Vaxfectin™ enhances immunogenicity and protective efficacy of P. yoelii circumsporozoite DNA vaccines

Vaccine 24 (2006) 1921–1927

VaxfectinTM enhances immunogenicity and protective efficacy of P. yoelii circumsporozoite DNA vaccines Martha Sedegah a,∗ , William O. Rogers a,1 , Arnel Belmonte a , Maria Belmonte a , Glenna Banania a , Noelle Patterson a , Marilyn Ferrari b , David C. Kaslow b , Daniel J. Carucci a , Thomas L. Richie a , Denise L. Doolan a a

Malaria Program, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, USA b Vical Inc., San Diego, CA 92121-4340, USA

Received 10 August 2005; received in revised form 14 October 2005; accepted 21 October 2005 Available online 2 November 2005

Abstract We evaluated the capacity of the cationic lipid based formulation, VaxfectinTM , to enhance the immunogenicity and protective efficacy of DNA-based vaccine regimens in the Plasmodium yoelii murine malaria model. We immunized Balb/c mice with varying doses (0.4–50 ␮g) of plasmid DNA (pDNA) encoding the P. yoelii circumsporozoite protein (PyCSP), either in a homologous DNA/DNA regimen (D-D) or a heterologous prime-boost DNA-poxvirus regimen (D-V). At the lowest pDNA doses, VaxfectinTM substantially enhanced IFA titers, ELISPOT frequencies, and protective efficacy. Clinical trials of pDNA vaccines have often used low pDNA doses based on a per kilogram weight basis. Formulation of pDNA vaccines in VaxfectinTM may improve their potency in human clinical trials. © 2005 Elsevier Ltd. All rights reserved. Keywords: Malaria; T-lymphocytes; Plasmid DNA (pDNA) vaccines

1. Introduction Plasmid DNA (pDNA)-based vaccines have been widely used in animal models of a variety of infectious diseases, including hepatitis B, HIV, and Ebola [1–3]. Phase 1/2a clinical trials of DNA prime/viral boost vaccination regimens for malaria and HIV have been conducted recently, and a large Phase 2b trial of a prime-boost HIV vaccine is underway in Thailand. Although such prime-boost regimens have proAbbreviations: Py, Plasmodium yoelii; Spz, sporozoites; IrrSpz, irradiated sporozoites; pDNA, plasmid DNA; ELISPOT, enzyme-linked immunospot; CSP, circumsporozoite protein; IFN-␥, interferon-gamma; IFAT, immunofluorescent antibody test; CTL, cytotoxic T-lymphocytes; SFCs, spot forming cells; MVA, modified vaccinia Ankara ∗ Corresponding author. Tel.: +1 301 319 7586; fax: +1 301 319 7545. E-mail address: [email protected] (M. Sedegah). 1 Current address: Naval Medical Research Unit 2, U.S. Embassy Jakarta, Unit 8132 NAMRU2, FPO AP 96520-8132, Indonesia. 0264-410X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2005.10.041

vided impressive immunogenicity and protective efficacy in a variety of animal models, preliminary human trials of similar vaccines for HIV and malaria have shown only modest immunogenicity [4–6]. In a small-scale challenge study, a pDNA prime/MVA boost vaccine provided minimal protection against challenge with P. falciparum sporozoites [7]. The pDNA doses used in human trials, 500–5000 ␮g or 7–70 ␮g/kg (70 kg human), are substantially lower on a host weight basis than those commonly used in mouse studies, 10–100 ␮g or 500–5000 ␮g/kg (20 g mouse). In some human studies, immune responses measured after DNA immunization were highest in volunteers receiving the highest doses [8,9]. The relatively poor immunogenicity of plasmid pDNA in previously reported human trials may be due, in part, to the low doses being used, rather than solely to speciesspecific differences in the response to pDNA. It is possible that approaches that improve the potency of very low doses of plasmid in mice (0.4–2.0 ␮g) will similarly improve

