Potentiating Functional Antigen-specific CD8+ T Cell Immunity by a Novel PD1 Isoform-based Fusion DNA Vaccine

Understanding and identifying new ways of mounting an effective CD8⁺ T cell immune response is important for eliminating infectious pathogens. Although upregulated programmed death-1 (PD1) in chronic infections (such as HIV-1 and tuberculosis) impedes T cell responses, blocking this PD1/PD-L pathway could functionally rescue the “exhausted” T cells. However, there exists a number of PD1 spliced variants with unknown biological function. Here, we identified a new isoform of human PD1 (Δ42PD1) that contains a 42-nucleotide in-frame deletion located at exon 2 domain found expressed in peripheral blood mononuclear cells (PBMCs). Δ42PD1 appears to function distinctly from PD1, as it does not engage PD-L1/PD-L2 but its recombinant form could induce proinflammatory cytokines. We utilized Δ42PD1 as an intramolecular adjuvant to develop a fusion DNA vaccine with HIV-1 Gag p24 antigen to immunize mice, which elicited a significantly enhanced level of anti-p24 IgG1/IgG2a antibody titers, and important p24-specific and tetramer⁺CD8⁺ T cells responses that lasted for ≥7.5 months. Furthermore, p24-specific CD8⁺ T cells remain functionally improved in proliferative and cytolytic capacities. Importantly, the enhanced antigen-specific immunity protected mice against pathogenic viral challenge and tumor growth. Thus, this newly identified PD1 variant (Δ42PD1) amplifies the generation of antigen-specific CD8⁺ T cell immunity when used in a DNA vaccine.     

A randomized trial of two postexposure prophylaxis regimens to reduce mother-to-child HIV-1 transmission in infants of untreated mothers

BACKGROUND: Single-dose nevirapine (NVP) prophylaxis to mother and infant is widely used in resource-constrained settings for preventing mother-to-child transmission (MTCT) of HIV-1. Where women do not access antenatal care or HIV testing, postexposure prophylaxis to the infant may be an important preventative strategy. METHODS: This multicentre, randomized, open-label clinical trial (October 2000 to September 2002) in South Africa compared single-dose NVP with 6 weeks of zidovudine (ZDV), commenced within 24 h of delivery among 1051 infants whose mothers had no prior antiretroviral therapy. HIV-1 infection rates were ascertained at birth, and at 6 and 12 weeks of age. Kaplan-Meier survival methods were used to estimate HIV-1 infection rates in an intention-to-treat analysis. RESULTS: Overall, 6 week and 12 week MTCT probability was 12.8% [95% confidence interval (CI),10.5-15.0] and 16.3% (95% CI,13.4-19.2), respectively. At 12 weeks, among infants who were not infected at birth, 24 (7.9%) infections occurred in the NVP arm and 41 (13.1%) in the ZDV arm (log rank P = 0.06). Using multivariate analysis, factors associated with infection following birth were ZDV use [odds ratio (OR), 1.8; 95% CI,1.1-3.2; P = 0.032), maternal CD4 cell count < 500 x 10(6) cells/l (OR, 2.5; 95% CI,1.3-5.0; P = 0.007), maternal viral load > 50 000 copies/ml (OR, 3.6; 95% CI,2.0-6.2; P < 0.0001) and breastfeeding (OR, 2.2; 95% CI,1.3-3.8; P = 0.006). CONCLUSION: A single-dose of NVP given to infants offers protection against HIV-1 infection and should be a strategy used in infants of mothers with untreated HIV infection.     

Evaluation of three substitutes for Percoll in sperm isolation by density gradient centrifugation

Silane-coated silica particle solutions (ISolate(TM) and PureSperm)TM)) and iodixanol (OptiPrep(TM)) were compared to polyvinylpyrrolidone (PVP)-coated silica particles (Percoll(TM)) in their efficacy to recover spermatozoa by gradient centrifugation for use in assisted reproductive procedures. Efficacy was assessed in terms of percentages of sperm recovery, sperm vitality and motility, normal sperm morphology and normal sperm chromatin condensation. No significant difference was found in the recovery of spermatozoa for men with both normal sperm counts and oligozoospermia, between PVP-coated and silane-coated particle solutions. Iodixanol had significantly lower sperm recovery compared to the other products. Sperm vitality, progressive motility, normal morphology and normal chromatin condensation did not differ significantly between any of the sperm isolation products.      

