Assessment of the immune response in kidney transplant patients.

Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2008.Background: Management of a transplant recipient involves the use of multiple immunosuppressant drugs. Currently there is no test that reflects the overall immune status of the patient. This results in under or over suppression of the immune system and consequently increases in morbidity and mortality rates. Evaluation of the proliferative response of PBMC's to a mitogen PHA by measurement of intracellular ATP was evaluated as a tool to assess the immune response in kidney transplant patients. Method: PBMC's were separated from the blood samples of healthy controls and kidney transplant patients on cyclosporine, sirolimus, and tacrolimus based regimens by density gradient centrifugation, cells were counted and incubated overnight with and without PHA. The luciferin-Iuciferase enzyme reaction which induces bioluminescence and the Turner Biosystem luminometer were used to measure intracellular ATP levels in relative light units (RLU). An A TP standard curve was generated for each test. Results: The ATP (nglml) levels measured in the transplant recipients were lower and statistically significantly different (p< 0.0001) than the healthy controls. No statistically significant difference was measured between the cycIosporine and sirolimus drug groups. Patients on tacrolimus gave a statistically significant (p<O.0001) stronger immune response than those receiving cyclosporine and sirolimus. Overall, the immune response results of kidney transplant patients were statistically significantly lower than the healthy control by 981 nglml. Linear regression analysis revealed no correlation between patient A TP (nglml) levels and therapeutic drug blood levels, immunosuppressant drug dosages, creatinine levels and white cell counts. The immune responses of patients who were diagnosed with infection or were clinically stable were characterised as low or moderate, of interest, one patient who was diagnosed with rejection was found to have a strong immune response (>501 nglml ATP). Conclusion: Future studies to determine the predictive value of the A TP assay in directing immunosuppressive therapy are required. The assay described in this study is simple, sensitive and rapid and has possible application in immunological monitoring in a variety of conditions that affects the immune system. Keywords: kidney transplantation, immunosuppression, bioluminescence, lymphocyte, Adenosine Triphosphate (A TP), Phytohemmagglutinin (PHA

Kidney transplant recipients possess less depressing plasma than healthy controls in KwaZulu-Natal (South Africa) – a paradoxical observation

Background: The reduced activation of lymphocytes of patients on immunosuppressive drugs is well documented. Human plasma has been reported previously to play a role in lymphocyte proliferation. Several factors, including alpha globulin and lipoproteins, have been proposed as modulators of lymphocyte proliferation. Aim: To measure the ATP response of peripheral blood mononuclear cells (PBMCs) from kidney transplant patients and healthy controls following phytohaemagglutinin (PHA) stimulation and to compare the effect of plasma of transplant recipients and of healthy controls on lymphocyte activation. Methods: Peripheral blood mononuclear cells from the blood of healthy controls and kidney transplant patients on regimens based respectively on cyclosporine, sirolimus and tacrolimus, were separated by density gradient centrifugation. Cells were counted and incubated overnight with and without PHA. The luciferin–luciferase enzyme reaction, which induces bioluminescence, and the Turner Biosystem luminometer were used to measure intracellular ATP levels in relative light units, which were converted to ng/mL using an ATP standard curve. A chi-squared test using the Instat 3 program (Graphpad®) was used to compare results. Results: PHA stimulation of PBMC from healthy individuals produced a 47% increase in ATP production. This increase was reduced to 31% when transplant patient plasma was added (P &lt; 0.05). However, when plasma from healthy controls was added instead, paradoxically, the ATP production decreased further to 14%. A similar difference between patient and control plasma was recorded using PBMCs from transplant patients. The reduction in ATP production was the greatest in PBMCs from transplant patients on the tacrolimus-based regimen (P = 0.0388). Receiver operator characteristic (ROC) for ATP level revealed an area under the curve of 0.986. The cut-off value of ATP level between kidney transplant and control using the Youden index was 595 ng/mL, with a sensitivity of 93.3% and specificity of 99.9% Conclusion: Plasma isolated from patients on immunosuppressive drugs suppressed the response of lymphocytes to PHA stimulation. Paradoxically, plasma from healthy controls suppressed T cell activation even more severely. If confirmed in a more extensive study, this observation may be used to influence the choice of replacement fluid in the practice of plasma exchange in transplantation. Keywords: plasma, peripheral blood mononuclear cells, T cell &nbsp

Genetic determinants of Nef-mediated CD4 and HLA class I down-regulation differences between HIV-1 subtypes B and C

BACKGROUND: HIV-1 subtype C Nef sequences have a significantly lower ability overall to down-regulate CD4 and HLA-I than subtype B Nef sequences. Here we investigated whether Nef amino acids differing in frequency between HIV-1 subtypes B and C explain lower CD4 and HLA-I down-regulation ability of subtype C. FINDINGS: Subtype-specific mutations were introduced into representative subtype B and C Nef sequences and the CD4 and HLA-I down-regulation ability of these mutants was measured by flow cytometry in a CD4+ T cell line. Subtype C consensus 20I and subtype B consensus 20M reduced and increased HLA-I down-regulation respectively, and the S88G immune escape mutation (which is significantly more frequent in subtype C than subtype B) reduced CD4 and HLA-I down-regulation. CONCLUSIONS: Our data suggest that these subtype-specific differences may partly contribute to inter-subtype functional differences, and identification of an immune escape mutation – S88G – that impairs Nef function is of relevance to vaccine design

