RESEARCH ARTICLE

Neither the HIV Protease Inhibitor

Lopinavir-Ritonavir nor the Antimicrobial
Trimethoprim-Sulfamethoxazole Prevent
Malaria Relapse in Plasmodium cynomolgiInfected Non-Human Primates
Charlotte V. Hobbs1*, Saurabh Dixit1, Scott R. Penzak2, Tejram Sahu1,
Sachy Orr-Gonzalez1, Lynn Lambert1, Katie Zeleski1, Jingyang Chen1,
Jillian Neal1, William Borkowsky3, Yimin Wu1, Patrick E. Duffy1
1. Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Rockville, Maryland, United States of America, 2. Department of
Pharmacotherapy, University of North Texas System College of Pharmacy, Fort Worth, Texas, 76107, United
States of America, 3. Department of Pediatrics, Division of Infectious Disease and Immunology, New York
University School of Medicine, New York, New York, United States of America
OPEN ACCESS
Citation: Hobbs CV, Dixit S, Penzak SR, Sahu T,
Orr-Gonzalez S, et al. (2014) Neither the HIV
Protease Inhibitor Lopinavir-Ritonavir nor the
Antimicrobial Trimethoprim-Sulfamethoxazole
Prevent Malaria Relapse in Plasmodium cynomolgi-Infected Non-Human Primates. PLoS
ONE 9(12): e115506. doi:10.1371/journal.pone.
0115506
Editor: Soren Gantt, University of British
Columbia, Canada
Received: July 31, 2014
Accepted: November 24, 2014
Published: December 26, 2014
This is an open-access article, free of all copyright,
and may be freely reproduced, distributed,
transmitted, modified, built upon, or otherwise used
by anyone for any lawful purpose. The work is
made available under the Creative Commons CC0
public domain dedication.
Data Availability: The authors confirm that all data
underlying the findings are fully available without
restriction. All relevant data are within the paper
and its Supporting Information files.
Funding: This work was supported by the National
Institutes of Health Division of Intramural Research.
The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.

*[email protected]

Abstract
Plasmodium vivax malaria causes significant morbidity and mortality worldwide,
and only one drug is in clinical use that can kill the hypnozoites that cause P. vivax
relapses. HIV and P. vivax malaria geographically overlap in many areas of the
world, including South America and Asia. Despite the increasing body of knowledge
regarding HIV protease inhibitors (HIV PIs) on P. falciparum malaria, there are no
data regarding the effects of these treatments on P. vivaxs hypnozoite form and
clinical relapses of malaria. We have previously shown that the HIV protease
inhibitor lopinavir-ritonavir (LPV-RTV) and the antibiotic trimethoprim
sulfamethoxazole (TMP-SMX) inhibit Plasmodium actively dividing liver stages in
rodent malarias and in vitro in P. falciparum, but effect against Plasmodium dormant
hypnozoite forms remains untested. Separately, although other antifolates have
been tested against hypnozoites, the antibiotic trimethoprim sulfamethoxazole,
commonly used in HIV infection and exposure management, has not been
evaluated for hypnozoite-killing activity. Since Plasmodium cynomolgi is an
established animal model for the study of liver stages of malaria as a surrogate for
P. vivax infection, we investigated the antimalarial activity of these drugs on
Plasmodium cynomolgi relapsing malaria in rhesus macaques. Herein, we
demonstrate that neither TMP-SMX nor LPV-RTV kills hypnozoite parasite liver
stage forms at the doses tested. Because HIV and malaria geographically overlap,

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Neither Lopinavir-RTV nor Trimethoprim-SMX Prevent Malaria Relapse

and more patients are being managed for HIV infection and exposure,
understanding HIV drug impact on malaria infection is important.