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the potency of commonly used doses in human clinical trials. Several reports have demonstrated that the cationic lipid/neutral co-lipid mixture VaxfectinTM , when complexed with a variety of antigen-encoding pDNA, is a potent inducer of antigen-specific antibodies. Levels of antibodies several fold (8–50-fold) higher than those associated with pDNA alone have been reported [10–16]. In studies conducted in mice and rabbits, VaxfectinTM , when used to deliver vaccine antigens for infectious diseases, including anthrax lethal factor (LF), anthrax protective antigen (PA) [13] and influenza [10] induced strong systemic, long-lived, and antigen specific antibody responses. Another member of this class of genedelivery compounds, DMRIE-DOPE, has undergone Phase 1, Phase 2, and Phase 3 testing in humans with candidate vaccines and immunotherapies for cancer (e.g. LeuvectinTM and Allovectin® ) [17,18] and have also been shown to induce immune response in large animals [19,20]. In the human cancer vaccine trials, only mild to moderate adverse events were associated with the gene-delivery/system, such as mild injection site discomfort and constitutional symptoms such as low-grade fever, malaise, and chills [17,18]. To assess the effect of VaxfectinTM , the Plasmodium yoelii murine malaria/model in Balb/c mice was used with a pDNA encoding the P. yoelii circumsporozoite protein, either alone, or as the priming agent in a heterologous prime-boost regimen. Antibody and IFN-␥ responses against PyCSP, and protection against challenge with P. yoelii sporozoites were selected as relevant immunological endpoints.

2. Materials and methods 2.1. Mice Six- to 8-week-old female Balb/c (H-2d) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). 2.2. Parasites for challenge P. yoelii (17XNL) clone 1.1 parasites were used [21]. Sporozoites for challenges were obtained by hand dissection of infected mosquito glands. The isolated sporozoites were suspended in M199 medium containing 5% normal mouse serum. All challenges were accomplished by injecting 50–100 sporozoites in the tail vein. For each batch of sporozoites used for the challenge, groups of na¨ıve mice were injected with serially diluted sporozoites to determine the ID50 of the sporozoite batch used. Protection in experimental groups was defined as lack of patent parasitemia during 14 days after challenge.

2.4. VaxfectinTM formulations Formulations were prepared by adding the appropriate concentration of VaxfectinTM in sterile water for injection (SWFI) to an equal volume of pDNA at twice the desired final concentration in 2× PBS (20 mM sodium phosphate, pH 7.2, 1.8% NaCl). Formulations were mixed by gentle inversion. The final molar ratio of pDNA/cationic lipid was 4:1. VaxfectinTM consists of VC1052: (±)-N-(3-Aminopropyl)-N,N -dimethyl -2,3 -bis(cis-9-tetradecenyloxy)-1-propanaminium bromide) and DPyPE: 1,2-Diphytanoyl-snglycero-3-phosphoethanolamine) at a 1:1 molar ratio. 2.5. Immunizations Mice received, at 4-week intervals, bilateral intramuscular injections (tibialis anterior muscle) of pDNA formulated in PBS or with VaxfectinTM . Insulin syringes (0.3 ml) with 29G1/2 gauge needles were used for all pDNA injections. Dose-levels of PyCSP pDNA included 0.4, 2, 5, 10, and 50 ␮g. After three injections with pDNA, the homologous immunization regimen groups received a fourth pDNA immunization, whereas the heterologous immunization groups received an intraperitoneal boost with 2 × 107 pfu COPACPyCSP recombinant virus. Controls included: (1) mice immunized four times with unmodified pDNA formulated in PBS or VaxfectinTM ; (2) mice immunized three times with unmodified pDNA formulated in PBS or VaxfectinTM and boosted with control virus; (3) mice immunized only with the COPACPyCSP boost; and (4) na¨ıve mice. In the protection studies involving pDNA priming followed by three booster injections, Balb/c mice were challenged with sporozoites 2 weeks after the last immunization (week 14). In these studies, two protection experiments were conducted, 14 mice per group were used (in the first experiment), and 34 mice per group were used (in the second). A third protection experiment was carried in which a shortened immunization regimen was followed. Mice received a single intramuscular injection of pDNA formulated in PBS or with VaxfectinTM . Dose-levels of PyCSP pDNA included 0.08, 0.4, and 2 ␮g. Four weeks later, all primed animals received an intraperitoneal boost with 2 × 107 pfu COPACPyCSP recombinant virus. Twenty-two mice per group were used. 2.6. Immuogenicity assays To detect antibodies against P. yoelii sporozoites, an indirect immunofluorescence assay (IFA) of serum collected 2 weeks after the final immunization was used as previously described [23]. The results were expressed as an IFA titer (IFAT).

