Improving the Yields and Reaction Rate in the Ethanolysis of Soybean Oil by Using Mixtures of Lipase CLEAs.

Due to the heterogeneity of oils, the use of mixtures of lipases with different activity for a large number of glycerol-linked carboxylic acids that compose the substrate has been proposed as a better alternative than the use of one specific lipase preparation in the enzymatic synthesis of biodiesel. In this work, mixtures of lipases from different sources were evaluated in their soluble form in the ethanolysis of soybean oil. A mixture of lipases (50% of each lipase, in activity basis) from porcine pancreas (PPL) and Thermomyces lanuginosus lipase (TLL) gave the highest fatty acid ethyl ester (FAEE) yield (around 20 wt.%), while the individual lipases gave FAEE yields 100 and 5 times lower, respectively. These lipases were immobilized individually by the cross-linked enzyme aggregates (CLEAs) technique, yielding biocatalysts with 89 and 119% of expressed activity, respectively. A mixture of these CLEAs (also 50% of each lipase, in activity basis) gave 90.4 wt.% FAEE yield, while using separately CLEAs of PPL and TLL, the FAEE yields were 84.7 and 75.6 wt.%, respectively, under the same reaction conditions. The mixture of CLEAs could be reused (five cycles of 6 h) in the ethanolysis of soybean oil in a vortex flow-type reactor yielding an FAEE yield higher than 80% of that of the first batch.

Serum 25-hydroxyvitamin D, vitamin D binding protein and risk of colorectal cancer in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial.

The potential role of vitamin D in cancer prevention has generated substantial interest, and laboratory experiments indicate several anti-cancer properties for vitamin D compounds. Prospective studies of circulating 25-hydroxyvitamin D [25(OH)D], the accepted biomarker of vitamin D status, suggest an inverse association with colorectal cancer risk, but with some inconsistencies. Furthermore, the direct or indirect impact of the key transport protein, vitamin D binding protein (DBP), has not been examined. We conducted a prospective study of serum 25(OH)D and DBP concentrations and colorectal cancer risk in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial, based on 476 colorectal cancer cases and 476 controls, matched on age, sex, race and date of serum collection. All subjects underwent sigmoidoscopic screening at baseline and once during follow-up. Conditional logistic regression estimated odds ratios (ORs) and 95% confidence intervals (CIs). Circulating 25(OH)D was inversely associated with colorectal cancer (OR = 0.60, 95% CI 0.38-0.94 for highest versus lowest quintile, p trend 0.01). Adjusting for recognized colorectal cancer risk factors and accounting for seasonal vitamin D variation did not alter the findings. Neither circulating DBP nor the 25(OH)D:DBP molar ratio, a proxy for free circulating 25(OH)D, was associated with risk (OR = 0.82, 95% CI 0.54-1.26, and OR = 0.79, 95% CI 0.52-1.21, respectively), and DBP did not modify the 25(OH)D association. The current study eliminated confounding by colorectal cancer screening behavior, and supports an association between higher vitamin D status and substantially lower colorectal cancer risk, but does not indicate a direct or modifying role for DBP.

Circulating 25-hydroxyvitamin D, vitamin D-binding protein and risk of prostate cancer.

We recently reported a significant positive association between 25-hydroxyvitamin D [25(OH)D], the accepted biomarker of vitamin D status, and prostate cancer risk. To further elucidate this association, we examined the influence of vitamin D-binding protein (DBP), the primary transporter of vitamin D compounds in the circulation. Prediagnostic serum concentrations of DBP were assayed for 950 cases and 964 matched controls with existing 25(OH)D measurements within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study of Finnish men. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs), and statistical tests were two sided. Serum DBP modified the association between serum 25(OH)D and prostate cancer, with higher risk for elevated 25(OH)D levels observed primarily among men having DBP concentrations above the median (OR = 1.81, 95% CI: 1.18-2.79 for highest vs. lowest quintile, p-trend = 0.001) compared to those with DBP below the median (OR = 1.22, 95% CI: 0.81-1.84, p-trend 0.97; p-interaction = 0.04). Serum DBP was not associated with prostate cancer risk overall (OR = 0.96, 95% CI: 0.70-1.33 for highest vs. lowest quintile); however, high serum DBP was associated with significantly decreased risk of prostate cancer in men with lower (

IL-15 administered by continuous infusion to rhesus macaques induces massive expansion of CD8+ T effector memory population in peripheral blood.