Introduction
Plasmodium vivax malaria causes significant morbidity and mortality worldwide
[1]. HIV and Plasmodium vivax malaria geographically overlap in many areas of
the world, including South America and Asia, and impact of drugs used in HIV
management on P. vivax remains understudied. In addition, treatment of P. vivax
is complicated by the ability of the parasite to assume a hypnozoite form in the
liver stages, which leads to relapsing malaria [24].
The World Health Organization (WHO) recommends HIV management with
combination antiretroviral therapy (ARV), with first line therapy including a nonnucleoside reverse transcriptase inhibitor (NNRTI) and 2 nucleoside reverse
transcriptase inhibitors (NRTIs) with few exceptions, and second line therapy
including an HIV protease inhibitor (HIV PI) and 2 NRTIs [5]. We have
previously shown that HIV PIs kill actively dividing liver stage forms in rodent
malaria models and in P. falciparum in vitro, whereas NNRTIs do not [6, 7]. In
addition, HIV PIs have been shown to kill P. vivax in ex vivo culture [4]. We and
others have demonstrated that HIV PIs kill malaria parasites in asexual and sexual
blood stages, and they can block transmission, in vitro [816]. In addition, a
recent clinical study showed a reduction in recurrent malaria events with the use
of the HIV PI lopinavir-ritonavir (LPV-RTV) in an area of high transmission
intensity where P. falciparum malaria is prevalent [17], with a reduced risk of
gametocytemia in HIV PI-treated patients on the day of malaria diagnosis as well
as 28 days later [18]. Separately, many clinical studies have shown the antibiotic,
trimethoprim-sulfamethoxazole (TMP-SMX), used to prevent opportunistic
infections in HIV-exposed infants and HIV-infected patients [5] can reduce
clinical malaria burden [3], and we have previously shown that TMP-SMX blocks
development of dividing liver stage Plasmodium parasites in rodent malaria
models [19] and in P. falciparum in vitro [19]. However, whether LPV-RTV or
TMP-SMX kill liver stage hypnozoites, or dormant liver stage form responsible for
malaria relapse, has not been tested. Drug effect on the hypnozoite is important to
investigate as this form perpetuates the cycle of transmission. Indeed, the only
clinically available drug which can target this stage is primaquine, which cannot be
used in patients with glucose 6 phosphate dehydrogenase (G6PD) deficiency or in
pregnant women, and which requires a 14 day course to kill hypnozoite forms in
the liver [20].
Given the overlap of HIV and malaria with HIV-infected and exposed patients
in endemic areas receiving these drugs, and given the shortage of drugs available
that offer radical cure for P. vivax malaria, we set out to investigate the effects of
the HIV PI lopinavir (here, LPV-RTV), the most potent antimalarial HIV PI with

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Neither Lopinavir-RTV nor Trimethoprim-SMX Prevent Malaria Relapse

other Plasmodium species [6, 12], and TMP-SMX, on P. cynomolgi liver stage
hypnozoite forms, an established animal model for P. vivax [20].

Materials and Methods

Non-Human Primates
All studies described herein were performed on an animal study proposal
approved by the NIAID Institutional Animal Care and Use Committee. Macaca
mulatta (rhesus macaque) monkeys of Indian origin maintained in facilities
accredited by the Association for Assessment and Accreditation of Laboratory
Animal Care were used, and all experiments were performed in accordance with
the guidelines of the NIAID Division of Intramural Research (DIR) Animal Care
and Use Committee (Permit Number ASP LMIV 9E). For the pilot experiments
(summarized in Table 1), 3 male monkeys were used with weight ranging from
8.19.36 kg. Six male and 3 female animals were used for the experiment testing
drug effects, with weight ranging from 4.94 to 10.34 kg. Animals were observed at
least twice per day with abnormalities reported to the staff veterinarian by the end
of the observation period. Animals were gavaged and infected under sedation with
ketamine (0.1 cc/kg 10% Ketamine intramuscularly, or IM), with close
monitoring for clinical and behavioral changes, and all efforts were made to
minimize suffering. Our study endpoint was the detection of parasites on malaria
smear, at which time animals were immediately treated (,1% parasitemia).

