
orthologs have been described in other parasitic protozoan and
helminthic species3235. Conceivably, this family of evolutionary
conserved proteins provides a generalized mechanism by which
parasites interfere with the adaptive response to maintain per-
sistence in the mammalian host and completion of their life
cycles.
Methods
Mice, parasites, and cell lines. All animals were maintained in specic pathogen-
free facility at Yale Animal Resource center (YARC). All animal procedures fol-
lowed federal guidelines and were approved by the Yale University Animal Care
and Use Committee (UACUC), approval number 2017-10929. Females. BALB/cJ
(CD45.2, CD45.1) and C57/BL6J mice between 6 and 10 weeks of age were used
and purchased from Jackson Laboratories. Cryopreserved wild-type P. berghei
ANKA (MR4) (PbAWT) and PbAmifparasites13 were passaged once through
Swiss Webster mice before infection in experimental animals. Eight to ten-week-
ab
de
c
PMIF or Con
mice
Cd45.2+
CD8+
cells
CFSE
CD45.1+
BALB/c
Rest
3 days
1 week
PbAspz infection
Parasitemia
CD4 proliferation
15
10
5
0
Radiance (p/s/cm2/sr)×105
CD8+CD45.2+
CD11ahiCSPtetrhi CFSElo ×106 cells
CD8+CD45.2+
CD11ahiCSPtetrhi IFN-γhi ×106 cells
Con PMIF
4
3
1
% Parasitemia
2
0
0246810
Days after infection
Con
PMIF
**
**
**
**
**
50
40
30
20
Counts
10
0
100101102
CFSE
103104
Con
PMIF
5
4
3
2
1
0Con PMIF Con PMIF
1.5
1.0
0.5
0.0
Fig. 8 Adoptively transferred liver CD8 T cells from PMIF-immunized mice confer protection to homologous sporozoite challenge. aBALB/cJ mice
immunized with replicons encoding Con RNA or PMIF RNA were infected with 2000 PbAWT sporozoites and cured by 6 days of chloroquine treatment
(days 712). Four weeks later, the mice were reinfected with 2000 PbAWT sporozoites and T cells from liver isolated 7 days after infection, incubated with
chloroquine to eliminate residual blood-stage parasites, and labeled with CFSE. Puried CD45.2+CD8 T cells (2 × 107) then were transferred into
naïve congenic CD45.1 BALB/cJ hosts and the mice infected 3 days later with 2000 PbAWT sporozoites. bLuminescence values of infected mice and
cparasitemia in mice adoptively transferred with liver CD8 T cells from Con RNA (white circle) or PMIF RNA (black circle) immunized mice. Results are
from two separate experiments. Bars represent the mean of 6 mice ± SD **p< 0.01 by two-way ANOVA. dRepresentative CFSE dilution histogram of
adoptively transferred (CD45.2) CD8 T cells from Con RNA or PMIF RNA immunized donors and enumeration of recovered CD45.2 CD8 T cells. eNumber
of proliferating CD45.2 CD8 T cells (CFSElo) producing IFN-γafter stimulation ex vivo with CSP peptide in the presence of Brefeldin A. Results are from
two separate experiments. Bars represent the mean of 6 mice ± SD;**p< 0.01 by two-tailed MannWhitney test, error bars denote ±SD
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old mice were infected intraperitoneally (i.p.) with 106PbA iRBCs and the ensuing
parasitemia assessed by enumeration of Giemsa-stained thin blood smears, ow
cytometry using a Tri-color method (TCM)36 and by qPCR for splenic PbA 18S
rRNA37. For sporozoite infection, PbAWT,PbAmif(Leiden Malaria Group) or
PbAWT-GFP (MR4) parasites were cycled between Swiss Webster mice and
Anopheles stephensi mosquitoes. Salivary gland sporozoites were extracted from
infected mosquitoes on day 19 post-blood meal infection. BALB/cJ mice were
infected by tail i.v. injection of 2000 PbA sporozoites and blood patency was
monitored beginning day 3 by blood smear, ow cytometry and when infected with
PbAWT-GFP parasites, the liver burden was monitored using an IVIS imaging
system.
Adoptive transfer of splenic CD4 T cells and liver CD8 T cells. Splenocytes or
liver lymphocytes were isolated from vaccinated and infected CD45.2+BALB/cJ
mice at day 7 after the second infec tion and incubated with 10 µM chloroquine for
2 h at 37 °C. Splenic CD4 T and liver CD8 T cells were puried with anti-CD4 or
anti-CD8 microbeads (CD4 (L3T4) and CD8a (Ly-2) Myltenyi Biotech) according
to manufacturers protocol. Puried CD4 T or CD8 T cells were labeled with 5 µM
CFSE (Life Technologies) and 2 ×107cells transferred i.v. into recipient CD45.1+
BALB/cJ mice. Recipient mice were infected with 1 × 106iRBCs or 2000 sporozoites
3 days after transfer and parasitemia monitored daily. At day 7 post-infection for
iRBC infection or at day 9 for sporozoites infection, mice were sacriced and donor
CD4 or CD8 CD45.2+T cells recovered, quantied, and proliferation assessed by
CFSE dilution.