IL-15 promotes activation and maintenance of natural killer (NK) and CD8(+) T effector memory (T(EM)) cells, making it a potential immunotherapeutic agent for the treatment of cancer and immunodeficiency states. Here we report the immunologic effects of 3 different IL-15 dosing strategies in Rhesus macaques. IL-15 at a dose of 20 µg/kg/d administered by continuous intravenous infusion for 10 days resulted in a massive (100-fold) expansion of CD8(+) T(EM) cells in the peripheral blood. In contrast, the administration of 20-40 µg/kg/d of IL-15 by subcutaneous injection resulted in a more modest (10-fold) expansion of CD8(+) T(EM) cells. NK expansion was similar in both the continuous intravenous and daily subcutaneous treatment groups. The observation that IL-15 administered by continuous intravenous infusion is able to induce markedly greater expansions of CD8(+) T(EM) cells than the same dose administered by other routes may have important implications for clinical development of this cytokine.

Noninvasive in vivo imaging of CD4 cells in simian-human immunodeficiency virus (SHIV)-infected nonhuman primates.

Since the earliest days of the HIV epidemic, the number of CD4(+) T cells per unit volume of blood has been recognized as a major prognostic factor for the development of AIDS in persons with HIV infection. It has also been generally accepted that approximately 2% of total body lymphocytes circulate in the blood. In the present study, we have used a nondepleting humanized anti-CD4 monoclonal antibody labeled with the gamma emitter indium-111 to visualize the CD4(+) T-cell pool in vivo in nonhuman primates with simian HIV infection. A strong correlation was noted between radiotracer uptake in spleen, tonsil, axillary lymph nodes, and peripheral blood CD4 T-cell counts (rho = 0.75, 0.93, and 0.85, respectively, P < .005). The relationship between radiotracer retention in lymphoid tissues and CD4(+) T-cell counts in the circulation was governed by an exponential law. These data provide an estimate for the total number of lymphocytes in the body as being between 1.9 and 2.9 x 10(12) and suggest that the partition between peripheral blood and lymphoid tissue is between 0.3% and 0.5%.

Supplementation with alpha-tocopherol or beta-carotene reduces serum concentrations of vascular endothelial growth factor-D, but Not -A or -C, in male smokers.

Evidence from the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study suggests that vitamin E and ß-carotene supplement use may influence the risk of several cancers. Vascular endothelial growth factors (VEGF) are proteins involved in angiogenesis, an important requirement for tumor growth and metastasis. Thus, vitamin E and ß-carotene may influence cancer risk through one or more VEGF. The ATBC Study was a randomized, double-blind, placebo-controlled, primary cancer prevention trial in which participants were assigned to 1 of 4 supplementation groups based on a 2 × 2 factorial design: 1) α-tocopherol (vitamin E); 2) ß-carotene; 3) both; or 4) placebo. For the present study, 100 cancer-free participants with follow-up serum available were randomly selected from each intervention group. VEGF-A, -C, and -D concentrations were measured by ELISA in serum obtained at baseline and after at least 2 y of supplementation. Differences in change in VEGF levels from baseline to follow-up between intervention groups were assessed using the ANOVA test. Change in VEGF-A and VEGF-C concentrations between baseline and follow-up did not differ by intervention group (P = 0.45 and 0.29, respectively). The decrease in the serum VEGF-D concentration was greater in the men supplemented with α-tocopherol (-9.7 ± 2.5%) or ß-carotene (-8.5 ± 2.7%) and tended to be greater in those supplemented with both (-6.8 ± 2.4%) compared to the placebo group, in which there was no change (-0.4 ± 3.0%) (P = 0.03). In this population of male smokers, supplementation with α-tocopherol or ß-carotene was associated with a decrease in VEGF-D levels over time. Although the mechanism through which these supplements affect cancer etiolog remains unclear, our results support the hypothesis that vitamin E and ß-carotene may influence cancer progression through VEGF-mediated lymphangiogenesis.