Monitoring and Treatment of Malaria Infections (Fig. 1)

This relapse study model was conducted as previously with few modifications
[2024]. No monkeys used in this study were previously involved in other malaria
vaccination or antimalarial drug testing studies, and monkeys had baseline CBC
and chemistries (including liver function tests) that were within normal limits two
weeks preceding the study. All drugs used were commercially available and
purchased, except quinine dihydrochloride (kind gift of Dr. Jetsumon
Sattabongkot Prachumsri, Mahidol University, Bangkok, Thailand).
Quinine dihydrochloride dosing was based on a mg/kg regimen used at the
United States Army Materiel Command-United States Army Armed Forces
Research Institute of Medical Sciences (USSAMC-AFRIMS, Bangkok, Thailand)
(Dr. Imerbsin Rawiwan, United States Army Material Command (pers. comm.)),
where quinine 20 mg/kg base dose IM BID for 7 days with a single dose of
artesunate 8 mg/kg IV times one is used for high parasitemias. From this, we
derived a dose of 32 mg/kg quinine dihydrochloride (conversion based on
approximation of clinical converting recommendations [25]), twice daily by
intramuscular (IM) injection for 5 days [26] to clear asexual parasite forms, and
to avoid possible pharmacokinetic interactions between quinine and LPV-RTV
[2730], Quinidine gluconate dosing was based on the quinine base 20 mg/kg
regimen and 26 mg/kg quinidine gluconate IM twice per day for 7 days was used

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Neither Lopinavir-RTV nor Trimethoprim-SMX Prevent Malaria Relapse

Table 1. Treatment with Quinidine gluconate in Rhesus Monkeys: Dose: 26 mg/kg IM Twice Per Day x 7 days.
Infection with P.cynomolgi

Result

Frozen vial infected red blood cells* (Animal CL4K)**

Parasite detected D4, treated, smear negative for 6 weeks

Frozen vial infected red blood cells at 0.9% parasitemia x2 on D0 and D7

(Animal CL4K, donor)**

Parasite detected D9, treated, smear negative for 6 weeks

Infected-Mosquito bites (Animal DBOB)

Primary attack D9: treated with quinidine; relapse #1 D29, #2

D46

Infected-Mosquito bites (Animal DB9P)

Primary attack D11: treated with quinidine; relapse #1 D29, #2

D45

*Kind gift of Bill Collins, PhD to Bob Gwadz, PhD.

**these experiments demonstrate curative efficacy of regimen in curing asexual blood stages since there is no possibility of relapse.
doi:10.1371/journal.pone.0115506.t001

(conversion based also on approximation of clinical converting recommendations

[31]).
Prior to the below experiment, pilot studies using P. cynomolgi infected red
blood cell stock were originally provided by Dr. Bill Collins (Centers for Disease
Control and Prevention, Atlanta, Georgia, USA), or from our own prepared

Fig. 1. Drug Efficacy Against Relapse Study Schematic. On Day 0, all monkeys were infected by with bites of P. cynomolgi-infected mosquitoes. Quinine
therapy was administered at parasitemia ,1%, to clear all asexual parasite forms, thereafter leaving only hypnozoite forms in the monkeys livers. After
parasitemia returned to zero and once the drugs were presumed safely eliminated, monkeys received trimethoprim-sulfamethoxazole (TMP-SMX), lopinavirritonavir (LPV-RTV), or no additional drug administration (control). Parasitemia was then monitored daily for relapse, and quinidine treatment was
administered to all groups when the first relapse was observed in the control group. Monkeys received primaquine and chloroquine for radical cure after the
second relapse in the control group.
doi:10.1371/journal.pone.0115506.g001