Flow cytometry. For splenic cell proling assessment, spleens were harvested at
the indicated days after infection, homogenized, and passed through a 70 μm
strainer to obtain single-cell suspensions. Red blood cells were lysed with ACK lysis
buffer and splenocytes were stained using uorophore-labeled antibodies (BD
Biosciences) directed against Ki67 as a surrogate marker of malaria-specic cells36,
CD3, CD4, IL-7Rα, CD62L, PD-1, CD45.2, and IFN-γfor memory CD4 T cells,
CXCR5, CD4, PD-1, CD62L, T-bet, Bcl6, and IFN-γfor Tfh or pre-Tfh cells, and
B220, CD19, CD138, IgM, Gl7, CD38, and IgD for the B cell lineage. For intra-
cellular cytokine staining, cells were stimulated ex vivo by co-culture of naïve
CD45.1 splenocytes with iRBC lysates or recombinant PMIF14 for 5 h in the pre-
sence of 1 μg/mL Brefeldin A (BD Bioscience). To assess the development of
memory immune responses in mice infected with sporozoites, perfused livers were
harvested at the indicated days after infection, homogenized, and the lymphocytes
isolated by centrifugation at 500gfor 15 min at RT using a 35% Percoll gradient;
splenocytes were isolated as described previously. Liver and spleen lymphocytes
were stained rst with CSP-specic tetramers (NIH Tetramer Facility) for 20 min
at 4 °C, followed by antibodies directed against CD3, CD8, CD69, KLGR1, CD44,
CD62L, CD45.1, and IFNγ(Biolegend) refer to Supplementary Table 1for anti-
bodies source and concentration. For intracellular cytokine staining, cells were
stimulated ex vivo by co-culture of naïve CD45.1 splenocytes with sporozoite
lysates for 5 h in the presence of 1 μg/mL of Brefeldin A before labeling with the
specied antibodies from Biolegend. For each staining, the corresponding Fluor-
escence Minus One (FMO) controls38 were performed (see Supplementary Fig-
ure 10 for representative analysis). Stained cells were analyzed on an LSR II ow
cytometer (BD Bioscience) and data processed with FlowJo software (TreeStar).
RNA synthesis, nanoparticle formulation, and vaccination. The synthesis of the
self-amplifying RNA (replicon) from a modied alphavirus encoding pmif or
control RNA was performed as previously described39. The control RNA com-
prised green uorescent protein (gfp)39, or in experiments requiring luminescent
Plasmodium detection, secreted placental alkaline phosphatase (seap)40. No dif-
ferences in background host responses were noted between the two different
controls (71,74). Briey, the codon optimized sequences were inserted into the
subgenomic reading frame of a modied DNA plasmid encoding the self-
amplifying RNA41. The plasmid was linearized by restriction enzyme digestion
immediately following the 3-end of the self-amplifying RNA sequence. Linearized
DNA was transcribed into RNA with the MEGAscript T7 Transcription Kit (Life
Technologies). Transcripts were puried by precipitation in the presence of 2.8 M
lithium chloride, capped using the ScriptCap m7G Capping System (CellScript),
and re-precipitated with lithium chloride. A cationic nanoemulsion was prepared
and characterized for particle size, RNase protection, and endotoxin as previously
described and allowed to complex for at least 30 min before immunization42.
Female BALB/cJ mice (810 weeks old) were injected i.m. in hind limbs on day 0
and on day 21 with 15 μg of the pmif or control RNA expressing replicon. Blood
was collected in both groups of animals by orbital bleeding of anesthetized mice at
day 0 pre-immunization, and at day 14 and day 35 post-immunization to titer the
anti-PMIF specic antibody response.
Western blotting and anti-PMIF and anti-Plasmodium ELISA. For the detection
of anti-PMIF antibody, microtiter plates (Nunc) were coated with 100 ng/mL of
recombinant PMIF or mouse MIF, incubated overnight, washed, and blocked with
assay diluent (eBioscience) for 1 h. Mouse sera were serially diluted and added to
wells for 2 h. Antibody binding to PMIF or mouse MIF14 was measured by addition
of HRP-labeled goat anti-mouse antibodies (1/1000, Southern Biotech). To
measure the titer of anti-Plasmodium-specic IgG, mouse sera were collected on
day 7 after infection and the microtiter plates were coated with 1 µg/mL of PbAWT
iRBC lysates as antigen43. Reciprocal endpoint titers were calculated as the reci-
procal of the dilution at which the O.D. was twice background observed in
uninfected mice.
