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Contents lists available at ScienceDirect

Vaccine

j ournal homepage: www.elsevier.com/locate/vaccine

1 Progress with sporozoite (PfSPZ)-based

2

Q1 a,∗ a a b

3 Thomas L. Richie , Peter F. Billingsley , B. Kim Lee Sim , Judith E. Epstein ,

c d e f

4 Kirsten E. Lyke , Benjamin Mordmüller , Pedro Alonso , Patrick E. Duffy ,

g h i j

5 Ogobara K. Doumbo , Robert W. Sauerwein , Marcel Tanner , Salim Abdulla ,

d k a

6 Peter G. Kremsner , Robert A. Seder , Stephen L. Hoffman

a

7 Sanaria Inc., Rockville, MD, United States

b

8 Naval Medical Research Center, Silver Spring, MD, United States

c

9 Center for Malaria Research, Institute for Global Health, University of Maryland School of Medicine, Baltimore, MD, United States

d

10 Institute of Tropical Medicine, University of Tübingen, Tübingen, Germany

e

11 World Health Organization Global Malaria Program, Geneva, Switzerland

f

12 Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, NIH, Rockville, MD, United States

g

13 Malaria Research and Training Center, University of Bamako, Bamako, Mali

h

14 Radboud University Medical Center, Nijmegen, The Netherlands

i

15 Swiss Tropical and Public Health Institute, Basel, Switzerland

j

16 Ifakara Health Institute, Bagamoyo, United Republic of Tanzania

k

17 Vaccine Research Center, National Institute of Allergy and Infectious Disease, NIH, Bethesda, MD, United States

18

3119 a r t i c l e i n f o a b s t r a c t

20

21 Article history: Sanaria Inc. has developed methods to manufacture, purify and cryopreserve aseptic Plasmodium falcip-

22 Available online xxx

arum (Pf) sporozoites (SPZ), and is using this platform technology to develop an injectable PfSPZ-based

23 vaccine that provides high grade, durable protection against infection with Pf malaria. Several candidate

24 Keywords:

vaccines are being developed and tested, including PfSPZ Vaccine, in which the PfSPZ are attenuated by

25

irradiation, PfSPZ-CVac, in which fully infectious PfSPZ are attenuated in vivo by concomitant adminis-

26 Plasmodium falciparum

tration of an anti-malarial drug, and PfSPZ-GA1, in which the PfSPZ are attenuated by gene knockout.

27 Sporozoite

Forty-three research groups in 15 countries, organized as the International PfSPZ Consortium (I-PfSPZ-

28 PfSPZ vaccine

C), are collaborating to advance this program by providing intellectual, clinical, and financial support.

29 PfSPZ-CVac

Fourteen clinical trials of these products have been completed in the USA, Europe and Africa, two are

30 PfSPZ challenge

underway and at least 12 more are planned for 2015–2016 in the US (four trials), Germany (2 trials), Tan-

zania, Kenya, Mali, Burkina Faso, Ghana and Equatorial Guinea. Sanaria anticipates application to license

a first generation product in late 2017, initially to protect adults, and in 2018 to protect all persons >6

months of age for at least six months. Improved vaccine candidates will be advanced as needed until

the following requirements have been met: long-term protection against natural transmission, excellent

safety and tolerability, and operational feasibility for population-wide administration. Here we describe

the three most developed whole PfSPZ vaccine candidates, associated clinical trials, initial plans for licen-

sure and deployment, and long-term objectives for a final product suitable for mass administration to

achieve regional malaria elimination and eventual global eradication.

© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND

license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

32 1. Background the SPZ during their journey from the mosquito proboscis to the 36

Q3 liver or during development in the liver (pre-erythrocytic stages 37

33 The Plasmodium sporozoite (SPZ), transmitted to the human host of the parasite life cycle), there would be no blood stage infection 38

34 by female Anopheles mosquitoes, is an attractive vaccine candi- and no production of gametocytes. This would address an urgent 39

35 date. If immune responses induced by such a vaccine could kill public health priority, namely protecting people in endemic areas 40

from clinical malaria, especially those susceptible to severe dis- 41

ease such as infants and young children. A pre-erythrocytic stage 42

∗ vaccine would also benefit travelers, obviating the difficulties of 43

Q2 Corresponding author. Tel.: +1 301 770 3222; fax: +1 301 770 5554.

http://dx.doi.org/10.1016/j.vaccine.2015.09.096

0264-410X/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.

0/).

Please cite this article in press as: Richie TL, et al. Progress with Plasmodium falciparum sporozoite (PfSPZ)-based malaria vaccines.

Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.09.096

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44 prophylactic drug compliance and side effects. Ultimately, a highly

45 effective pre-erythrocytic stage vaccine, serving as a vaccine to Box 1: Targeting the SPZ.

Whole SPZ were one of the first malaria immunogens tested

46 interrupt malaria transmission (VIMT), would be the ideal tool to

as a malaria vaccine, described by Sergent and Sergent in a

47 eliminate malaria and contain spread of malaria parasites from

report published in 1910 by the Academy of Sciences, Paris,

48 defined geographic regions, leading to global eradication [1].

on the avian parasite Plasmodium relictum [2]. In the early

1940s, Mulligan, Russell and Mohan at the Pasteur Institute

in Coonoor, India partially protected fowl against another bird

49 1.1. Targeting the sporozoite

parasite, P. gallinaceum, by administering five intravenous

injections of a crude preparation of dried, ground thoraces

50Q4 Several approaches have been taken to induce protective immu-

from infected mosquitoes (220 thoraces per fowl) [3,4]. Vacci-

51 nity against SPZ. Forty years ago injection of attenuated whole

nated animals and controls were challenged by the bites of two

52 SPZ was shown to protect rodents against malaria infection, fol-

infected Aëdes albopictus mosquitoes, and while all immu-

53 lowed quickly by proof-of-concept that the same approach works

nized fowl developed asexual blood stage infections (thus

54

in humans (Box 1). However, the SPZ had to be administered to protection against infection was nil), prior SPZ vaccination

55 humans by mosquito bite, limiting feasibility as a vaccine approach. halved the parasite density in the blood, and reduced mortal-

56 The alternative, producing large numbers of infected mosquitoes, ity from 55% to 30% or less. Similar results were obtained with

57 harvesting the SPZ, and purifying them from contaminating sali- SPZ dissected from salivary glands and inactivated by a 30-min

exposure to ultraviolet light. The partial protection by killed

58 vary gland antigens, also appeared to be difficult if not impossible,

SPZ in the avian model was confirmed in subsequent stud-

59 especially as the purified sporozoites would have to be aseptic for

ies [5]. It was shown that protected fowl were still susceptible

60 injection. All in all, immunization with whole sporozoites was felt

to blood stage challenge, indicating that the partially effective

61 to be “too crude and impractical to produce a vaccine for wide

immune response induced by whole killed SPZ vaccination

62 application” [27].

targeted the pre-erythrocytic stages.