2.3. Immunogens 2.7. IFN-γ ELISPOT in P. yoelii vaccine study The pDNA encoding P. yoelii circumsporozoite protein (PyCSP) and the recombinant, attenuated vaccinia virus, COPACPyCSP, have been described previously [22].

In the T cell studies, spleen cells from immunized BALB/c mice were harvested for use in IFN-␥ Elispot assays after

M. Sedegah et al. / Vaccine 24 (2006) 1921–1927

the last immunization. There were eight mice per group in the T cell experiment and mice were assayed individually. Two weeks after last immunization, the number of PyCSP CTL epitope (aa 280–288, SYVPSAEQI) -specific, IFN-␥-secreting, spot forming cells (SFC) was determined, as previously described [22]. Briefly, triplicate wells were tested in all assays; unpulsed target cells served as controls; the number of IFN-␥-secreting cells, recognized as spots, was determined using an automated EliSpot Reader manufactured by AutoImmun Diagnostika (AID), GmbH, Strassberg, Germany. The results were expressed as the number of IFN-␥-secreting SFC cells per million spleen cells. 2.8. Statistical analysis IFAT and SFC frequencies were not, in general, normally distributed; however, when the data were log-transformed, they approximately followed a normal distribution. All calculations of confidence intervals were carried out with logtransformed data. For IFAT, we report the mean of the log of the titer in each group and the difference in the mean log titer between corresponding VaxfectinTM and PBS groups. For IFN-␥ ELISPOT, we report the geometric mean SFC frequencies as SFC/106 splenocytes and the ratios of the frequencies in the VaxfectinTM groups compared with the frequencies in the corresponding PBS groups. Finally, we report mean and 95% confidence intervals around the proportion of mice protected against sporozoite challenge in corresponding groups in which the PyCSP pDNA was formulated in VaxfectinTM

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or in PBS. Statistical analysis was performed with Stata/SE version 8.0.

3. Results 3.1. Enhancement of antibody and IFN-γ ELISPOT responses to PyCSP pDNA In three independent experiments, we immunized groups of Balb/c mice with varying doses of the PyCSP pDNA formulated either in VaxfectinTM or in PBS. In the first two experiments, two regimens were assessed, one in which four doses of pDNA were administered at 4-week intervals (D-D regimen); and one in which three injections of pDNA were administered at 4-week intervals followed 4 weeks later by a single boost with recombinant attenuated pox virus expressing the PyCSP (D-V regimen). In the third experiment only the D-V regimen was used, and only a single priming dose of pDNA was given. Table 1 shows the effect of formulating the pDNA in VaxfectinTM rather than PBS. For the D-D regimens, IFAT were substantially higher in the VaxfectinTM groups. The greatest effects, 10–200-fold increases in titer, were observed at the lower doses of pDNA, 2.0 and 0.4 ␮g. The mean log titer, 4.00 (95% CI, 3.85, 4.16), induced by immunization with 0.4 ␮g pDNA formulated with VaxfectinTM was similar to the mean log titer induced by a 10 ␮g dose delivered in PBS, 3.86 (95% CI, 3.71, 4.00). A similar, though less marked, finding was observed in the heterologous

Table 1 Anti-sporozoite IFA titers Regimen

Plasmid dose (␮g)

Log IFA titer (95% CI) Experiment 1 (n = 14) Vaxfectin

D-D

0.4 2.0 10 50

D-V

PBS

Experiment 2 (n = 34) Difference (Vax-PBS)

N.D. 3.89 (3.75, 4.03) 3.94 (3.72, 4.16) 4.35 (4.18, 4.53)

0.08

3.15 (3.00 3.30) 3.15 (2.97, 3.33) 3.62 (3.46, 3.78)

0.74 (0.55, 0.94) 0.79 (0.53, 1.06) 0.73 (0.50, 0.96)

10 50 a

Vaxfectin

PBS

Difference (Vax-PBS)a

4.00 (3.85, 4.16) 4.31 (4.18, 4.44) 4.49 (4.33, 4.65)

1.74 (1.62, 1.87) 3.12 (3.01, 3.22) 3.86 (3.71, 4.00) N.D.

2.26 (2.06, 2.46) 1.19 (1.03, 1.36) 0.63 (0.42, 0.84)

N.D.

N.D.