For western blotting, 100 ng of recombinant PMIF or mouse MIF29 were
electrophoresed by SDS-PAGE in Tris-glycine gel (Bio-Rad) and then transferred to a
PVDF membrane (Millipore). The PMIF and mouse MIF proteins were probed for
serum antibody responses by the addition of PMIF or GFP (control) immune serum
(1/1000) and detection with rabbit anti-PMIF antibodies or goat anti-mouse MIF
antibodies (1/1000, Santa Cruz Biotech.) as previously described14. The signal was
developed by ECL HRP substrate and the membrane exposed to a lm (Amersham).
For the study of PMIF expression by merozoites, iRBC were lysed with saponin
lysis buffer (0.03% saponin in PBS) and merozoites pelleted by centrifugation at
1500×gfor 5 min at 4 °C. Merozoites were lysed with lysis buffer (25 mM TrisHCl
pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) for 30min
in ice44. The lysates were centrifugated at 15,000×gfor 15 min (4 °C) and the
supernatant (cytosolic fraction) and pellet (membrane fraction) were separated and
mixed with SDS-PAGE loading buffer (BioRad). Protein extracts were separated by
gel electrophoresis and transferred to PVDF membrane (Millipore). The
membrane was probed with rabbit anti-PMIF14 (1/1000) or anti-MSP-1 IgG (1/
1000) MRA-667, MR4 ATTC, and after incubation with anti-rabbit IgG-RD800 (1/
15000, Li-Cor), the membrane was imaged with an Odyssey system (Li-Cor). All
uncropped Western Blot scans are included in Supplementary Figure 12.
To measure the titer of anti-Plasmodium-specic IgG14, sera were collected on
day 7 after infection and microtiter plates coated with 1 µg/mL of PbA iRBC lysates
or 100 μg/mL of CSP peptide as antigen43. Reciprocal endpoint titers were
calculated as the reciprocal of the dilution at which the O.D. was twice background
observed in uninfected mice.
Histology. To examine GC architecture, spleens were removed from mice and
xed in 10% formalin (Sigma-Aldrich). For immunostaining, parafn sections
where deparafned, hydrated and treated with antigen retrieval buffer (Dako). The
sections then were permeabilized by 4 min treatment with Triton X-100 (0.1% in
PBS) and incubated for 90 min with blocking buffer (Dako) followed by an over-
night incubation with primary antibodies: rat anti-mouse B220 and hamster anti-
mouse CD3e (1/1000, BD Pharmingen). For immunohistochemistry, antibodies
were detected with AP-conjugated goat anti-Armenian hamster IgG or HRP-
conjugated donkey anti-rat IgG (1/10000, Jackson ImmunoResearch Laboratories).
HRP was reacted with DAB (Peroxidase Substrate Kit; Vector), and alkaline
phosphatase with Fast Blue/Napthol AS-MX (Sigma-Aldrich). Levamisole (Sigma)
was used to block endogenous alkaline phosphatase activity and slides were
mounted in Crystal Mount (Electron Microscopy Sciences). Sections were viewed
under a Nikon Microphot FXA light microscope and photographs were taken with
a Spot Insight Camera, using 10× and 40× objectives, then analyzed using Spot
Advanced software (Diagnostic Instruments) and ImageJ (NIH).
Statistical analysis. All data were expressed as a mean ± SD of at least two
independent experiments. Differences in parasitemia involving repeated mea-
surements were analyzed using a two-way ANOVA. Mouse survival times were
analyzed by the Mantel-Cox log-rank test. All other data were rst tested for
Gaussian distribution of values using a DAgostino-Pearson normality test. The
statistical signicance of differences was assessed using the MannWhitney Utest
for non-parametric data distribution or Studentst-test for parametric data. All
statistical analysis was performed using Software Prism v.6.0, (GraphPad). pValues
of less than 0.05, 0.01, or 0.001 were used to indicate statistical signicance.
Ethics approval. All animal procedures followed federal guidelines and were
approved by the Yale University Animal Care and Use Committee, approval
number 2017-10929
Data availability. The data that support the ndings of this study are available
from the corresponding author upon reasonable request.
Received: 16 September 2017 Accepted: 13 June 2018
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Acknowledgments
This work was funded by National Institutes of Health Grants AI 5R01-51306-05, AI
2R01-042310-12, and Novartis Vaccines, Inc. This study was supported by the Deutsche
Forschungsgemeinschaft grant SFB1123/A03 to J.B. We thank Michelle Chan and Nisha
Chandler for coordinating the delivery of formulations for the animal studies. We thank
MR4 for providing us with malaria parasites provided by Mark F. Wisser, Andy Waters,
and Victor Nussenzweig.
Author contributions
A.B.G., A.G., J.B. and R.B. conceived and designed the experiments. A.B.G., E.S., V.E. and
T.S. performed the experiments. L.B., A.H., G.O. and J.U. contributed with RNA vector
and vaccine production. C.J.J., E.F. and K.A. provided reagents. A.B.G. and R.B. analyzed
the data and wrote the paper.>
Additional information
Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467-
018-05041-7.
Competing interests: Yale University and Novartis AG have led a joint patent
application describing the potential utility of a pmif encoding RNA replicon. R.B. and
A.G. are co-inventors on this application. The remaining authors declare no competing
interests.