A significant improvement in protection was reported by

Nussenzweig and colleagues in 1967 by using attenuated,

63 1.2. Whole sporozoites for parenteral injection

rather than killed SPZ, this time in a rodent model. X-irradiation

was used to damage P. berghei SPZ dissected from infected

64 Sanaria Inc., a biotechnology company in Rockville, Maryland,

Anopheles stephensi mosquitoes [6]. SPZ receiving this sub-

65 spent the last 10 years addressing the challenges of production,

lethal irradiation remained metabolically active and motile,

66

purification and cryopreservation, and now routinely manufactures could invade hepatocytes and round up (a morphological

67 vials of highly purified, aseptic P. falciparum (Pf) SPZ that are in com- change signifying the earliest stage of development in the

68 pliance with regulatory standards for purity, potency, safety and hepatocyte), but could not develop into hepatic trophozoites

or schizonts. When mice immunized by IV administration

69 consistency [28]. 2082 doses of PfSPZ (doses as high as 2.2 million

of 75,000 SPZ irradiated with 80–100 gray (Gy) were chal-

70 PfSPZ) have now been administered to 789 adults by the intrader-

lenged by IV injection of non-irradiated SPZ two weeks after

71 mal (ID), subcutaneous (SC), intramuscular (IM), intravenous (IV) or

immunization, infection rates were 37% compared to 90% in

72 direct venous inoculation (DVI) routes, and have shown excellent

controls, indicating that more than half were sterilely immune.

73 safety and tolerability. In blinded, randomized, placebo-controlled

Those animals becoming infected despite vaccination showed

74 trials, the PfSPZ have caused no detectable systemic reactogenicity

delays in the prepatent period. Eleven protected mice were

75 (Sissoko et al., unpublished; Mordmüller et al., unpublished) [29]

re-challenged at 36 days, and most were again protected, indi-

76 and no allergic responses, indicating that PfSPZ may be suitable for cating a degree of durability and also ruling out non-specific

77 mass administration campaigns for malaria elimination. protection mediated by the innate immune system. The murine

78 Because Plasmodium is a eukaryotic organism, the PfSPZ need model thus established that attenuated, metabolically active

parasites could induce antigen-specific, sterile immunity to

79 to be cryopreserved and stored in liquid nitrogen vapor phase

malaria.

80 (LNVP) to maintain viability, like other cellular therapies or prod-

The radiation-attenuated SPZ approach was rapidly translated

81 ucts (mammalian sperm, eggs, embryos, cellular cancer vaccines),

to humans in a series of studies in the early 1970s carried

82 as well as eleven veterinary vaccines including the Theileria parva

out by Clyde and colleagues [7–11], and Rieckmann and col-

83 vaccine used in Africa. Indeed, LNVP storage may prove to be advan-

leagues [12–14] and to non-human primates [15]. A review of

84 tageous, enhancing delivery to remote areas since no electricity is

these studies and subsequent work by the US military [16]

85

needed to maintain the cold chain (compared to refrigerated vac- and the University of Maryland [17,18] by Hoffman et al. [19]

86 cines), and the vaccine can remain stable for weeks to months in concluded that administration of more than 1000 bites from

87 a free-standing container. LN is widely available across Africa and irradiated, infected mosquitoes consistently protected >90%

(13/14) of recipients undergoing CHMI by mosquito bite within

88 other tropical areas, with infrastructure in place to support veteri-

10 weeks of immunization, and 5/6 volunteers were still pro-

89 nary vaccine applications, artificial insemination of cattle, oil and

tected on repeat CHMI up to 42 weeks later. Furthermore, 4

90 mining applications, and the brewing industry. Distribution of the

subjects underwent CHMI with 7 heterologous strains of Pf,

91 vaccine in LNVP is feasible in malaria endemic countries using a

and there was 100% protection. 15 Gy was established as a

92 hub and spoke model, and projected costs are roughly equivalent

sufficiently attenuating radiation dose to prevent blood stage

93 to those required for adding a new vaccine to current distribution

infection without over-irradiating the parasites.

94 networks for refrigerated (2–8 C) vaccines [30].

This work provided proof-of-concept for high-grade protection

95 PfSPZ, the core Sanaria product, are manufactured in accordance against parasitemia in humans by targeting the SPZ (and/or the

96 with Title 21 of the Code of Federal Regulations (21 CFR) and in early liver stage parasites they become), and inspired efforts

97 accordance with International Conference on Harmonization (ICH) to develop pre-erythrocytic stage malaria vaccines. It was rea-

soned that if the antigenic components of whole SPZ could

98 guidelines. Manufacturing of PfSPZ products is performed follow-

be identified and suitably formulated as vaccines, it should

99 ing current Good Manufacturing Practice (cGMP) guidelines where

be possible for subunits to reproduce the high-grade protec-

100 processes are defined and controlled to ensure consistency and

tion induced by whole SPZ. The identification and cloning

101 compliance with specifications. PfSPZ products have excellent sta-

in 1983/1984 of the major surface protein of the P. knowlesi

102 bility profiles when stored for more than 4 years. Sanaria maintains

and P. falciparum (Pf) SPZ, the circumsporozoite protein (CSP)

103 Biologics Master Files with the US FDA, and all clinical trials are

[20,21], galvanized this effort, and led to the clinical testing of

104 conducted with US FDA oversight under investigational new drug

Please cite this article in press as: Richie TL, et al. Progress with Plasmodium falciparum sporozoite (PfSPZ)-based malaria vaccines.