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Neither Lopinavir-RTV nor Trimethoprim-SMX Prevent Malaria Relapse

stocks, were used to infect a splenectomized monkey to test the quinidine

gluconate regimen used as described below. This monkey also served as a donor
(as described below) for a mosquito bite infection study in which the quinidine
regimen used was also tested. In the former, the splenectomized monkey was
infected and tested positive for parasites by D4D9 after infection and was cured
with quinidine gluconate 26 mg/kg IM BID x 7 days with smears remaining
negative for 6 weeks. In the latter, two monkeys were infected by the bites of
mosquitoes and one had a primary attack D9, relapse #1 D29, #2 D46; the second
had Primary attack D11, relapse #1 D29, #2 D45 (Table 1).
To produce infected mosquitoes, laboratory-reared Anopheles stephensi
mosquitoes (Laboratory of Malaria Vector Research, NIAID) were allowed to feed
on a splenectomized donor rhesus monkey that had been infected with P.
cynomolgi B strain by intravenous (IV) injection of 16106 frozen infected RBCs
(derived from frozen vial of 0.9% parasitemia) thawed from frozen stock. When
mature sporozoites reached the salivary glands at day 10 to 12, the infected
mosquitoes were allowed to feed on 9 malaria-naive monkeys for 30 min, with 34
to 51 bites recorded used to infect each monkey (Day 0, or D0 5 Day of
infection of test monkeys with sporozoites). After feeding, mosquitoes not
engorged with blood were removed, and mosquito bite counts represent the
number of mosquitoes with blood (Table 2). Approximately 7 to 8 days after
feeding, parasites were detected in peripheral blood, and the monkeys were treated
with quinine dihydrochloride (32 mg/kg) twice daily by intramuscular (IM)
injection for 5 days to clear all asexual parasite forms, thereafter presumably
leaving only hypnozoite forms in the monkeys livers since quinine (and
quinidine) only kill blood stage parasite forms [20, 21].
Nine monkeys were divided into three groups after completion of quinine
therapy, which was used to cure the primary attack due to concerns for drug
interaction with a higher potential frequency of cardiac side effects if quinidine
had been used immediately preceding LPV-RTV [2729, 32]. All drugs were
administered by gavage; the third group received no additional treatment after
quinine therapy and served as a control. Test drugs were administered after
quinine to test the effect of drug on hypnozoites, which would remain after
quinine treatment. TMP-SMX was administered as 4 mg/kg TMP +20 mg/kg of
SMX twice daily orally derived from prophylaxis dosing information for
Pneumocystis jirovecii [33]. LPV-RTV was administered as12 mg/kg lopinavir
+3 mg/kg ritonavir twice daily orally based on a prior study in which this dose was
deemed safe in monkeys [34]. Monkeys received TMP-SMX for 4 days starting on
D17 or LPV-RTV for 7 days, starting on D21. Parasitemia was monitored daily to
observe relapse patterns. Due to shortage of quinine, quinidine gluconate
treatment (26 mg/kg twice daily for 7 days IM [21]) was applied to all groups
when the first relapse was observed in the control group once the animals reached
1%, and one control animal (DB9H) received a dose of artesunate 8mg/kg IV
times one due to a rapid rise in parasitemia (from 0.55% to 4.75%) within two
smear readings. At the end of the study, all monkeys received radical cure by
gavage with primaquine 0.5 mg base/kg once per day for 14 days and chloroquine

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Neither Lopinavir-RTV nor Trimethoprim-SMX Prevent Malaria Relapse

Table 2. Bites Counted Per Monkey in Experiment Assessing Effects of the HIV Protease Inhibitor Lopinavir-ritonavir and the Antimicrobial TrimethoprimSulfamethoxazole on Relapse in Plasmodium cynomolgi-infected Rhesus Monkeys.
Group

Number of Bites

Control: EZD, DB36, D89H

51, 34, 24

TMP-SMX: DC04, DBZ8, DA8G

40, 33, 16

LPV-RTV: CL84, FLM, FZ8

34, 30, 21

doi:10.1371/journal.pone.0115506.t002

25 mg base/kg once per day for 3 days after the second relapse in the control
group [2024].
Giemsa stain thin smears were monitored during the study daily once parasites
were observed to every other day if parasitemia had resolved (up to D90, to ensure
radical cure), and 10,000 red cells per smear were examined before being declared
negative.
Based on the pharmacokinetic profiles of the drugs used in this investigation,
and the assumption that 45 half-lives are required to both reach steady state and
for drug elimination to occur [29, 3537], plasma concentrations were assumed to
reach steady state as well as for drug elimination to occur during a period in
which hypnozoites would be present [21, 26, 38]. Pharmacokinetics of TMP and
SMX parallel what is seen in humans [35]. Plasma concentrations of LPV and
RTV were expected to be below clinically relevant concentrations [39].