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... Plasmodium secretes a cytokine called macrophage migrating inhibitory factor (PMIF) that prevents the development of T cell long-term memory [222]. An experimental mRNA vaccine encoding PMIF improved the induction of T helper cells and memory development and elicited Plasmodium-specific IgG antibodies [223]. Moreover, the T cells induced by this vaccination were revealed to be protective for unvaccinated mice against challenge with Plasmodium sporozoites [223]. ...
... An experimental mRNA vaccine encoding PMIF improved the induction of T helper cells and memory development and elicited Plasmodium-specific IgG antibodies [223]. Moreover, the T cells induced by this vaccination were revealed to be protective for unvaccinated mice against challenge with Plasmodium sporozoites [223]. In a second study, another protein from Plasmodium falciparum was used as a target for mRNA expression (Plasmodium (P.) falciparum glutamic-acid-rich protein (PfGARP)). ...
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Many bacterial infections are major health problems worldwide, and treatment of many of these infectious diseases is becoming increasingly difficult due to the development of antibiotic resistance, which is a major threat. Prophylactic vaccines against these bacterial pathogens are urgently needed. This is also true for bacterial infections that are still neglected, even though they affect a large part of the world’s population, especially under poor hygienic conditions. One example is typhus, a life-threatening disease also known as “war plague” caused by Rickettsia prowazekii, which could potentially come back in a war situation such as the one in Ukraine. However, vaccination against bacterial infections is a challenge. In general, bacteria are much more complex organisms than viruses and as such are more difficult targets. Unlike comparatively simple viruses, bacteria possess a variety of antigens whose immunogenic potential is often unknown, and it is unclear which antigen can elicit a protective and long-lasting immune response. Several vaccines against extracellular bacteria have been developed in the past and are still used successfully today, e.g., vaccines against tetanus, pertussis, and diphtheria. However, while induction of antibody production is usually sufficient for protection against extracellular bacteria, vaccination against intracellular bacteria is much more difficult because effective defense against these pathogens requires T cell-mediated responses, particularly the activation of cytotoxic CD8+ T cells. These responses are usually not efficiently elicited by immunization with non-living whole cell antigens or subunit vaccines, so that other antigen delivery strategies are required. This review provides an overview of existing antibacterial vaccines and novel approaches to vaccination with a focus on immunization against intracellular bacteria.
... Several studies have also been conducted towards the development of an mRNA vaccine against malaria. The first is an saRNA-based [151] encoding for Plasmodiumsecreted cytokine macrophage migrating inhibitory factor, which had previously shown efficacy [152]. In this study, anti-Plasmodium antibodies and protective T-cell memory were achieved [151]. ...
... The first is an saRNA-based [151] encoding for Plasmodiumsecreted cytokine macrophage migrating inhibitory factor, which had previously shown efficacy [152]. In this study, anti-Plasmodium antibodies and protective T-cell memory were achieved [151]. In another study, Plasmodium falciparum acid-rich protein (PfGARP) mRNA-encoded LNPs were dosed to infected aotus monkeys, showing reduced levels of the parasite after three doses [153]. ...
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The year 2020 was a turning point in the way society perceives science. Messenger RNA (mRNA) technology finally showed and shared its potential, starting a new era in medicine. However, there is no doubt that commercialization of these vaccines would not have been possible without nanotechnology, which has finally answered the long-term question of how to deliver mRNA in vivo. The aim of this review is to showcase the importance of this scientific milestone for the development of additional mRNA therapeutics. Firstly, we provide a full description of the marketed vaccine formulations and disclose LNPs’ pharmaceutical properties, including composition, structure, and manufacturing considerations Additionally, we review different types of lipid-based delivery technologies currently in preclinical and clinical development, namely lipoplexes and cationic nanoemulsions. Finally, we highlight the most promising clinical applications of mRNA in different fields such as vaccinology, immuno-oncology, gene therapy for rare genetic diseases and gene editing using CRISPR Cas9.
... The mRNA platform offers enhanced stability and targeted antigen expression and has already proven successful against challenging diseases where conventional technology has failed. To date, mRNA vaccines against three different single-celled parasites (Plasmodium malaria [112,113], Leishmania donovani [114] and Toxoplasma gondii [115]) have been developed. Anti-Plasmodium mRNA vaccines targeting the circumsporozoite protein (PfCSP) and cytokine macrophage migration inhibitory factor (PMIF) have been successfully tested; they induce strong specific CD4+ T cell responses and high titer IgG antibodies, resulting in the generation of protective immunity against malaria infection in mice [112,113]. ...