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a series of recombinant constructs [22,23], the most successful the highest total dose (Table 1) demonstrated that the PfSPZ were 149

being RTS,S, a recombinant protein containing a portion of potent and provided the critical proof-of principle that the model 150

PfCSP fused to hepatitis B surface antigen, produced in yeast,

of protection by mosquito bite with radiation-attenuated SPZ (Box 151

assembled into virus-like particles, and formulated in adjuvant.

1) could indeed be translated into a human injectable product. 152

RTS,S has been extensively tested in Africa, and the results of

There was a clear dose-threshold effect, with the three lower doses 153

a large Phase 3 trial recently reported [24]. In addition to RTS,S,

showing limited, but dose-related protection, and the highest dose 154

several other subunit approaches using pre-erythrocytic stage

conferring high-grade sterile immunity (Table 1). There was a dose 155

antigens are being pursued, including gene-based approaches;

response for antibody and cellular immune responses, and antibody 156

some, like RTS,S, have sterilely protected volunteers against

157

CHMI [25,26]. While these subunit approaches are promising, responses to PfSPZ were associated with protection. The most sig-

they present the immune system with only a small number of nificant association was with antibodies to PfSPZ measured by the 158

pre-erythrocytic immunogens, and have provided only modest inhibition of invasion into hepatocytes (ISI) assay [36]. 159

protection. With the development by Sanaria of technologies

for the manufacture and administration of whole SPZ, many

2.2. PfSPZ challenge 160

in the malaria vaccine development field have begun to direct

their efforts back toward this more empiric whole organism

®

161

approach (see Box 2). Sanaria PfSPZ challenge is manufactured identically to PfSPZ

Vaccine except that the PfSPZ are not irradiated, and are therefore 162

fully infectious. CHMI with injectable PfSPZ can replace traditional 163

CHMI by the bite of mosquitoes to measure vaccine and drug 164

105 applications (IND), and with appropriate host nation regulatory

efficacy, as well as to increase understanding of factors (genetic, 165

106 oversight.

immune) that affect Pf infectivity. The optimal dose and route 166

for administering PfSPZ Challenge were worked out in a series of 167

107 2. Products

eight clinical trials conducted from 2010 to 2014, with the first five 168

testing ID and IM routes. These were conducted at Radboud Uni- 169

108 Sanaria’s first PfSPZ products are based on the NF54 strain of

versity Medical Center (RUMC), Nijmegen, the Netherlands [37], 170

109 Pf, isolated in 1981 from a Dutch farmer living near Amsterdam’s

the University of Oxford, Oxford, UK [38], the Ifakara Health Insti- 171

110 Schiphol airport and originating in Africa [31,32]. Pf NF54 strain

tute (IHI), Bagamoyo, Tanzania [29], UMB CVD (Lyke et al., in 172

111 is chloroquine sensitive. More recently, Sanaria has manufactured

press, AJTMH), and the Kenya Medical Research Institute (KEMRI), Q5 173

112 PfSPZ derived from a clone of a Brazilian isolate, 7G8 [33], as well

Nairobi, Kenya [39,40]. The aim of all these studies was to admin- 174

113 as PfSPZ from a Cambodian clone, NF135.C10 [34].

ister a dose of PfSPZ Challenge that reproduced the infectivity 175

114 Controlled human malaria infection (CHMI) and exposure to

and prepatent period of five PfSPZ-infected mosquitoes, namely 176

115 natural transmission have been used to test the efficacy of Sanaria’s

100% of volunteers infected and a prepatent period by thick smear 177

116 vaccine products. CHMI has been performed by mosquito bite, using

microscopy of <12 days. ID administration has achieved 100% infec- 178

117 mosquitoes infected with NF54, 3D7 (a clone of NF54) or 7G8, or

tion rates, but not the target prepatent period. IM administration of 179

118 by the direct injection of infectious PfSPZ (NF54) manufactured 4

7.5 × 10 PfSPZ achieved both attributes [41]. IV inoculation using 180

119 by Sanaria. When NF54 or 3D7 are used, the CHMI is considered

an in-dwelling intravenous catheter was first tested at the Uni- 181

120 homologous, since the parasite is identical or highly similar to the

versity of Tübingen, Germany, and achieved 100% infection rate 182

121 vaccine. When other strains are used to assess efficacy, the CHMI is 3

after administering only 3.2 × 10 PfSPZ [42]. The geometric mean 183

122 considered heterologous, and a potentially better predictor of effi-

(GM) prepatent period was 11.2 days (range 10.5–12.5 days). These 184

123 cacy under conditions of natural transmission, where mosquitoes

results were then reproduced at the Barcelona Centre for Interna- 185

124 harbor highly diverse strains of Pf. 3

tional Health (CRESIB), Spain [41,42], establishing 3.2 × 10 PfSPZ 186

of PfSPZ Challenge as the new gold standard for “mosquito-free” 187

125 2.1. PfSPZ vaccine

CHMI. In Barcelona the PfSPZ were administered by DVI, insert- 188

ing the 25 g needle of a tuberculin syringe directly into a vein 189

126 The first PfSPZ product developed using Sanaria’s manufactur-

® and rapidly injecting the PfSPZ in a volume of 0.5 mL (Fig. 1). This 190

127 ing technology was Sanaria PfSPZ Vaccine, which is composed of

standard dose and DVI administration were used by the Lambaréné 191

128 aseptic, purified, live (metabolically active), radiation attenuated,

129 cryopreserved PfSPZ. Several clinical trials of PfSPZ Vaccine have

130 been completed in the USA. In the first trial, conducted by the

131 Naval Medical Research Center (NMRC), the University of Mary-

132 land, Baltimore Center for Vaccine Development (UMB CVD), and

133 the Walter Reed Army Institute of Research (WRAIR), the vaccine

134 was administered ID or SC, was poorly immunogenic and protected

135 at best only 2 of 16 volunteers at one dosage against CHMI [35]. A

136 concurrent study in non-human primates (NHP) at the NIH Vac-

137 cine Research Center (VRC) administered the vaccine SC, as in the

138 clinical trial, and by direct venous inoculation (DVI), as done in

139 the original rodent experiments (Box 1). DVI induced far supe-

140 rior immune responses: 3.2% of CD8+ T cells in the livers of the

141 three NHPs immunized by DVI responded with IFN␥ production to

142 PfSPZ stimulation, compared with low to undetectable frequencies

143 in SC-immunized monkeys [35]. This finding provided the proof

144 of concept for the second trial, conducted at the VRC, where the Fig. 1. Direct venous inoculation (DVI) of PfSPZ [42]. Once the injection is prepped

with an antiseptic such as 70% isopropyl alcohol, needle insertion, loosening the

145 vaccine was given IV through an in-dwelling catheter. This trial

tourniquet and vaccine injection taken only about 10–15 s. Note the small size of

146 escalated through five increasing doses of the vaccine, adminis-

the needle (25 gauge) and syringe (1 mL), and blood flashback confirming successful

147 tering four to six injections to the volunteers over the course of

puncture of, in this case, the median cubital or cephalic vein. Veins in the back of

148 20–26 weeks. Sterile protection in 6/6 (100%) subjects receiving the hand or side of the wrist may also be used. Photo by B. Mordmüller.

