Infection with <i>P. vivax</i> Brazil VII sporozoites induces antibodies to VMP001.

<p>Control monkeys immunized with <i>Pf</i>LSA-1 did not generate antibodies against <i>P. vivax</i> (denoted as “pre challenge” in the figure). Following challenge with <i>P. vivax</i> sporozoites, the control monkeys developed anti-<i>P. vivax</i> CSP antibodies, as assessed by ELISA reactivity to VMP001, that were significantly boosted following rechallenge (2-tailed Mann-Whitney U test). Data is represented as Box (25–75 percentile) and Whisker (minimum and maximum) plot with horizontal line at the median value.</p

Loss of protective efficacy following rechallenge could be attributed to a drop in anti-repeat antibody titers.

<p>Kaplan-Meier survival curves were generated by pooling data from the low (15 µg) and high (50 µg) dose groups (from <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003268#pntd-0003268-g003" target="_blank">Figure 3</a>), and the combined VMP001 immunized group was compared to the control group. <b>6A</b>. Following primary challenge, 12 of 16 immunized monkeys and 2 of 8 control monkeys remained uninfected, resulting in a calculated vaccine efficay of 66.7% (p = 0.004). <b>6B</b>. Following rechallenge, one immunized monkey remained uninfected and several others had a delay compared to the controls (<i>p</i> = .04). <b>6C</b>. Sera drawn two weeks prior to the first challenge from protected monkeys (n = 12) had significantly higher anti-repeat antibody titers compared to sera drawn pre-rechallenge from the non-protected monkeys (n = 13); p = 0.02 (2-taled Mann Whitney U-test). Titers indicate serum dilution that gives an OD₄₁₄ of 1.0. Each dot represents an individual monkey. Horizontal bar represents the median titer for each group.</p

<i>Aotus nancymaae</i> immunized with VMP001 generated antigen-specific antibodies that were boosted following each immunization.

<p>Groups of 8 monkeys were immunized three times with either 15 µg (panel A) or 50 µg (panel B) of VMP001 formulated with 200 µg CpG 10104 and Montanide ISA 720. Sera, drawn at two week intervals, were tested for presence of antibodies to VMP001. Antibody titers waned over time, but were boosted with each immunization. Titers are defined as serum dilution that gives an OD₄₁₄ of 1.0. Each symbol represents an individual monkey. Some time points have less than eight data points due to the unavailibility of serum sample at that time point. Horizontal bars represent the median titer for each group at the given time point.</p

Protective efficacy of VMP001 in <i>Aotus</i> monkeys challenged intravenously with <i>P. vivax</i> Brazil VII strain post primary and rechallenge.

<p>Monkeys were immunized with either 15 µg or 50 µg of VMP001. Control monkeys were immunized with 50 µg of <i>P. falciparum</i> LSA-1 (<i>Pf</i>LSA-1). All antigens were formulated with Montanide ISA 720 and CpG 10104. Kaplan-Meier survival curves represent percentage of uninfected monkeys over time. Vaccine Efficacy was calculated as a ratio between the vaccinated and control groups (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003268#s3" target="_blank">results</a>). <b>3A</b>. Six weeks following the third immunization, all monkeys were challenged intravenously with 10,000 <i>P. vivax</i> Brazil VII parasites. <b>3B</b>. Eight weeks after the primary challenge, monkeys were rechallenged with 15,000 <i>P. vivax</i> Brazil VII parasites.</p

Onset and magnitude of antibody response to VMP001 is similar in both the low (15 µg) and high (50 µg) dose groups.

<p>In the presence of a strong adjuvant formulation the differences in anti-VMP001 titers between the two immunized groups were not stastically significant (ns = <i>p</i>>0.05; 2-tailed Mann Whitney U test) at any timepoint. Data is represented as Box (25–75 percentile) and Whisker (minimum and maximum) plot with horizontal line at the median value.</p

Protected monkeys from both the low- and high dose groups had significantly higher anti-repeat antibodies compared to the non-protected monkeys.

<p>Pre-primary challenge sera from immunized monkeys from the low (15 µg) and high (50 µg) dose groups were pooled from the protected (n = 12) and non-protected (n = 4) groups. Antibody reactivity between the protected and non-protected groups were similar for VMP001, N- and C-terminal regions. However, anti-repeat antibodies were significantly different between the protected and non-protected groups (<i>p</i> = 0.034, 2-tailed Mann Whitney U test). Data is represented as Box (25–75 percentile) and Whisker (minimum and maximum) plot with horizontal line at the median value. Data also represented in a tabular form to demonstrate correlation bewteen protected and non-protected groups.</p

No change in infectivity after cryopreservation of Pv spz.

<p>25,000 Pv spz (Fresh or thawed after cryopreserved in LNVP for one year) were added to wells containing 20,000 hepatoma cells and cultured for 3 days. The numbers of early liver stage trophozoites (3 day assay) were counted by immunofluorescence microscopy after staining with the anti-PvCSP mAb, NVS3, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014275#s2" target="_blank">Materials and Methods</a>.</p

<i>In-vitro</i> development of late liver stage schizonts expressing PvMSP1.

<p>20,000 HepG2-A16 cells were infected with 50,000 Pv spz. Three hrs later uninfected Pv spz were washed off and the culture was maintained for 9 days with daily media changes. Mature Pv liver stage schizonts (400 X magnification) in HepG2-A16 cells were stained with (A) the mAb to the PvCSP, NVS3 (20 µg/ml) or (B) with a mAb against Pv merozoite surface protein 1(PvMSP1), 3F8.A2 (1∶50 dilution). As a negative control, uninfected HepG2-A16 cells were incubated with the individual mAbs and labeled secondary antibodies. There was no evidence of staining in these negative control cultures (data not shown).</p

Are these hypnozoites?

<p>HepG2-A16 cells were infected, maintained for 9 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014275#pone-0014275-g004" target="_blank">Fig 4</a> and stained with PvCSP mAb. Approximately 10% of the Pv liver stage parasites that expressed PvCSP were similar in size to 3-day trophozoites, and much smaller than the 9-day schizonts. Are these hypnozoites?</p

Multiple Pv liver stage parasites were seen in single hepatocyte in a 3 Day culture.

<p>HepG2-A16 cells were infected with Pv spz and the liver stage trophozoites were stained with the anti-PvCSP mAb, NVS3. Some HepG2-A16 cells were seen with multiple liver stage parasites (400X magnification). <b>N</b>: Nucleus of hepatocyte, C: Cytoplasm of hepatocyte, White Arrows: Individual 3 Day hepatocyte stage Pv.</p