... To date, mRNA vaccines against three different single-celled parasites (Plasmodium malaria [112,113], Leishmania donovani [114] and Toxoplasma gondii [115]) have been developed. Anti-Plasmodium mRNA vaccines targeting the circumsporozoite protein (PfCSP) and cytokine macrophage migration inhibitory factor (PMIF) have been successfully tested; they induce strong specific CD4+ T cell responses and high titer IgG antibodies, resulting in the generation of protective immunity against malaria infection in mice [112,113]. Heterologous mRNA has also been used to vaccinate mice against L. donovani infection, resulting in a significant reduction in liver parasite burden through inducing strong IFN-γ secretion and antigen-specific Th1 responses by splenocytes [114]. A self-amplifying mRNA-LNPs approach was also utilized to develop an effective vaccine against T. gondii infection in mice [115]. ...
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Schistosomiasis, caused by human trematode blood flukes (schistosomes), remains one of the most prevalent and serious of the neglected tropical parasitic diseases. Currently, treatment of schistosomiasis relies solely on a single drug, the anthelmintic praziquantel, and with increased usage in mass drug administration control programs for the disease, the specter of drug resistance developing is a constant threat. Vaccination is recognized as one of the most sustainable options for the control of any pathogen, but despite the discovery and reporting of numerous potentially promising schistosome vaccine antigens, to date, no schistosomiasis vaccine for human or animal deployment is available. This is despite the fact that Science ranked such an intervention as one of the top 10 vaccines that need to be urgently developed to improve public health globally. This review summarizes current progress of schistosomiasis vaccines under clinical development and advocates the urgent need for the establishment of a revolutionary and effective anti-schistosome vaccine pipeline utilizing cutting-edge technologies (including developing mRNA vaccines and exploiting CRISPR-based technologies) to provide novel insight into future vaccine discovery, design, manufacture and deployment.
... RNA-based vaccines have been used to deliver bacterial and parasite genes but, except for vaccine candidates for Chlamydia trachomatis [110], most of these remain in preclinical or early clinical stages of development (e.g., those against the protozoan Toxoplasma gondii [111], Plasmodium [112], and Leishmania donovani [113]). Recently, Raj et al. [114] developed an RNA vaccine for the expression of the glutamic-acid-rich protein (PfGARP) of Plasmodium falciparum and showed that it induced antibody formation in in vitro assays and in a non-human primate challenge model. ...
... Vaccines 2021, 9, 1345 9 of 38 plasma gondii [111], Plasmodium [112], and Leishmania donovani [113]). Recently, Raj et al. [114] developed an RNA vaccine for the expression of the glutamic-acid-rich protein (PfGARP) of Plasmodium falciparum and showed that it induced antibody formation in in vitro assays and in a non-human primate challenge model. ...
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In recent years, vaccine development using ribonucleic acid (RNA) has become the most promising and studied approach to produce safe and effective new vaccines, not only for prophylaxis but also as a treatment. The use of messenger RNA (mRNA) as an immunogenic has several advantages to vaccine development compared to other platforms, such as lower coast, the absence of cell cultures, and the possibility to combine different targets. During the COVID-19 pandemic, the use of mRNA as a vaccine became more relevant; two out of the four most widely applied vaccines against COVID-19 in the world are based on this platform. However, even though it presents advantages for vaccine application, mRNA technology faces several pivotal challenges to improve mRNA stability, delivery, and the potential to generate the related protein needed to induce a humoral- and T-cell-mediated immune response. The application of mRNA to vaccine development emerged as a powerful tool to fight against cancer and non-infectious and infectious diseases, for example, and represents a relevant research field for future decades. Based on these advantages, this review emphasizes mRNA and self-amplifying RNA (saRNA) for vaccine development, mainly to fight against COVID-19, together with the challenges related to this approach.
Chapter
mRNA vaccines have been increasingly recognized as a powerful vaccine platform since the FDA approval of two COVID-19 mRNA vaccines, which demonstrated outstanding prevention efficacy as well as great safety profile. Notably, nucleoside modification and lipid nanoparticle-facilitated delivery has greatly improved the immunogenicity, stability, and translation efficiency of mRNA molecule. Here we review the recent progress in mRNA vaccine development, including nucleoside modification, in vitro synthesis and product purification, and lipid nanoparticle vectors for in vivo delivery and efficient translation. We also briefly introduce the clinical application of mRNA vaccine in preventing infectious diseases and treating inflammatory diseases including cancer.
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The first malaria vaccine has been recently approved for children living in malaria-endemic areas. While this is long-awaited and welcome news, the modest efficacy of the vaccine highlights several areas that require further attention. Here, we describe the likely impact of the vaccine and where clinical and basic discovery research will still be required.
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Messenger RNA (mRNA) vaccines have been studied for decades, but only recently, during the COVID-19 pandemic, has the technology garnered noteworthy attention. In contrast to traditional vaccines, mRNA vaccines elicit a more balanced immune response, triggering both humoral and cellular components of the adaptive immune system. However, some inherent hurdles associated with stability, immunogenicity, in vivo delivery, along with the novelty of the technology, have generated scepticism in the adoption of mRNA vaccines. Recent developments have pushed to bypass these issues and the approval of mRNA-based vaccines to combat COVID-19 has further highlighted the feasibility, safety, efficacy, and rapid development potential of this platform, thereby pushing it to the forefront of emerging therapeutics. This review aims to demystify mRNA vaccines, delineating the evolution of the technology which has emerged as a timely solution to COVID-19 and exploring the immense potential it offers as a prophylactic option for other cryptic diseases.