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Table 1

Summary of protective efficacy and antibody responses in volunteers immunized in the VRC 312 clinical trial with PfSPZ vaccine [36].

a

Dosage regimen Anti-PfCSP Antibodies (OD 1.0) Number of volunteers Protective efficacy

5 5

Dose (PfSPZ × 10 ) Number of doses Maximum total dose (PfSPZ × 10 ) Challenged Protected

0.075 4 or 6 0.45 13 6 0 0%

0.3 4 or 6 1.8 324 11 1 9%

b

1.35 4 5.4 3454 9 6 60%

1.35 5 6.75 6716 6 6 100%

a

Anti-PfCSP antibody level 2 weeks after the last dose and before CHMI by ELISA (geometric mean of the inverse of the serum dilution at which the optical density (OD)

was 1.0).

b

5/6 controls developed parasitemia.

192 Centre for Medical Research in Gabon to study the effect of natu- route was by far the most efficient for infecting volunteers. Thus 243

193 rally acquired immunity and sickle cell trait on the growth rate of for each objective – inducing protective immunity with PfSPZ Vac- 244

194 Pf in vivo (Lell et al., unpublished). These studies were performed cine or the PfSPZ-CVac approach, or inducing infection with PfPSZ 245

195 using different PfSPZ Challenge lots, some of which were manu- Challenge – there was a multi-fold difference in potency between 246

196 factured years apart. In summary, these studies of PfSPZ Challenge the IV/DVI routes of administration and the more traditional ID, SC 247

197 demonstrated that the purified, cryopreserved PfSPZ produced by and/or IM routes. 248

198 Sanaria were potent (infectious) and highly stable, and that IV or

199 DVI routes were the most efficient means of administration.

2.4. Comparative potency of PfSPZ vaccine and PfSPZ-CVac 249

200 Having established a standard route and dose, injections of

3

201 3.2 × 10 PfSPZ of PfSPZ Challenge, administered by DVI, have now

Our cumulative experience using PfSPZ Vaccine shows that it 250

202 been used successfully in seven CHMIs in Tanzania and Germany

requires several hundred thousand PfSPZ to induce high-grade 251

203 to test the efficacy of PfSPZ Vaccine and PfSPZ-CVac respectively,

protection, while the same or better can be achieved using the 252

204 infecting all control volunteers (n = 43) (Mordmüller et al., unpub-

PfSPZ-CVac approach using a fraction of the dose. This parallels ear- 253

205 lished; Shekalaghe et al., unpublished). CHMI by PfSPZ Challenge

lier experience using mosquito bite immunization: with CPS, it took 254

206 reduces costs and streamlines the logistics compared with CHMI

exposure to ∼45 PfSPZ-infected mosquitoes to achieve durable, 255

207 by mosquito bite. It also allows repeated CHMIs and adaptive clin-

high level protective efficacy. In contrast, it required exposure to 256

208 ical trial designs, since the timing of CHMI is independent of the

the bites of at least 1000 mosquitoes carrying radiation-attenuated 257

209 complex process needed to produce a batch of infected mosquitoes.

PfSPZ to consistently achieve high-level protection (Box 1). The 258

210 Since each inoculation is identical, PfSPZ Challenge also standard-

likely reason for this is that radiation-attenuated PfSPZ invade 259

211 izes the dose of infectious PfSPZ, which cannot be done with CHMI

hepatocytes and begin the process of development, and although 260

212 by mosquito bite.

they express ∼1000 proteins their replication arrests early in liver 261

stage development. In contrast, the infectious PfSPZ in PfSPZ-CVac 262

invade hepatocytes, but then replicate 10,000–40,000-fold and 263

213 2.3. PfSPZ-CVac

express ∼4500 different proteins, including blood stage proteins. 264

Thus there are dramatically more parasites and antigens presented 265

214 In a seminal study of chemoprophylaxis with SPZ (CPS)

to the immune system per PfSPZ injected with PfSPZ-CVac than 266

215 conducted at RUMC in Nijmegen [43], 3 × 12–15 bites from

with PfSPZ Vaccine. 267

216 non-irradiated Anopheles stephensi mosquitoes harboring PfSPZ

217 administered to malaria-naïve adults concurrently taking chloro-

2.5. PfSPZ-GA1 268

218 quine resulted in 100% sterile protection against CHMI (10/10

219 volunteers protected). The protection persisted for at least 28

®

Sanaria PfSPZ-GA1 consists of purified, aseptic, cryopreserved 269

220 months in the majority of volunteers undergoing a second

Pf sporozoites (NF54 strain) genetically attenuated by removal of 270

221 CHMI, with 4/6 sterilely protected and 2/6 showing prolonged

the b9 and slarp genes to halt development in the early liver stages 271

222 prepatent periods [44]. The Sanaria team and collaborators rea-

[46,47]. The parasite line was generated by the Leiden University 272

223 soned that PfSPZ Challenge should be able to substitute for the

Medical Center (LUMC) and RUMC in collaboration with Sanaria. 273

224 mosquito bites, and accordingly, PfSPZ Challenge was tested as the

®

Pfb9slarp parasites invade hepatocytes but are incapable of sus- 274

225 immunogen in a CPS vaccine approach called Sanaria PfSPZ-CVac

taining liver stage development, similar to radiation-attenuated 275

226 (CVac = Chemoprophylaxis Vaccine).

