Protection of Rhesus Monkeys by a DNA Prime/Poxvirus Boost Malaria Vaccine Depends on Optimal DNA Priming and Inclusion of Blood Stage Antigens

(Pk) malaria. This is a multi-stage vaccine that includes two pre-erythrocytic antigens, PkCSP and PkSSP2(TRAP), and two erythrocytic antigens, PkAMA-1 and PkMSP-1(42kD). The present study reports three further experiments where we investigate the effects of DNA dose, timing, and formulation. We also compare vaccines utilizing only the pre-erythrocytic antigens with the four antigen vaccine.In three experiments, rhesus monkeys were immunized with malaria vaccines using DNA plasmid injections followed by boosting with poxvirus vaccine. A variety of parameters were tested, including formulation of DNA on poly-lactic co-glycolide (PLG) particles, varying the number of DNA injections and the amount of DNA, varying the interval between the last DNA injection to the poxvirus boost from 7 to 21 weeks, and using vaccines with from one to four malaria antigens. Monkeys were challenged with Pk sporozoites given iv 2 to 4 weeks after the poxvirus injection, and parasitemia was measured by daily Giemsa stained blood films. Immune responses in venous blood samples taken after each vaccine injection were measured by ELIspot production of interferon-γ, and by ELISA.1) the number of DNA injections, the formulation of the DNA plasmids, and the interval between the last DNA injection and the poxvirus injection are critical to vaccine efficacy. However, the total dose used for DNA priming is not as important; 2) the blood stage antigens PkAMA-1 and PkMSP-1 were able to protect against high parasitemias as part of a genetic vaccine where antigen folding is not well defined; 3) immunization with PkSSP2 DNA inhibited immune responses to PkCSP DNA even when vaccinations were given into separate legs; and 4) in a counter-intuitive result, higher interferon-γ ELIspot responses to the PkCSP antigen correlated with earlier appearance of parasites in the blood, despite the fact that PkCSP vaccines had a protective effect

Summary of Parasitemia Data in Experiment #1

<p>‘Day 1^st parasitemia’ is the day after sporozoite challenge when parasites were first seen on malaria smear. ‘Day >1% parasitemia’ is the day of drug treatment (‘unknown’ means that monkeys were drug treated before reaching 1% parasitemia). Priming with 3 doses of DNA in PBS gave better protection than priming with 1 dose of DNA in PBS or 3 doses of DNA on PLG (<b>*</b>p = 0.04 Fisher's Exact test).</p

Panels A–D show the % parasitemia for individual monkeys by vaccine group according to the day after sporozoite challenge for Experiment #1.

<p>Data shows the first day parasites were detected and continues until the animal was drug treated when parasitemia exceeded 1% ( 3 animals inadvertently treated at lower parasitemias have open symbols). For comparison, in each panel the grey line shows the mean parasitemia for the Control group. In panel C, one animal never became parasitemic as indicated by x-x. Panel E shows the geometric mean parasitemias for vaccine groups for all days in which at least three animals had not been drug treated. (The monkey from the DNA Pk4×3/COPAK group which did not become infected was excluded from the average).</p

Panel A. Interferon-γ ELIspot was tested for only two antigens: PkCSP and PkMSP1.

<p>Spots in medium controls were subtracted from antigen test wells, and the results averaged. Priming with DNA on PLG gave a stronger interferon-γ response than DNA in PBS but this was not significant (p = 0.58). Panel B. Geometric mean antibody titers by ELISA at time of sporozoite challenge for each of the four malaria vaccine antigens. Priming with 3 doses of DNA gave higher antibody levels than did a single DNA priming dose for PkCSP, PkMSP1 and PkAMA1 antigens (p<0.05). Priming with 3 doses of DNA in PBS produced higher serum antibody titers than did priming with 3 doses of DNA on PLG for PkCSP, PkSSP2, and PkAMA1 antigens (p<0.05).</p

Summary of parasitemia data in Experiment #2

<p>‘Day 1^st parasite’ is the day after sporozite challenge when a parasite was first seen on malaria smear. ‘Day >2% parasitemia’ is the time of drug treatment. The group receiving the four antigen vaccine and the single antigen PkCSP vaccine had a delay in the appearance of parasites in the blood (<b>*</b>p = 0.03 vs Control group, Fisher's Exact Test). The group receiving high doses of PkCSP DNA priming was not as well protected. Co-immunization with PkSSP2 and PkCSP was worse than immunization with PkCSP alone (p = 0.03). Only the monkeys receiving all 4 antigens were protected against high parasitemias (<b>**</b>p<0.001 vs Control group).</p

Immune response to PkCSP antigen at time of challenge for individual animals by day to first parasite seen in blood.

<p>Data from Experiments #1, 2, and 3 are plotted. Data from Control animals are not included. Panel A shows PkCSP endpoint ELISA titers where there is no significant correlation. Panel B shows PkCSP interferon-γ ELIspot titers with a linear regression line included. There is a significant negative correlation (p = 0.04), animals with lower ELIspot responses having longer times to the appearance of first parasite in the blood.</p

Immunization schedule for Experiment #2

<p>Five monkeys in each of five vaccine groups were immunized and challenged with Pk malaria sporozoites. The four antigen vaccine given Group 1 was identical to that given to Group 1 in Experiment 1. Group 2 received only the PkCSP components of the vaccine given to Group 1. Group 3 received a ten-fold larger dose of the PkCSP DNA than Group 2 and the same dose of PkCSP COPAK. Group 4 received only the PkCSP and PkSSP2 components of the vaccine given to Group 1. Group 5, the Control group, received a mock vaccine consisting of plasmid DNA without malaria antigen inserts followed by parental COPAK without malaria inserts. (See text for more details).</p

Panels A–C show the % parasitemias for individual monkeys by vaccine group according to the day after sporozoite challenge for Experiment #3.

<p>Data shows the first day parasites were detected and continues until the animal was treated with anti-malaria drugs. For comparison, in each panel the grey line shows the mean parasitemia for the Control group. Panel D shows the geometric mean parasitemias for all the vaccine groups for all days in which at least three animals had not been drug treated. For each day 8–11 the mean parasitemia for the group receiving the booster dose at the 21 week interval was lower than the other groups (p<0.05, Student's T test).</p