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Gene therapy has emerged as a potential platform for treating several dreaded and rare diseases that would otherwise not be possible with traditional therapies. Due to their ability to transport genomes to cells, Viral vectors have been a platform of choice in gene delivery applications. However, since their delivery is not precision based, the application has led to off-target toxicities. As such, various strategies in the form of non-viral gene delivery vehicles have been explored and are being developed. In this review, we discuss the opportunities lipid nanoparticles (LNPs) present for gene delivery, efficiently and precisely. We also discuss synthesis strategies via microfluidics used for high throughput fabrication of such non-viral gene delivery vehicles. Finally, the application of these vehicles for the delivery of different genetic materials such as peptides and RNA for different diseases ranging from more common diseases to rare diseases are explored.
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Infectious diseases are a leading cause of death worldwide, and vaccines are the cheapest and efficient approach to preventing diseases. Use of conventional vaccination strategies such as live, attenuated, and subunit has limitations as it does not fully provide protection against many infectious diseases. Hence, there was a need for the development of a new vaccination strategy. Use of nucleic acids—DNA and RNA—has emerged as promising alternative to conventional vaccine approaches. Knowledge of mRNA biology, chemistry, and delivery systems in recent years have enabled mRNA to become a promising vaccine candidate. One of the advantages of a mRNA vaccine is that clinical batches can be generated after the availability of a sequence encoding the immunogen. The process is cell-free and scalable. mRNA is a noninfectious, nonintegrating molecule and there is no potential risk of infection or mutagenesis. mRNA is degraded by normal cellular processes, and its in vivo half-life can be regulated by different modifications and delivery methods. The efficacy can be increased by modifications of the nucleosides that can make mRNA more stable and highly translatable. Efficient in vivo delivery can be achieved by formulating mRNA into carrier molecules, allowing rapid uptake and expression in the cytoplasm. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and spread globally, prompting an international effort to accelerate development of a vaccine. The spike (S) glycoprotein mediates host cell attachment and is required for viral entry; it is the primary vaccine target for many candidate SARS-CoV-2 vaccines. Development of a lipid nanoparticle encapsulated mRNA vaccine that encodes the SARS-CoV-2 S glycoprotein stabilized in its prefusion conformation conferred 95% protection against Covid-19.
Chapter
Throughout the world, billions of people are infected with various diseases and continue to suffer from them despite various treatments. Vaccination is commonly regarded as one of the most advanced approaches to disease prevention. RNA-based innovations have sparked widespread interest in the production of prophylactic and therapeutic vaccines over the last two decades. Because of their high efficacy, safe administration, and low manufacturing cost, mRNA vaccines have emerged as a promising tool for disease prevention. In animal models and humans, mRNA vaccines can induce a healthy, long-lasting cellular and humoral immune response. Furthermore, mRNA is an intrinsically secure vector that is just a transient carrier of information that does not interfere with the genome and provides full production versatility. Following the outbreak of COVID-19 in December 2020, mRNA-based vaccines made headlines in 2020. This chapter covers mRNA vaccines (both traditional and alternative), their delivery, immune responses elicited by them, and mRNA vaccines for infectious disease prevention.
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Naturally acquired immunity to malaria develops only after years of repeated exposure to Plasmodium parasites. Despite the key role antibodies play in protection, the cellular processes underlying the slow acquisition of immunity remain unknown. Using mouse models, we show that severe malaria infection inhibits the establishment of germinal centers (GCs) in the spleen. We demonstrate that infection induces high frequencies of T follicular helper (Tfh) cell precursors but results in impaired Tfh cell differentiation. Despite high expression of Bcl-6 and IL-21, precursor Tfh cells induced during infection displayed low levels of PD-1 and CXCR5 and co-expressed Th1-associated molecules such as T-bet and CXCR3. Blockade of the inflammatory cytokines TNF and IFN-γ or T-bet deletion restored Tfh cell differentiation and GC responses to infection. Thus, this study demonstrates that the same pro-inflammatory mediators that drive severe malaria pathology have detrimental effects on the induction of protective B cell responses.
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Malaria-specific antibody responses are short lived in children, leaving them susceptible to repeated bouts of febrile malaria. The cellular and molecular mechanisms underlying this apparent immune deficiency are poorly understood. Recently, T follicular helper (Tfh) cells have been shown to play a critical role in generating long-lived antibody responses. We show that Malian children have resting PD-1+CXCR5+CD4+ Tfh cells in circulation that resemble germinal center Tfh cells phenotypically and functionally. Within this population, PD-1+CXCR5+CXCR3− Tfh cells are superior to Th1-polarized PD-1+CXCR5+CXCR3+ Tfh cells in helping B cells. Longitudinally, we observed that malaria drives Th1 cytokine responses, and accordingly, the less-functional Th1-polarized Tfh subset was preferentially activated and its activation did not correlate with antibody responses. These data provide insights into the Tfh cell biology underlying suboptimal antibody responses to malaria in children and suggest that vaccine strategies that promote CXCR3− Tfh cell responses may improve malaria vaccine efficacy.