The Fitness Landscape of HIV-1 Gag: Advanced Modeling Approaches and Validation of Model Predictions by In Vitro Testing

Viral immune evasion by sequence variation is a major hindrance to HIV-1 vaccine design. To address this challenge, our group has developed a computational model, rooted in physics, that aims to predict the fitness landscape of HIV-1 proteins in order to design vaccine immunogens that lead to impaired viral fitness, thus blocking viable escape routes. Here, we advance the computational models to address previous limitations, and directly test model predictions against in vitro fitness measurements of HIV-1 strains containing multiple Gag mutations. We incorporated regularization into the model fitting procedure to address finite sampling. Further, we developed a model that accounts for the specific identity of mutant amino acids (Potts model), generalizing our previous approach (Ising model) that is unable to distinguish between different mutant amino acids. Gag mutation combinations (17 pairs, 1 triple and 25 single mutations within these) predicted to be either harmful to HIV-1 viability or fitness-neutral were introduced into HIV-1 NL4-3 by site-directed mutagenesis and replication capacities of these mutants were assayed in vitro. The predicted and measured fitness of the corresponding mutants for the original Ising model (r = −0.74, p = 3.6×10−6) are strongly correlated, and this was further strengthened in the regularized Ising model (r = −0.83, p = 3.7×10−12). Performance of the Potts model (r = −0.73, p = 9.7×10−9) was similar to that of the Ising model, indicating that the binary approximation is sufficient for capturing fitness effects of common mutants at sites of low amino acid diversity. However, we show that the Potts model is expected to improve predictive power for more variable proteins. Overall, our results support the ability of the computational models to robustly predict the relative fitness of mutant viral strains, and indicate the potential value of this approach for understanding viral immune evasion, and harnessing this knowledge for immunogen design

No evidence for selection of HIV-1 with enhanced Gag-Protease or Nef function among breakthrough infections in the CAPRISA 004 tenofovir microbicide trial

BACKGROUND: Use of antiretroviral-based microbicides for HIV-1 prophylaxis could introduce a transmission barrier that inadvertently facilitates the selection of fitter viral variants among incident infections. To investigate this, we assessed the in vitro function of gag-protease and nef sequences from participants who acquired HIV-1 during the CAPRISA 004 1% tenofovir microbicide gel trial. Methods and RESULTS: We isolated the earliest available gag-protease and nef gene sequences from 83 individuals and examined their in vitro function using recombinant viral replication capacity assays and surface protein downregulation assays, respectively. No major phylogenetic clustering and no significant differences in gag-protease or nef function were observed in participants who received tenofovir gel versus placebo gel prophylaxis. CONCLUSION: Results indicate that the partial protective effects of 1% tenofovir gel use in the CAPRISA 004 trial were not offset by selection of transmitted/early HIV-1 variants with enhanced Gag-Protease or Nef fitness

No evidence for selection of HIV-1 with enhanced gag-protease or nef function among breakthrough infections in the CAPRISA 004 tenofovir microbicide trial.

CAPRISA, 2013.Background: Use of antiretroviral-based microbicides for HIV-1 prophylaxis could introduce a transmission barrier that inadvertently facilitates the selection of fitter viral variants among incident infections. To investigate this, we assessed the in vitro function of gag-protease and nef sequences from participants who acquired HIV-1 during the CAPRISA 004 1% tenofovir microbicide gel trial. Methods and Results: We isolated the earliest available gag-protease and nef gene sequences from 83 individuals and examined their in vitro function using recombinant viral replication capacity assays and surface protein down regulation assays, respectively. No major phylogenetic clustering and no significant differences in gag-protease or nef function were observed in participants who received tenofovir gel versus placebo gel prophylaxis. Conclusion: Results indicate that the partial protective effects of 1% tenofovir gel use in the CAPRISA 004 trial were not offset by selection of transmitted/early HIV-1 variants with enhanced Gag-Protease or Nef fitness

Nef-mediated down-regulation of CD4 and HLA class I in HIV-1 subtype C infection: association with disease progression and influence of immune pressure.

CAPRISA, 2014.Abstract available in pdf

Translating HIV Sequences into Quantitative Fitness Landscapes Predicts Viral Vulnerabilities for Rational Immunogen Design

A prophylactic or therapeutic vaccine offers the best hope to curb the HIV-AIDS epidemic gripping sub-Saharan Africa, but it remains elusive. A major challenge is the extreme viral sequence variability among strains. Systematic means to guide immunogen design for highly variable pathogens like HIV are not available. Using computational models, we have developed an approach to translate available viral sequence data into quantitative landscapes of viral fitness as a function of the amino acid sequences of its constituent proteins. Predictions emerging from our computationally defined landscapes for the proteins of HIV-1 clade B Gag were positively tested against new in vitro fitness measurements and were consistent with previously defined in vitro measurements and clinical observations. These landscapes chart the peaks and valleys of viral fitness as protein sequences change and inform the design of immunogens and therapies that can target regions of the virus most vulnerable to selection pressure.Ragon Institute of MGH, MIT and HarvardNational Institutes of Health (U.S.) (Director's Pioneer Award)Ragon Institute of MGH, MIT and Harvard (Postdoctoral Fellowship