PfSPZ. The potential advantages of PfSPZ-GA1 are that (1) the PfSPZ 276

227 The first PfSPZ-CVac trial was conducted at RUMC; PfSPZ Chal-

are homogenous and have a precisely characterized genetic basis 277

228 lenge was administered ID with chloroquine as the drug partner.

for attenuation; (2) manufacturing PfSPZ-GA1 cannot result in acci- 278

229 Disappointingly, but in hindsight not unexpectedly, three or four ID

dental exposure of staff to clinical malaria, thereby simplifying 279

230 administrations of 75,000 PfSPZ induced minimal immunogenicity

the approach to safety of operators and lowering costs. PfSPZ-GA1 280

231 and little or no protection [45]. Once the superiority of the IV route

also has the potential to induce more efficient protection than the 281

232 was demonstrated for PfSPZ Vaccine in the VRC314 trial, however,

radiation-attenuated PfSPZ Vaccine due to a different pattern of 282

233 there was justification for a second trial of PfSPZ-CVac, which was

developmental arrest and antigen expression. 283

234 conducted at the University of Tübingen. PfSPZ were administered

235 by DVI rather than ID, chloroquine was retained as the partner drug,

236 and this time the outcome was dramatically reversed [Mordmüller 2.6. Safety and tolerability 284

237 et al., unpublished]. The transformation from low- to high-grade

238 immunogenicity and efficacy mirrored the similar transformation 2082 doses of cryopreserved PfSPZ from Sanaria products have 285

239 for PfSPZ Vaccine when the route of administration was changed been administered to 789 adult volunteers in 17 clinical trials via 286

240 from ID or SC to IV. The finding that IV or DVI administration was a variety of routes (Richie et al., unpublished). Several of these 287

241 needed to reveal the potency of PfSPZ for immunizing volunteers trials included randomized, double-blind allocation to PfSPZ or 288

242 parallels experience with PfSPZ Challenge for which the IV or DVI normal saline (NS) placebo, and data are available from three 289

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290 such trials, involving 97 PfSPZ recipients and 68 placebo recipi- by repeated malaria infections. The specific results from these trials 352

291 ents in total. The two groups were compared in each trial after will be published by the investigators. 353

292 unblinding the adverse event data; there were no differences in Stage 2 of the clinical development plan, launching in mid-2015, 354

293 adverse event profiles between volunteers receiving PfSPZ and will address the following objectives: 355

294 placebo during the first seven days after injection [29] (Sissoko

295 et al., unpublished; Mordmüller et al., unpublished). Moreover,

(1) Demonstrate high-grade sterile protection in malaria-naïve 356

296 there have been no allergic reactions to PfSPZ, nor any serious

adults, including durable (≥6 month) protection against het- 357

297 adverse events attributed to PfSPZ. Experience includes 30 IV injec-

5 6 erologous CHMI following a simplified (e.g., three dose) 358

298 tions of 9 × 10 PfSPZ and 36 IM injections of 2.2 × 10 PfSPZ (Lyke

regimen. These studies will finalize the regimen for licensure 359

299 et al., unpublished). The absence of clinically significant side effects

to protect travelers, including military personnel, during stays 360

300 linked to PfSPZ administration applies also to laboratory abnormal-

in malarious areas. 361

301 ities, including liver function tests.

(2) Demonstrate high-grade sterile protection in malaria-exposed 362

302 DVI is rapid and efficient (Fig. 1). If the veins in the arm are

African adults, including durable (6 month) protection against 363

303 suitable for obtaining blood via standard venipuncture, they are

naturally transmitted malaria following a similarly simplified 364

304 suitable for administration of PfSPZ by DVI. The procedure involves

regimen. These studies are aimed at defining a regimen that 365

305 the insertion of a 25 g needle into the vein, a slight withdrawal

can be used in malaria elimination campaigns to halt infection 366

306 on the plunger to demonstrate blood “flashback,” loosening of the

and transmission. 367

307 restricting band and immediate injection of 0.5 mL of diluted PfSPZ

(3) Evaluate the tolerability, immunogenicity and efficacy of trun- 368

308 (or placebo), typically taking less than 10 s. More than half (in some

cated regimens: 0, 1, and 4 weeks; 0 and 1 week; 0, 2, 4 and 369

309 trials >90%) of subjects have rated the injections as painless on a

6 days or even a single immunization. These studies aim to 370

310 four-point scale (painless, mild pain, moderate pain, severe pain).

improve the operational feasibility of using the vaccine for all 371

311 DVI may cause slight bruising at the injection site if there is extrava-

indications. 372

312 sation of blood from the vein, but there are no persistent local signs

(4) Demonstrate safety, immunogenicity and protection in African 373

313 or symptoms such as tenderness, erythema or induration, since

infants 5 months or older and in young children. These stud- 374

314 the vaccine is dispersed on injection. ID and SC injections are also

ies aim to protect the most vulnerable age group from malaria 375

315 well tolerated, indicating that if any inoculum is deposited into the

and will optimize dose of vaccine with respect to age and body 376

316 surrounding tissues during DVI, it does not affect tolerability. The

weight. We hypothesize that because African infants have had 377

317 vaccine contains no adjuvant or pro-inflammatory material.

5 limited exposure to malaria, vaccinations will result in better 378

318 The demonstrated safety of PfSPZ at doses up to 9 × 10 PfSPZ by

protective responses than for African adults thereby providing 379

319 DVI has enabled plans for further dose escalation, in order to max-

an effective vaccine for the most vulnerable populations. 380

320 imize the degree and duration of sterile immunity, and this will be

(5) Demonstrate safety in the elderly and in HIV-infected individ- 381

321 conducted for both PfSPZ Vaccine and PfSPZ-CVac. An interesting

uals. These studies aim to show that screening for diminished 382

322 aspect of the latter approach when using chloroquine as the anti-

health or immunodeficiency will not be required when 383

323 malarial is that 5.5–7 days after injection merozoites are released

conducting mass administration campaigns. Since radiation- 384

324 into the blood and are detectable by qPCR, providing a transient

attenuated parasites cannot replicate, they should prove safe 385

325 low-grade parasite density that is rapidly cleared by chloroquine.