Study Endpoints
An increase in the time to detection of parasites in the presumed first and second
relapses as detected by smear were each used to deduce liver stage reductions, and
appearance of parasites after treatment with quinine and quinidine were
considered relapses, as previously described [20, 23]. Study outcomes are
presented as Kaplan-Meier survival curves, and statistics are descriptive [40].

Results
By smear, all control monkeys had time to first detection of blood stage parasites
of 912 days (first parasitemias detected on D912 post infection) (Fig. 2). Only
2/3 control monkeys had detectable blood stage parasites (presumed first relapse)
by smear by D22. All 3 monkeys did have another (second) relapse on D39, D39,
D44.
The TMP-SMX-treated monkeys did not have a first relapse at all, but did have
second relapses Day 43, D41, D45.
LPV-RTV-treated monkeys all had first and second relapses which occurred at
parallel time points (1st relapse 5D25, D22, D22; 2nd relapse: D37, D40, D42)
compared with the control monkeys, suggesting that there was no impact on
hypnozoites at this dose.

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Neither Lopinavir-RTV nor Trimethoprim-SMX Prevent Malaria Relapse

Fig. 2. TMP-SMX and LPV-RTV plus TMP-SMX failed to prevent or delay P.cynomolgi relapses at given
doses. Relapse patterns in Plasmodium cynomolgi B strain infected rhesus monkeys after 5 days of quinine
treatment to clear the initial parasitemia, as outlined in Fig. 1. Panels are as follows: (A) control monkeys,
treated with quinine only; (B) monkeys treated with quinine and then with 4 mg/kg of TMP +20 mg/kg of SMX
of commercially available suspension twice per day D1720 (C) monkeys treated with quinine and then with
lopinavir-ritonavir (LPV-RTV) 12 mg lopinavir; 3 mg ritonavir/kg D2127. Smears were obtained as outlined in
Fig. 1.
doi:10.1371/journal.pone.0115506.g002

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Neither Lopinavir-RTV nor Trimethoprim-SMX Prevent Malaria Relapse