0.4 2.0

5.32 (5.06, 5.58) 5.26 (5.03, 5.49) 4.33 (4.19, 4.48)

4.66 (4.36, 4.95) 4.55 (4.25, 4.85) 4.70 (4.49, 4.91)

Experiment 3 (n = 22)

0.66 (0.29, 1.04) 0.71 (0.35, 1.08) −0.37 (−0.61, −0.12)

4.39 (4.27, 4.51) 4.02 (3.88, 4.16) 4.16 (4.05, 4.26) 4.29 (4.18, 4.40)

A difference in log titer of 2.0 corresponds to a 100-fold difference in titer.

3.38 (3.22, 3.54) 3.77 (3.55, 3.99) 3.97 (3.87, 4.08) 4.22 (4.07, 4.37)

1.01 (0.82, 1.21) 0.25 (−0.01, 0.51) 0.18 (0.03, 0.33) 0.07 (−0.11, 0.26)

Vaxfectin

PBS

Difference (Vax-PBS)

N.D.

2.65 (2.44, 2.87) 3.38 (3.25, 3.51) 3.50 (3.36, 3.65)

1.75 (1.62, 1.89) 2.07 (1.92, 2.22) 2.33 (2.17, 2.49) N.D.

0.90 (0.69, 1.17) 1.31 (1.14, 1.48) 1.18 (0.99, 1.36)

M. Sedegah et al. / Vaccine 24 (2006) 1921–1927

1924 Table 2 IFN-␥ ELISPOT responses Regimen

D-D

Plasmid dose (␮g)

0.4 2.0 10 50

D-V

GeoMean ELISPOT frequency (95% CI) (SFC/106 splenocytes) Experiment 1 (n = 9) Vaxfectin

PBS

166 (110, 251) 347 (251, 490) 263 (170,417)

N.D. 41 (19, 89) 200 (126, 316) 245 (191, 309)

0.08

Experiment 3 (n = 8) Ratio (Vax/PBS)

10 50

240 (138, 407) 617 (513, 741) 794 (589, 1072)

immunization regimen (D-V); again the largest effects were seen at the lowest pDNA doses. Mean log IFN-␥ ELISPOT frequencies were higher in mice immunized with PyCSP plasmid in VaxfectinTM (Table 2). The greatest effect, a 9.8-fold increase (95% CI, 5.1- to 18.2-fold) in ELISPOT frequency, was seen at the lowest dose of DNA, 0.08 ␮g in the D-V regimen (Table 2, experiment 3). The mean log IFN-␥ ELISPOT frequency induced by immunization with 2 ␮g PyCSP in VaxfectinTM (Table 2, experiment 1), 166 IFN-␥ SFC/106 splenocytes, (95% CI, 110, 251) was similar to that induced by 10 ␮g delivered in PBS, 200 SFC/106 (95% CI, 126, 316). In the heterologous immunization regimen, VaxfectinTM enhanced the IFN-␥ ELISPOT response only at priming doses of pDNA less than 10 ␮g. 3.2. Enhancement of protection by formulation of PyCSP pDNA in VaxfectinTM Two challenge experiments were performed in the multiple priming regimen. Because the virulence of individual preparations of P. yoelii sporozoites may vary, serial dilutions of sporozoites were inoculated into groups of na¨ıve mice to determine the ID50 of the sporozoites used in each challenge. The challenge doses were determined to have been 10 ID50 units in the first challenge and 50 ID50 units in the second. In each case, 100% of na¨ıve mice and mice immunized with control plasmid became infected. Across all tested regimens, the proportion of protected mice was lower in the second experiment, consistent with the higher challenge dose in the second experiment. Table 3 (experiments 1 and 2) shows the proportion of mice protected against P. yoelii sporozoite challenge in groups of mice immunized with homologous (D-D) or heterologous (D-V) immunization regimens with varying doses

Ratio (Vax/PBS)

N.D.

N.D.

708 (575, 871) 724 (575,933) 550 (407, 759)

PBS

4.1 (1.8, 9.1) 1.7 (1.0, 3.0) 1.1 (0.7, 1.7)

0.4 2.0

Vaxfectin

3.0 (1.7, 5.0) 1.2 (0.9, 1.6) 0.7 (0.5, 1.0)

977 (631, 1514) 1549 (1318, 1820) 1622 (1202, 2291)

102 (59, 174) 417 (224, 759) 955 (661, 1349) N.D.