in all individuals, including the immunocompromised. 386

326 The kinetics of transient parasitemia allows an estimate of the

(6) Evaluate efficacy against P. vivax by CHMI and natural trans- 387

327 number of infected hepatocytes, which can be used to correlate

mission. Pv and Pf share tens of thousands of minimal T cell 388

328 immunogen dose (number of sporozoites infecting hepatocytes)

epitopes, and PfSPZ may induce cross-protective cell mediated 389

329 with protective efficacy.

immunity [48,49]. 390

(7) Establish immunological correlates of infection; all immune 391

responses measured in these trials will be assessed as potential 392

330 3. Clinical development plan

correlates. 393

(8) Continue with operational research in preparation for phase 394

331 3.1. PfSPZ vaccine

3 clinical trials and elimination campaigns with the licensed 395

vaccine. 396

332 Springboarding off the two completed trials of PfSPZ Vaccine

333 [35,36], particularly following the high-grade protection in VRC 312

334 and ongoing studies, there are five trials completed or underway of There are eight funded trials that will address these objectives 397

335 this product that constitute “Stage 1” of the PfSPZ Vaccine clinical in the USA, Germany (2 trials), Tanzania, Mali, Burkina Faso, Kenya 398

336 development plan (CDP) (Fig. 2). Each of these trials was initiated by and Equatorial Guinea (Table 2), plus one additional trial of a new 399

337 the primary performing institutions (Table 2), which also provided strain of PfSPZ Challenge. A program for studying PfSPZ Vaccine in 400

338 funding and developed protocols in close partnership with Sanaria. pregnancy is in the planning stages. 401

339 Preliminary analysis of the data has shown that these studies have Stage 3 of the CDP will include expanded safety testing and large 402

340 reproduced the high-grade efficacy seen in VRC 312, demonstrated scale CHMI trials in malaria-naïve adults using one or more het- 403

341 that PfSPZ Vaccine induces heterologous and durable (6 month) erologous parasites for CHMI, and will also include large-scale field 404

342 protection against CHMI and against naturally transmitted malaria, efficacy trials in malaria endemic areas including all age groups 405

343 and that a three-dose regimen can be highly protective. We have older than six months. These studies are planned for 2016–18. 406

344 also learned that malaria-naïve individuals in the U.S. respond The studies in malaria-naïve adults will support the targeted sub- 407

345 better to the vaccine than malaria-exposed individuals in Africa mission of a biologics license application (BLA) late in 2017 for a 408

346 after receiving an identical dose and regimen, exhibiting multifold traveler’s vaccine, and the studies in endemic areas will support an 409

347 higher titers of antibodies to PfCSP by ELISA. This indicates that additional indication for use in endemic areas subsequently. Opera- 410

348 increased doses of PfSPZ, and potentially interval changes between tionally we intend to initially target infants, age 6–12 months, to 411

349 doses, will be required to achieve high-level immunogenicity and reduce morbidity and mortality, and mass administration projects 412

350 sterile protection in malaria-exposed individuals. This difference intended to achieve halting of transmission and elimination of 413

351 in responsiveness may result from the immune modulation caused malaria will follow. 414

Please cite this article in press as: Richie TL, et al. Progress with Plasmodium falciparum sporozoite (PfSPZ)-based malaria vaccines.

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Fig. 2. Clinical development plan for PfSPZ vaccine. Current activities fall into Stage 2.

415 3.2. PfSPZ-CVac regimen of the partner drug. Development is being prioritized 418

and accelerated because of PfSPZ-CVac’s increased potency com- 419

416 The CDP for PfSPZ-CVac parallels that of PfSPZ Vaccine with pared to PfSPZ Vaccine. One clinical trial is ongoing (TÜCHMI-002 420

417 the added necessity of optimizing the administration, dose and trial at the University of Tübingen), and four additional trials are 421

Table 2

Summary of Stage 1 and Stage 2 PfSPZ Vaccine Clinical Trials. The performing institutions are core members of the expanding International PfSPZ Consortium (see Table 4).

Stage 1

Country Protocol name Primary performing institutions ClinTrials.gov Identifier

USA VRC 314 NIAID Vaccine Research Center (VRC), UMB CVD NCT02015091

Mali 14-I-N010 Malaria Research & Training Center (MRTC), University of Bamako NCT01988636

NIAID Laboratory for Malaria Immunology and Vaccinology (LMIV)

Tanzania BSPZV01 Ifakara Health Institute (IHI) NCT02132299

Swiss Tropical and Public Health Institute (Swiss TPH)

USA WRAIR 2080 NMRC NCT02215707

WRAIR

Equatorial Guinea EGSPZV1 Equatorial Guinea Ministry of Health and Social Welfare (MOHSW) NCT02418962 IHI

Swiss TPH

Medical Care Development International (MCDI)

La Paz Hospital, Malabo, EG

Stage 2

Country Primary performing institutions Objectives Challenge method

Germany University of Tübingen Condensed regimens in adults CHMI – PfSPZ

Challenge

Comparison with PfSPZ-CVac CHMI – PfSPZ

Challenge

Tanzania IHI Age de-escalation to infants CHMI – PfSPZ Challenge

Swiss TPH

Mali MRTC Dose escalation and efficacy in adults CHMI – PfSPZ

Challenge + Natural exposure

LMIV

Burkina Faso CNRFP Dose escalation and efficacy in adults Natural exposure

UMB CVD

USA NMRC Finalized regimen for adult travelers CHMI – Mosquito bite UMB-CVD

WRAIR

Equatorial Guinea Equatorial Guinea MOHSW Efficacy in adults CHMI – PfSPZ

Challenge

IHI Age escalation

Swiss TPH Safety in HIV positive subjects

MCDI

Kenya KEMRI Safety and efficacy in infants Natural exposure

US Centers for Disease Control (CDC)

NIAID VRC

Please cite this article in press as: Richie TL, et al. Progress with Plasmodium falciparum sporozoite (PfSPZ)-based malaria vaccines.

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Table 3

Summary of current and planned PfSPZ-CVac clinical trials.