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Author Summary The importance of antibody and B-cell responses for control of the erythrocytic-stage of the malaria parasite, Plasmodium, was first described when immune serum, passively transferred into Plasmodium falciparum-infected children, reduced parasitemia. This was later confirmed in experimental models in which mice deficient in B cells were unable to eliminate erythrocytic-stage infections. The signals required to activate these protective long-lasting B cell responses towards Plasmodium have not been investigated. IL-21 has been shown to be important for development of B-cell responses after immunization; however, a direct requirement for IL-21 in the control of infection via B-cell dependent mechanisms has never been demonstrated. In this paper, we have used mouse models of erythrocytic P. chabaudi and P. yoelii 17X(NL) infections in combination with IL-21/IL-21R deficiency to show that IL-21 from CD4+ T cells is required to eliminate Plasmodium infection by activating protective, long-lasting B-cell responses. Disruption of IL-21 signaling in B cells prevents the elimination of the parasite resulting in sustained high parasitemias, with no development of memory B-cells, lack of antigen-specific plasma cells and antibodies, and thus no protective immunity against a second challenge infection. Our data demonstrate the absolute requirement of IL-21 for B-cell control of this systemic infection. This has important implications for the design of vaccines against Plasmodium.
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Nucleic acid-based vaccines such as viral vectors, plasmid DNA, and mRNA are being developed as a means to address a number of unmet medical needs that current vaccine technologies have been unable to address. Here we describe a cationic nanoemulsion delivery system developed to deliver a self-amplifying mRNA vaccine. This non-viral delivery system is based on Novartis's proprietary adjuvant MF59, which has an established clinical safety profile and is well tolerated in children, adults and the elderly. We show that non-viral delivery of a 9 kb self-amplifying mRNA elicits potent immune responses in mice, rats, rabbits, and non-human primates comparable to a viral delivery technology, and demonstrate that, relatively low doses (75 µg) induce antibody and T-cell responses in primates. We also show the cationic nanoemulsion-delivered self-amplifying mRNA enhances the local immune environment through recruitment of immune cells similar to an MF59 adjuvanted sub-unit vaccine. Lastly, we show that the site of protein expression within the muscle and magnitude of protein expression is similar to a viral vector. Given the demonstration that self-amplifying mRNA delivered using a cationic nanoemulsion is well tolerated and immunogenic in a variety of animal models, we are optimistic about the prospects for this technology.Molecular Therapy (2014); doi:10.1038/mt.2014.133.
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Malaria is a significant global burden but after >30 years of effort there is no vaccine on the market. While the complex life cycle of the parasite presents several challenges, many years of research have also identified several mechanisms of immune evasion by Plasmodium spp. Recent research on malaria, has investigated the programmed cell death-1 (PD-1) pathway which mediates exhaustion of T cells, characterized by poor effector functions and recall responses and in some cases loss of the cells by apoptosis. Such studies have shown exhaustion of CD4(+) T cells and an unappreciated role for CD8(+) T cells in promoting sterile immunity against blood stage malaria. This is because PD-1 mediates up to a 95% reduction in numbers and functional capacity of parasite-specific CD8(+) T cells, thus masking their role in protection. The role of T cell exhaustion during malaria provides an explanation for the absence of sterile immunity following the clearance of acute disease which will be relevant to future malaria-vaccine design and suggests the need for novel therapeutic solutions. This review will thus examine the role of PD-1-mediated T cell exhaustion in preventing lasting immunity against malaria.
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The timing of vaccine availability is essential for an effective response to pandemic influenza. In 2009, vaccine became available after the disease peak, and this has motivated the development of next generation vaccine technologies for more rapid responses. The SAM® vaccine platform, now in pre-clinical development, is based on a synthetic, self-amplifying mRNA, delivered by a synthetic lipid nanoparticle (LNP). When used to express seasonal influenza hemagglutinin (HA), a SAM vaccine elicited potent immune responses, comparable to those elicited by a licensed influenza subunit vaccine preparation. When the sequences coding for the HA and neuraminidase (NA) genes from the H7N9 influenza outbreak in China were posted on a web-based data sharing system, the combination of rapid and accurate cell-free gene synthesis and SAM vaccine technology allowed the generation of a vaccine candidate in 8 days. Two weeks after the first immunization, mice had measurable hemagglutinin inhibition (HI) and neutralizing antibody titers against the new virus. Two weeks after the second immunization, all mice had HI titers considered protective. If the SAM vaccine platform proves safe, potent, well tolerated and effective in humans, fully synthetic vaccine technologies could provide unparalleled speed of response to stem the initial wave of influenza outbreaks, allowing first availability of a vaccine candidate days after the discovery of a new virus.