9.8 (5.1, 18.2) 3.8 (2.1, 6.8) 1.7 (1.1, 2.7)

of PyCSP pDNA formulated in VaxfectinTM or PBS. As previously reported, protection with the D-D regimen was less marked than with the D-V regimen [22]. With lower doses of DNA, in either the D-D or D-V regimen, the proportion of protected mice was higher in the VaxfectinTM groups than the PBS groups. This effect was most pronounced in the 0.4 ␮g D-V regimen in which the difference between the proportion of protected mice in the VaxfectinTM and PBS groups was 0.50 (95% CI, 0.31, 0.69). One challenge experiment was performed using a single priming regimen. Table 3 (experiment 3) shows the proportion of mice protected against P. yoelii sporozoite challenge in groups of mice immunized with the heterologous (D-V) immunization regimen using reduced doses of PyCSP pDNA formulated in VaxfectinTM or PBS. Using low pDNA doses in a single priming regimen, mice primed with pDNA formulated in PBS had no significant level of protection, an effect similar to what is usually obtained with a single injection with the recombinant pox virus only (0–10%). The challenge dose was determined to be 15 ID50 units, and 100% of na¨ıve mice became infected. In the groups that received the single priming dose of pDNA formulated with VaxfectinTM , a significant level of protection was seen in all groups. To determine the role of CD8+ T cells in this protection, some protected mice from the 0.4 ␮g PyCSP pDNA formulated in VaxfectinTM were depleted of CD8+ T cells by intraperitoneal injections of anti-CD8 mAb 3 weeks after the first challenge (and 5 weeks after the boost). This procedure removed over 99% of the CD8+ T cells as indicated in FACS analysis. Upon re-challenge, five of five previously protected mice became infected while only two of the seven control depleted mice became infected. All 15 na¨ıve control mice became infected with the 9 ID50 challenge dose. These results indicated that

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Table 3 Protective efficacy against sporozoite challenge Regimen

Plasmid dose (␮g)

Proportion protected (95% CI) Experiment 1 (n = 14) Vaxfectin

D-D

0.4 2.0 10 50

D-V

PBS

Experiment 2 (n = 34) Difference (Vax-PBS)

N.D. 0.54 (0.27, 0.81) 0.42 (0.17, 0.69) 0.29 (0.05, 0.52)

0.08

0.14 (0.0, 0.33) 0.07 (0.0, 0.21) 0.29 (0.05, 0.52)

0.40 (0.07, 0.72) 0.36 (0.06, 0.65) 0.0 (−0.33, 0.33)

10 50

Vaxfectin

PBS

Difference (Vax-PBS)

0.06 (0.0, 0.14) 0.0 (0.0, 0.09) 0.09 (0.0, 0.18)

0.03 (0.0, 0.09) 0.0 (0.0, 0.09) 0.06 (0.0, 0.14)

0.03 (−0.07, 0.13) 0.0 (−0.13, 0.13) 0.03 (−0.09, 0.15)

0.93 (0.79, 1.0) 0.77 (0.54, 1.0) 1.0 (0.79, 1.0)

0.77 (0.54, 1.0) 0.86 (0.67, 1.0) 0.64 (0.39, 0.89)

N.D.

0.16 (−0.11, 0.43) −0.09 (−0.38, 0.20) 0.36 (0.11, 0.61)

Vaxfectin

PBS

Difference (Vax-PBS)

N.D.

N.D.

N.D.

0.4 2.0

Experiment 3 (n = 22)

0.59 (0.42, 0.75) 0.86 (0.73, 0.97) 0.62 (0.45, 0.78) 0.79 (0.66, 0.93)

the mechanism of protection with this prime-boost regimen, in which low dose pDNA in VaxfectinTM is used for priming was similar to that induced by high dose PyCSP pDNA in PBS alone or in a prime-boost regimen [22].

4. Discussion We assessed the ability of VaxfectinTM to enhance the immunogenicity and protective efficacy of a pDNA vaccine encoding PyCSP, either as a stand alone vaccine or as the priming component of a heterologous prime-boost regimen with recombinant attenuated vaccinia. In all regimens, VaxfectinTM substantially increased anti-sporozoite IFAT, PyCSP-specific IFN-␥ ELISPOT, and protection against sporozoite challenge. The effect of VaxfectinTM was most marked in those vaccination regimens which were inherently less immunogenic, lower priming doses of pDNA, one rather than three priming doses, and pDNA rather than recombinant poxvirus as the boost. In all three experiments, the lowest doses of pDNA (0.08, 0.4, and 2.0 ␮g), when formulated in VaxfectinTM , induced levels of antibodies, IFN-␥ responses, and protection, similar to those induced by 5–25-fold higher doses of pDNA formulated in PBS. In contrast, at high pDNA doses (10 and 50 ␮g), VaxfectinTM had little effect on any of the measured outcomes. Although the enhancement mechanism is unclear, we think it is possible that at very low DNA doses tested, VaxfectinTM could possibly be acting as an adjuvant through stimulation of the innate immune system. Plasmid DNA complexed with cationic lipid has been shown to enhance