Discussion
We have demonstrated that TMP-SMX and LPV-RTV do not have significant
effect on relapse in the P. cynomolgi rhesus macaque model. This is the first
evaluation of LPV-RTV and TMP-SMX in vivo in non-human primates against
relapses of P. cynomolgi as a model for P.vivax.
Time to first detection of blood stage parasites, or prepatent period, was 9-12
days in 2 control monkeys, consistent with previous reports [22, 26]. Although
only 2/3 control monkeys had detectable blood stage parasites (presumed first
relapse) by smear by D22, all 3 monkeys had relapses on D39, D39, D44. The
absence of first relapse (in one animal) and variability of parasitemia is within the
relapse response range observed in other reports [26].
LPV-RTV did not increase prepatent period, nor did it prevent relapse. LPVRTV-treated monkeys all had first and second relapses which occurred at parallel
time points (1st relapse 5D25, D22, D22; 2nd relapse: D37, D40, D42) compared
with the control monkeys, suggesting that there was no impact of the HIV PI on
hypnozoites. HIV PIs have been shown to kill a multitude of Plasmodium species
and life cycle stages, including P. vivax ex vivo [4], as well as actively dividing liver
stage rodent malaria parasites [6] and P. falciparum [7]. Although the mechanism
by which the HIV protease inhibitors kill the malaria parasite remains unknown,
it is hypothesized that they interfere with aspartyl protease, or plasmepsin,
function in the malaria parasite [7]. It is possible that, although it is not known if
these or one of these proteases is the target of HIV PIs in malaria, the target may
not be important or active in the hypnozoite stage. Indeed the metabolic roles of
the plasmepsins, especially in P. vivax, have not been fully described [41, 42]. Of
note LPV-RTV dosing as used in this study would achieve levels far below clinical
significance [39]. Thus it is also possible that the doses used of the HIV PIs were
not high enough to exert antimalarial effect.
The TMP-SMX-treated monkeys did not have a first relapse at all, but did have
relapses Day 43, D41, and D45. As for TMP-SMX, delay to the first appearance of
parasitemia but no difference between relapse in treated compared with control
suggests there was killing of the actively dividing liver stage parasites, but not
hypnozoites [43]. Antifolate-killing of actively dividing liver stage parasites is
consistent with data we have previously published in rodent malaria models, in P.
falciparum in vitro, and in P. knowlesi in vivo [19, 39]). These data are also
consistent with prior work in which it has been demonstrated that other
antifolates such as pyrimethamine and proguanil, when administered between the
time of sporozoites inoculation and invasion of the blood, may simply eliminate
growing liver schizonts and thereby delay primary parasitemia, that is, increase
prepatent period, which directly correlates to reduction of actively dividing
parasites in the liver [43]. If hypnozoites are spared, however, which they have
been shown to be with pyrimethamine and proguanil with P. cynomolgi in vivo,
primary parasitemia itself could be delayed and relapses still occur. These
antifolates have also been shown to kill the exoerythrocytic stages of P. cynomolgi
and P. knowlesi in vitro [40, 4448], all consistent with what we observed.

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Neither Lopinavir-RTV nor Trimethoprim-SMX Prevent Malaria Relapse

Trimethoprim alone, and at a much higher dose (50 mg/kg BID compared to the
4 mg/kg BID we used) has been shown to have no anti-hypnozoite effect, nor an
effect on delaying primary parasitemia, when used in combination with subtherapeutic doses of primaquine [48]. Reduction of liver stage burden may
translate to delay in blood stage infections in the field, but absence of antihypnozoite effect in P. cynomolgi indicates TMP-SMX would offer no benefit in
treating or preventing P. vivax malaria.
An alternative explanation is that delay to the first appearance of parasitemia in
this TMP-SMX treated group could be due to incomplete effectiveness of a
shortened regimen of quinine fully killing asexual stage parasites, with TMP-SMX
killing asexual stages. Other antifolates can kill P. cynomolgi asexual stage [49].
In either case, the parasites seen on D43, 41, 45 are relapses because all animals
were treated for the first parasitemia observed after the primary attack with
quinidine, and this regimen was proven effective at eradicating asexual stages in
our pilot experiments (Table 1). Therefore, based on our pilot data, what we call
the second relapse we believe is a true relapse since we demonstrated our
quinidine was effective at killing asexual blood stages (Fig. 1 and Table 1).
Because HIV and malaria overlap, further studies are required to validate
whether PIs and TMP-SMX offer benefit in reducing clinical malaria through
anti-liver stage, although possibly not anti-hypnozoite, effect. If drugs used in
HIV management can reduce malaria burden, their administration can be tailored
or timed to maximize those benefits.

Acknowledgments
We thank: the Animal and Insectary Management Teams for their work in this
study; Boris Skopets, DVM, and Tom Thomas, DVM, for their support and
suggestions in this study; Bob Gwadz, PhD, Montip Gettayacamin, DVM, Parag
Kumar, PharmD, and Brian Kirmse, MD, for their review and suggestions for this
paper.

Author Contributions
Conceived and designed the experiments: CVH SD TS JN JC WB YW PED.
Performed the experiments: SD SOG LL KZ TS JN. Analyzed the data: CVH SD
WB PED. Contributed reagents/materials/analysis tools: CVH SD SRP YW PED.
Wrote the paper: CVH SD SRP PED.

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