Feasibility of direct venous inoculation of the radiation-attenuated Plasmodium falciparum whole sporozoite vaccine in children and infants in Siaya, western Kenya.

PfSPZ Vaccine, composed of radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum sporozoites, is administered by direct venous inoculation (DVI) for maximal efficacy against malaria. A critical issue for advancing vaccines that are administered intravenously is the ability to efficiently administer them across multiple age groups. As part of a pediatric safety, immunogenicity, and efficacy trial in western Kenya, we evaluated the feasibility and tolerability of DVI, including ease of venous access, injection time, and crying during the procedure across age groups. Part 1 was an age de-escalation, dose escalation trial in children aged 13 months-5 years and infants aged 5-12 months; part 2 was a vaccine efficacy trial including only infants, using the most skilled injectors from part 1. Injectors could use a vein viewer, if needed. A total of 1222 injections (target 0.5 mL) were initiated by DVI in 511 participants (36 were 5-9-year-olds, 65 were 13-59-month-olds, and 410 infants). The complete volume was injected in 1185/1222 (97.0%) vaccinations, 1083/1185 (91.4%) achieved with the first DVI. 474/511 (92.8%) participants received only complete injections, 27/511 (5.3%) received at least one partial injection (<0.5 mL), and in 10/511 (2.0%) venous access was not obtained. The rate of complete injections by single DVI for infants improved from 77.1% in part 1 to 92.8% in part 2. No crying occurred in 51/59 (86.4%) vaccinations in 5-9-year-olds, 25/86 (29.1%) vaccinations in 13-59-month-olds and 172/1067 (16.1%) vaccinations in infants. Mean administration time ranged from 2.6 to 4.6 minutes and was longer for younger age groups. These data show that vaccination by DVI was feasible and well tolerated in infants and children in this rural hospital in western Kenya, when performed by skilled injectors. We also report that shipping and storage in liquid nitrogen vapor phase was simple and efficient. (Clinicaltrials.gov NCT02687373).

Accuracy of DNA amplification from archival hematological slides for use in genetic biomarker studies.

Archival slides are a potentially useful source of DNA for mutation analyses in large population-based studies. However, it is unknown whether specimen age or histological stains alter the accuracy of Taq polymerase or induce secondary mutations in sample DNA. To address this question, we evaluated five methods for extraction of genomic DNA from archival bone marrow slides of 17 leukemia patients and analyzed exons 1 and 2 of the N- and K-ras genes for the presence of mutations. Of the five methods, optimal DNA purification was achieved by boiling and phenol:chloroform extraction. N-and K-ras exons 1 and 2 were independently amplified using 35 cycles of PCR, and 6-12 clones for each exon were isolated and individually sequenced for each patient. Mutations were confirmed by repeat extraction, cloning, and sequencing. Sixteen of 17 patient samples were successfully amplified (94%), including slides up to 29 years old. Twelve slides had been stained with Wright-Giemsa, I stained with toluidine blue, and 4 were unstained. A total of 16 single-base mutations were identified of 33,840 nucleotides sequenced. No insertions or deletions were identified. Six of 16 single-base mutations were previously described activating mutations in codon 13 of N-ras exon 1. The 10 other mutations were in other regions of the N- and K-ras genes and were not reproduced after repeat extraction, cloning, and sequencing. The frequency of these other alterations was I of 3384 bp. This value is comparable with the inherent error frequency for Taq polymerase. Our findings suggest that high fidelity DNA amplification can be achieved using archival hematological slides as old as 29 years and can be reliably used in genetic analyses.