0.09 (0.0, 0.18) 0.59 (0.42, 0.75) 0.79 (0.65, 0.93) 0.79 (0.66, 0.93)

0.50 (0.31, 0.69) 0.26 (0.06, 0.47) −0.17 (−0.39, 0.04) 0.0 (−0.19, 0.19)

0.23 (0.05, 0.40) 0.67 (0.47, 0.87) 0.82 (0.66, 0.98)

0.09 (0.0, 0.21) 0.05 (0.0, 0.13) 0.05 (0.0, 0.13) N.D.

0.15 (−0.07, 0.37) 0.62 (0.40, 0.84) 0.77 (0.60, 0.96)

the immune responses to pDNA vaccines in animals by the generation of cytokines like IFN-␥, IL-12, and IL-6. It is possible that VaxfectinTM delivers the pDNA to multiple cell types within muscle tissue including antigen-presenting cells while unformulated pDNA transfected mainly muscle fibers [10,24]. Previous studies in the P. yoelii challenge model have shown that immunization with heterologous pDNA prime/pox virus boost regimens is more effective than immunization with DNA alone [22]. In the current study, immunization with 0.4 ␮g PyCSP pDNA formulated in VaxfectinTM as a D-D regimen induced higher antisporozoite IFAT titers than 0.4 ␮g plasmid in PBS as part of a D-V regimen (difference of mean log titers, 0.62 log units; 95% CI, 0.40, 0.84). Similarly, the mean log IFN-␥ ELISPOT response in mice immunized with 2 ␮g PyCSP pDNA formulated with VaxfectinTM as a D-D regimen, (2.22 log units, 95% CI, 2.04, 2.40), was similar to that in mice immunized with 2 ␮g plasmid in PBS as part of a D-V regimen (2.38 log units, 95% CI, 2.15, 2.61). Finally, in the first challenge study (challenge dose 10 ID50 units), the proportion of protected mice immunized with 2 ␮g PyCSP pDNA formulated with VaxfectinTM as a D-D regimen, (0.54, 95% CI 0.27, 0.81) was only slightly smaller than that in the mice immunized with 2 ␮g plasmid in PBS as part of a D-V regimen (0.77, 95% CI, 0.54, 1.0). Thus at low doses, the effect of formulating the pDNA in VaxfectinTM is similar to that of adding a recombinant vaccine boost to the immunization regimen. In preliminary human clinical trials, pDNA vaccines have induced low to no antibody responses and modest cytotoxic T-lymphocyte and IFN-␥ responses [8,9,25]. Addition of a

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recombinant poxvirus boost to the immunization regimen has improved immunogenicity [4–7] but preliminary efficacy studies with a pDNA prime/MVA boost malaria vaccine gave only minimal protection [7]. These results contrast markedly with the immunogenicity and efficacy of pDNA and pDNA prime/virus boost malaria vaccine regimens in animal models [22,26]. The pDNA doses used in most human trials are, on a per kilogram basis, 10–300-fold lower than those used in many animal models. We used relatively low dose immunization in this murine malaria model to mimic the pDNA dose per kilogram used in recent clinical trials. At these low doses, formulation of the PyCSP pDNA with VaxfectinTM substantially improved its immunogenicity and protective efficacy. Similar results using even lower pDNA doses and pDNA encoding P. falciparum candidate vaccine antigens would support use of VaxfectinTM in a DNA-based malaria vaccine clinical trial.

Acknowledgements This work was supported by the Naval Medical Research and Development Command work units 61102A 3M161102.BS13 AK111 and 62787A 3M162787.A870 AN121 and under CRADA with Vical Inc., entitled “DNA Vaccine Delivery Optimization”, Agreement Number: NCRADA-NMRC-03-1561. The assertions herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the U.S. Navy or the Naval service at large. The experiments reported herein were conducted according to the principles set forth in the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animal research, National Research Council, National Academy Press (1996).

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