Country Primary performing institutions Objectives Challenge method

Germany University of Tübingen Condensed regimens CHMI – PfSPZ Challenge

(ClinicalTrials.gov NCT02115516) Alternative drug partner

USA LMIV Alternative partner drug CHMI – PfSPZ Challenge

NIAID Division of Microbiology and Infectious Disease (DMID) Dose escalation Condensed regimen CHMI – PfSPZ Challenge

Seattle Group Health, Seattle BioMed

Ghana Kintampo Health Research Centre US Navy Dose escalation CHMI – PfSPZ Challenge

Indonesia Eijkman Institute Field efficacy Natural exposure

Indonesian Military Pv cross-protection

Table 4

Box 2, Table 1 Members of the International PfSPZ Consortium. Partners and funding organizations are listed by country and do not necessarily match.

Location Collaborative and funding partners Funding organizations

USA NMRC, DoD Military Infectious Disease Research Program (MIDRP)

WRAIR, DoD US Navy Advanced Medical Development Program

a

VRC, NIAID, NIH USAMMDA

a

LMIV, NIAID, NIH DMID, NIAID, NIH

University of Maryland Baltimore, CVD PATH MVI (BMGF)

Centers for Disease Control Marathon Oil Corporation

Medical Care Development International Noble Energy

AMPCO

Institute for OneWorld Health (BMGF)

Europe European Vaccine Initiative

a

Switzerland Swiss TPH Swiss State Secretariat for Education, Research and Innovation

a

Germany University of Tübingen German Centre for Infection Research

The Netherlands Radboud University Medical Center (RUMC), Top Institute Pharma

Leiden University Medical Center

a

Spain ISGlobal, Barcelona Centre for International Health Research (CRESIB) CRESIB, Spanish Government

a,b

UK Jenner Institute, Oxford University The Wellcome Trust

Africa

Tanzania Ifakara Health Institute (IHI) Tanzania Commission on Science and Technology (COSTECH)

Equatorial Guinea Ministry of Health and Social Welfare Government of Equatorial Guinea

Equatorial Guinea LNG

Kenya Kenya Medical Research Institute

Wellcome Trust Laboratories

Centre for Research in Therapeutic Sciences (CREATES)

Gabon Centre de Recherches Médicales de Lambaréné, Albert Schweitzer Hospital

Mozambique Manhica Center

Ghana Kintampo Health Research Center Ghana Ministry of Health

Mali University of Bamako (MRTC)

Burkina Faso Centre National de Recherche et de Formation sur le Paludisme (CNRFP)

Asia

Indonesia Eijkman-Oxford Clinical Research Unit (EOCRU), Jakarta

a

Invested institutional funds in clinical trials of PfSPZ-based products.

b

Only involved with PfSPZ Challenge.

422 planned for 2015–2016 (Table 3). These include trials of condensed immediately after release of parasites into the blood is also being 442

423 regimens (as few as 3 doses in 10 days) and alternative drug part- actively pursued, as such parasites will mimic PfSPZ-CVac without 443

424 ners (atovaquone/proguanil). Close attention is being paid to safety the need for administering anti-malarial drugs. 444

425 considerations, since the PfSPZ-CVac approach includes injecting

426 healthy individuals with a human pathogen, notwithstanding the 3.4. International SPZ consortium 445

427 fact that PfSPZ Challenge (NF54) is highly sensitive to chloroquine

428 and other antimalarials. Any regimen will require ingestion and A key aspect of the CDPs for PfSPZ Vaccine, PfSPZ-CVac and 446

429 retention of a protective drug before the PfSPZ are administered. PfSPZ-GA1 are their reliance on an informed and proactive consor- 447

430 This will be done under direct observation. The challenge during tium of research and funding institutions that together constitute 448

431 development is to demonstrate that vaccination with PfSPZ-CVac the International PfSPZ Consortium (I-PfSPZ-C) (Table 4). Mem- 449

432 is as safe as any other approach to vaccination. PfSPZ-CVac may bers meet periodically to share and critique data, and to discuss 450

433 be most appropriate for use in elimination campaigns in endemic plans. The most recent meeting was held 11–12 March 2015, in 451

434 areas, where the population is already exposed to natural malaria Tübingen (Box 2). The enthusiasm and aggressive research strate- 452

435 transmission. gies of the I-PfSPZ-C have propelled innovative approaches and 453

greatly accelerated the development and testing of PfSPZ-based 454

436 3.3. PfSPZ-GA1 products. Sanaria acts as sponsor for nearly all the trials, which 455

are conducted under US FDA oversight, as well as the oversight 456

437 Clinical lots of PfSPZ-GA1 have been manufactured. A proposal of regulatory authorities in the countries outside the US where 457

438 for the first trial has been submitted for funding. The first step will the trials are conducted. Sanaria also serves as lead organizer for 458

439 be to establish adequate attenuation and to compare protective the I-PfSPZ-C and as a repository for information. Of particular 459

440 efficacy with PfSPZ Vaccine. A vaccine based on genetically atten- importance is the dissemination of clinical data among sites, to 460

441 uated Pf designed to arrest development late in the liver stage or provide alerts in case of adverse events, and to inform all partners 461

Please cite this article in press as: Richie TL, et al. Progress with Plasmodium falciparum sporozoite (PfSPZ)-based malaria vaccines.

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underlying protection. Moreover, the whole organism approach 501

Box 2: The International PfSPZ Consortium. mirrors that of many other live, attenuated vaccine products, nearly 502

The International PfSPZ Consortium (I-PfSPZ-C) has evolved

all of which are highly protective. 503

to integrate the planning and governance of development

The fact remains, however, that there are no vaccines licensed 504

among the organizations collaborating to take PfSPZ Vaccine

to protect humans against parasites, which are far more complex 505

and PfSPZ-CVac through to licensure. The I-PfSPZ-C has an

than viral or bacterial pathogens. It is therefore to be expected that 506

inclusive structure in which all individuals, groups, institutions

numerous innovations have been required, and these have included 507

and funding organizations involved in studies with Sanaria’s

novel manufacturing process steps for the production of highly 508

PfSPZ-based products participate in the planning of trials, and

509

reviewing data on safety, immunogenicity and protective effi- PfSPZ-infected, aseptic mosquitoes, harvesting infected glands, and

cacy of PfSPZ vaccines. They also contribute to the efforts to purifying and cryopreserving the PfSPZ. On the clinical side, it has 510

raise funds to support the program. The I-PfSPZ-C has met been necessary to develop new immunization regimens, and to 511

two to three times per year since November 2011, including

develop DVI as a method for efficient PfSPZ administration. Fur- 512

sessions organized in association with the annual meeting

ther process refinements are anticipated in the coming years after 513

of the American Society of Tropical Medicine and Hygiene.