FcR blocking activity in serum of actively enhanced rat renal allograft recipients due to IgG anti-class II MHC alloantibody

In some rat strain combinations, pre-operative donor-specific blood transfusion produces long-term renal allograft survival, although the underlying mechanisms are unclear. This study has examined whether Fc receptor (FcR)-blocking activity could be detected in the serum of unmodified PVG strain recipients bearing a rejecting renal allograft and in recipients bearing an actively enhanced graft following pre-operative blood transfusion. Serum harvested on Day 5 from actively enhanced PVG recipients of DA rat renal allografts was shown to specifically inhibit erythrocyte-antibody (EA) rosette formation with donor strain, but not third-party, splenocytes, while the levels of EA rosette inhibition (EAI) in Day 5 serum from rejecting rats remained markedly lower. This FcR-blocking activity was present in enhanced serum fractions, prepared by discontinuous density gradient centrifugation, which corresponded to the 7 S peak. Purified IgG prepared from enhanced serum was also found to inhibit EA rosette formation with donor splenocytes, and absorption of the IgG preparations with donor strain erythrocytes failed to abrogate EA rosette inhibition. Further experiments, in which absorbed IgG from enhanced animals was tested for FcR blocking activity against splenocytes of defined major histocompatability complex (MHC) subregion specificities, established that FcR-blocking activity was mediated by IgG alloantibodies directed against donor MHC class II antigens. Whether the presence of such antibodies early after transplantation contributes to the beneficial effect of blood transfusion on graft survival remains to be determined.     

Comparison of selective media for isolation of Campylobacter jejuni/coli

A comparison of Skirrow's, Butzler's, Blaser's, Campy-BAP and Preston media for Campylobacter spp was made using human, animal and environmental specimens. Butzler's medium gave the lowest isolation rate and Preston medium, which was the most selective, the highest isolation rate. Enrichment culture using Preston enrichment broth gave a higher isolation rate than direct plating onto Preston medium.     

The ventrolateral medulla of the cat mediates vestibulosympathetic reflexes

Extracellular recordings were made from 94 neurons located in the ventrolateral medulla (VLM) whose firing rate was affected by vestibular nerve (VN) stimulation; 50 of these units were in the subretrofacial (SRF) nucleus, which contains cells that make direct excitatory connections with sympathetic preganglionic neurons. The sample included 12 SRF cells which were antidromically driven from the upper thoracic spinal cord and had conduction velocities of 10 m/s or less; the effect of VN stimulation on all but one of these units was inhibition. The onset latency of the response to VN stimulation was long [20.3 +/- 3.7 (S.E.M.) ms, n = 9, for the antidromically activated neurons and 12.1 +/- 1.2 ms, n = 73, for the others], suggesting that the effects were predominantly polysynaptic. In addition, most of the spontaneously active units tested (33/36) received convergent inputs from the carotid sinus nerve (CSN), as would be expected for neurons which influence sympathetic outflow. Vestibular-elicited inhibition of SRF neurons with projections to the intermediolateral cell column could account for late, long duration inhibition of sympathetic discharges produced by labyrinth stimulation.     

Vitamin D3 analog, EB1089, inhibits growth of subcutaneous xenografts of the human colon cancer cell line, LoVo, in a nude mouse model

PURPOSE: In this study, we investigated the effect of the vitamin D ₃ analog, EB1089, on the growth of subcutaneous xenografts of the human colon cancer cell line, LoVo, in a nude mouse model. METHODS: BALB/c Nu/Nu nude mice were inoculated subcutaneously with 10 ⁶ LoVo cells. EB1089 dissolved in isopropanol was administered intraperitoneally and orally on alternate days at doses of 0.1, 0.5, and 2.5 g/kg/day. Control animals received isopropanol alone. Tumor volumes estimated using the formula 0.5×length×(width). The tumor kinetic index was determined by immunohistochemical detection of proliferating cell nuclear antigen. RESULTS: Significant dose-dependent inhibition of tumor growth was seen. After 20 days of treatment with 0.1 g/kg/day EB1089, mean tumor volume in treated mice was 41 to 49 percent less than that in control animals (P <0.01). Significant inhibition of tumor growth was also seen with 0.5 g/kg/day EB1089 after 22 days of treatment (51 percent of control P <0.01). Treatment with 2.5 g/kg/day resulted in weight loss that required termination of this group; these mice were subsequently found to be hypercalcemic. The tumor kinetic index was significantly lower in tumors treated with 0.1 g/kg/day EB1089 compared with that for control tumors (8 vs.30 percent in controls). CONCLUSION: These findings suggest that the vitamin D ₃ analog, EB1089, is a potent antiproliferative agent for some human colon cancers.The human colonic adenocarcinoma cancer cell line, LoVo, was a gift from CRC Laboratories, Nottingham, United Kingdom.Read at the Royal Australasian College of Surgeons Annual Scientific Congress, Melbourne, Australia, June 5 to 10, 1996.