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Interleukin-27 (IL-27) is known to control primary CD4+ T cell responses during a variety of different infections, but its role in regulating memory CD4+ T responses has not been investigated in any model. In this study, we have examined the functional importance of IL-27 receptor (IL-27R) signaling in regulating the formation and maintenance of memory CD4+ T cells following malaria infection and in controlling their subsequent reactivation during secondary parasite challenge. We demonstrate that although the primary effector/memory CD4+ T cell response was greater in IL-27R-deficient (WSX-1−/−) mice following Plasmodium berghei NK65 infection than in wild-type (WT) mice, there were no significant differences in the size of the maintained memory CD4+ T population(s) at 20 weeks postinfection in the spleen, liver, or bone marrow of WSX-1−/− mice compared with WT mice. However, the composition of the memory CD4+ T cell pool was slightly altered in WSX-1−/− mice following clearance of primary malaria infection, with elevated numbers of late effector memory CD4+ T cells in the spleen and liver and increased production of IL-2 in the spleen. Crucially, WSX-1−/− mice displayed significantly enhanced parasite control compared with WT mice following rechallenge with homologous malaria parasites. Improved parasite control in WSX-1−/− mice during secondary infection was associated with elevated systemic production of multiple inflammatory innate and adaptive cytokines and extremely rapid proliferation of antigen-experienced T cells in the liver. These data are the first to demonstrate that IL-27R signaling plays a role in regulating the magnitude and quality of secondary immune responses during rechallenge infections.
Article
RNA-based vaccines have recently emerged as a promising alternative to the use of DNA-based and viral vector vaccines, in part because of the potential to simplify how vaccines are made and facilitate a rapid response to newly emerging infections. SAM vaccines are based on engineered self-amplifying mRNA (SAM) replicons encoding an Ag, and formulated with a synthetic delivery system, and they induce broad-based immune responses in preclinical animal models. In our study, in vivo imaging shows that after the immunization, SAM Ag expression has an initial gradual increase. Gene expression profiling in injection-site tissues from mice immunized with SAM-based vaccine revealed an early and robust induction of type I IFN and IFN-stimulated responses at the site of injection, concurrent with the preliminary reduced SAM Ag expression. This SAM vaccine-induced type I IFN response has the potential to provide an adjuvant effect on vaccine potency, or, conversely, it might establish a temporary state that limits the initial SAM-encoded Ag expression. To determine the role of the early type I IFN response, SAM vaccines were evaluated in IFN receptor knockout mice. Our data indicate that minimizing the early type I IFN responses may be a useful strategy to increase primary SAM expression and the resulting vaccine potency. RNA sequence modification, delivery optimization, or concurrent use of appropriate compounds might be some of the strategies to finalize this aim.
Article
In recent years, various intervention strategies have reduced malaria morbidity and mortality, but further improvements probably depend upon development of a broadly protective vaccine. To better understand immune requirement for protection, we examined liver-stage immunity after vaccination with irradiated sporozoites, an effective though logistically difficult vaccine. We identified a population of memory CD8⁺ T cells that expressed the gene signature of tissue-resident memory T (Trm) cells and remained permanently within the liver, where they patrolled the sinusoids. Exploring the requirements for liver Trm cell induction, we showed that by combining dendritic cell-targeted priming with liver inflammation and antigen recognition on hepatocytes, high frequencies of Trm cells could be induced and these cells were essential for protection against malaria sporozoite challenge. Our study highlights the immune potential of liver Trm cells and provides approaches for their selective transfer, expansion, or depletion, which may be harnessed to control liver infections or autoimmunity.
Article
Leishmania major encodes 2 orthologs of the cytokine macrophage migration inhibitory factor (MIF), whose functions in parasite growth or in the host-parasite interaction are unknown. To determine the importance of Leishmania-encoded MIF, both LmMIF genes were removed to produce an mif(-/-) strain of L. major. This mutant strain replicated normally in vitro but had a 2-fold increased susceptibility to clearance by macrophages. Mice infected with mif(-/-) L. major, when compared to the wild-type strain, also showed a 3-fold reduction in parasite burden. Microarray and functional analyses revealed a reduced ability of mif(-/-) L. major to activate antigen-presenting cells, resulting in a 2-fold reduction in T-cell priming. In addition, there was a reduction in inflammation and effector CD4 T-cell formation in mif(-/-) L. major-infected mice when compared to mice infected with wild-type L. major. Notably, effector CD4 T cells that developed during infection with mif(-/-) L. major demonstrated statistically significant differences in markers of functional exhaustion, including increased expression of IFN-γ and IL-7R, reduced expression of programmed death-1, and decreased apoptosis. These data support a role for LmMIF in promoting parasite persistence by manipulating the host response to increase the exhaustion and depletion of protective CD4 T cells.-Holowka, T., Castilho, T. M., Baeza Garcia, A., Sun, T., McMahon-Pratt, D., Bucala, R. Leishmania-encoded orthologs of macrophage migration inhibitory factor regulate host immunity to promote parasite persistence.