licensure of the first generation PfSPZ vaccine(s), and these include 514

The most recent meeting was at the University of Tübingen,

robotic extraction of PfSPZ from mosquitoes and in vitro develop- 515

Germany, in March 2015. The meeting was attended by 81

ment of PfSPZ from sexual stage parasites. Although the worldwide 516

individuals from 35 different institutions based in 14 different

need for a malaria vaccine can be met using current methods for 517

countries in the USA, Europe and Africa. In addition to clin-

manufacture, such innovations will simplify scale-up and reduce 518

ical investigators, participants included representatives from

African governments, policy agencies, regulatory authorities the cost of goods. 519

and funding agencies. The rapid progress achieved by the I-PfSPZ-C would not have 520

been possible without the open-minded and creative approaches 521

adopted by Sanaria and collaborators. Members of the I-PfSPZ- 522

C have provided leadership in key developments including DVI 523

462 regarding outcomes, which may impact the design and manage-

administration, condensed immunization regimens, and novel 524

463 ment of other trials. In the case of the meeting in Tübingen, results

vaccine concepts such as PfSPZ-CVac. Remarkably, our clinical 525

464 of five clinical trials were reported to members long before publi-

experience has demonstrated excellent safety and tolerability, 526

465 cation in a scientific journal.

regardless of route of administration. This allows for the testing 527

of higher doses, which appear needed to achieve our objectives in 528

466 3.5. Malaria elimination campaigns

those with prior malaria exposure. As new technologies for man- 529

ufacturing, formulation, cryopreservation and administration are 530

467 A remarkable aspect of the PfSPZ development story has been

developed, and as indicated by the results of ongoing clinical test- 531

468 the financial support by African partner countries, first Tanzania,

ing, optimized vaccine candidates and immunization regimens will 532

469 then Equatorial Guinea and, most recently, Ghana; this is an impor-

be advanced under appropriate regulatory guidance. The long-term 533

470 tant milestone in malaria vaccine development. A striking example

goal is durable, cross-strain, sterile immunity in >90% of vaccine 534

471 is occurring in Equatorial Guinea, which will support four trials

recipients with the fewest numbers of PfSPZ in the fewest num- 535

472 of PfSPZ Vaccine and associated operational research in Equato-

bers of doses in the shortest period of time. The target product 536

473 rial Guinea and Tanzania to optimize vaccine administration for

must also demonstrate operational, safety and tolerability char- 537

474 later use in malaria elimination campaigns. The Equatorial Guinea

acteristics suitable for use in mass administration campaigns. The 538

475 Government, Marathon Oil Corporation, Noble Energy, AMPCO,

I-PfSPZ-C is working toward these long-term objectives, aiming for 539

476 Medical Care Development International (MCDI) and Sanaria have

a PfSPZ vaccine to be the cornerstone for malaria elimination and 540

477 partnered to conduct a malaria elimination campaign after a PfSPZ-

eradication. 541

478 based vaccine is licensed for this indication. The collaboration in

479 Equatorial Guinea involves not only Equatorial Guinea Ministry of

480 Health and Social Welfare and Sanaria investigators, but also a team

Acknowledgements 542

481 from the Ifakara Health Institute in Bagamoyo, Tanzania, and collab-

482 orators from the Swiss Tropical and Public Health Institute, Medical

We warmly thank the many institutions, investigators and 543

483 Care Development International (MCDI), and La Paz Medical Cen-

their teams who were or are involved in the conduct of com- 544

484 tre. The first phase of the demonstration will target elimination

pleted, ongoing and planned trials of PfSPZ products, for their 545

485 of malaria from Bioko Island. The population of >250,000 endure

collaboration and, in many cases, financial support (listed by insti- 546

486 ongoing malaria transmission and significant malaria-related mor-

tution in alphabetical order): Armed Forces Research Institute of 547

487 bidity and mortality despite a well-funded and executed malaria

Medical Sciences, Bangkok, Thailand: Ratawan Ubalee; Center for 548

488 control program [50]. Plans for this campaign will be developed

Malaria Research, Institute for Global Health, University of Mary- 549

489 during the coming years, anticipating launch of the campaign

land School of Medicine, Baltimore, MD, United States: Matthew 550

490 immediately after licensure.

Laurens, Andrea Berry, Robert Edelman, Christopher Plowe; CER- 551

MEL (Centre de Recherches Médicales de Lambaréné), Lambaréné, 552

491 4. Conclusions Gabon: Jean-Claude Dejon Agobe, Jeannot Zinzou, Bertrand Lell; 553

Centers for Disease Control, Atlanta, GA, United States: Mary Hamel, 554

492 With the development of PfSPZ-based products for parenteral Laurence Slutsker; Centre National de Recherche et de Forma- 555

493 injection, the field of malaria vaccines is returning to principles tion sur le Paludisme (CNRFP), Burkina Faso: Sodiomon Sirima; 556

494 of highly protective immunization first established in birds in the Eijkman-Oxford Clinical Research Unit (EOCRU), Jakarta, Indonesia: 557

495 early 1900s, in mice in the 1960s and in humans in the 1970s Kevin Baird; Equatorial Guinea Ministry of Health and Social Wel- 558

496 (Box 1). The focus has been to reproduce the same durable protec- fare, Malabo, Equatorial Guinea: Diosdado Nsue Milang, Vicente 559

497 tive immunity using an injectable product that is safe for human Urbano Nsue Ndong Nchama, Eka Ondó Martín; Fred Hutchin- 560

498 use. This approach, unencumbered by a priori restrictions on vac- son Cancer Research Center: James Kublin Group Health, Seattle, 561

499 cine design, has led to rapid progress, and should translate into a WA, United States: Lisa Jackson Ifakara Health Institute, Bagamoyo, 562

500 more thorough understanding of the immunological mechanisms United Republic of Tanzania: Seif Shekalaghe, Said Abdallah Jongo, 563

Please cite this article in press as: Richie TL, et al. Progress with Plasmodium falciparum sporozoite (PfSPZ)-based malaria vaccines.

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