Protection of rhesus monkeys by a DNA prime/poxvirus boost malaria vaccine depends on optimal DNA priming and inclusion of blood stage antigens.

BACKGROUND: We have previously described a four antigen malaria vaccine consisting of DNA plasmids boosted by recombinant poxviruses which protects a high percentage of rhesus monkeys against Plasmodium knowlesi (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. METHODOLOGY: 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 i.v. 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-gamma, and by ELISA. CONCLUSIONS: 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-gamma 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.

Response of lightly and highly pigmented porcine skin (Sus scrofa domestica) to single 3.8-microm laser radiation pulses.

The purpose of this study was to determine the effect of melanin on skin response to single 3.8-microm, 8-micros laser pulses and the difference in lesion formation thresholds. Our hypothesis was that pigmentation would play a significant role in skin energy absorption at 3.8 microm. Previous studies comparing pigmented and lightly pigmented porcine skin with human skin found that compared with Yorkshire pigs, Yucatan minipigs were a superior model for laser skin exposure because of their higher pigmentation levels. In the current study, 10 pigs under general anesthesia were exposed to 3.8-microm laser pulses ranging from 0.01 J/cm2 to 93 J/cm2. Gross examinations and skin biopsies were done 24 h after laser exposure, and histologic examinations were conducted on these tissue samples. The 24-h effective dose (ED50) was determined to be 4.5 J/cm2 for Yucatan mini-pigs and 2.6 J/cm2 for Yorkshire pigs. As deposited energy was increased, the lesion presentation progressed from desiccation of the superficial layer of epidermis (4 J/cm2) to desiccation with inflammatory centers (14 J/cm2), and finally to replacement of inflammatory areas with an epidermal ulcerated central area (=21 J/cm2). Therefore we found no statistical difference between the 24-h ED50 of the 2 breeds of pigs, nor was there any difference in histologic presentation at 24 h postexposure.