A pilot study of consolidative immunotherapy in patients with high-risk pediatric sarcomas.

PURPOSE: Patients with metastatic or recurrent Ewing's sarcoma family of tumors and alveolar rhabdomyosarcoma have <25% 5-year survival in most studies. This study administered a novel immunotherapy regimen aimed at consolidating remission in these patients. EXPERIMENTAL DESIGN: Fifty-two patients with translocation positive, recurrent, or metastatic Ewing's sarcoma family of tumors or alveolar rhabdomyosarcoma underwent prechemotherapy cell harvest via apheresis for potential receipt of immunotherapy. Following completion of standard multimodal therapy, 30 patients ultimately initiated immunotherapy and were sequentially assigned to three cohorts. All cohorts received autologous T cells, influenza vaccinations, and dendritic cells pulsed with peptides derived from tumor-specific translocation breakpoints and E7, a peptide known to bind HLA-A2. Cohort 1 received moderate-dose recombinant human interleukin-2 (rhIL-2), cohort 2 received low-dose rhIL-2, and cohort 3 did not receive rhIL-2. RESULTS: All immunotherapy recipients generated influenza-specific immune responses, whereas immune responses to the translocation breakpoint peptides occurred in 39%, and only 25% of HLA-A2(+) patients developed E7-specific responses. Toxicity was minimal. Intention-to-treat analysis revealed a 31% 5-year overall survival for all patients apheresed (median potential follow-up 7.3 years) with a 43% 5-year overall survival for patients initiating immunotherapy. CONCLUSIONS: Consolidative immunotherapy is a scientifically based and clinically practical approach for integrating immunotherapy into a multimodal regimen for chemoresponsive cancer. Patients receiving immunotherapy experienced minimal toxicity and favorable survival. The robust influenza immune responses observed suggest that postchemotherapy immune incompetence will not fundamentally limit this approach. Future studies will seek to increase efficacy by using more immunogenic antigens and more potent dendritic cells.

1,3-Regiospecific ethanolysis of soybean oil catalyzed by crosslinked porcine pancreas lipase aggregates.

The preparation of crosslinked aggregates of pancreatic porcine lipase (PPL-CLEA) was systematically studied, evaluating the influence of three precipitants and two crosslinking agents, as well as the use of soy protein as an alternative feeder protein on the catalytic properties and stability of the immobilized PPL. Standard CLEAs showed a global yield (CLEA' observed activity/offered total activity) of less than 4%, whereas with the addition of soy protein (PPL:soy protein mass ratio of 1:3) the global yield was approximately fivefold higher. The CLEA of PPL prepared with soy protein as feeder (PPL:soy protein mass ratio of 1:3) and glutaraldehyde as crosslinking reagent (10 µmol of aldehyde groups/mg of total protein) was more active mainly because of the reduced enzyme leaching in the washing step. This CLEA, named PPL-SOY-CLEA, had an immobilization yield around 60% and an expressed activity around 40%. In the ethanolysis of soybean oil, the PPL-SOY-CLEA yielded maximum fatty acid ethyl ester (FAEE) concentration around 12-fold higher than that achieved using soluble PPL (34 h reaction at 30°C, 300 rpm stirring, soybean oil/ethanol molar ratio of 1:5) with an enzyme load around 2-fold lower (very likely due to free enzyme inactivation). The operational stability of the PPL-SOY-CLEA in the ethanolysis of soybean oil in a vortex flow type reactor showed that FAEE yield was higher than 50% during ten reaction cycles of 24 h. This reactor configuration may be an attractive alternative to the conventional stirred reactors for biotransformations in industrial plants using carrier-free biocatalysts. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:910-920, 2018.

Lymphocyte subsets in hemophilic patients with hepatitis C virus infection with or without human immunodeficiency virus co-infection: a nested cross-sectional study.

BACKGROUND: With chronic infection, hepatitis C virus (HCV) RNA can be detected in B cells and associated with B-cell disorders, but these are not well defined. METHODS: The relationship between HCV infection and lymphocyte subpopulations was evaluated rigorously in 120 asymptomatic hemophilic patients, randomly selected from a prospective cohort study. CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD56+ NK cells were quantified by flow cytometry using cryopreserved peripheral blood mononuclear cells of 24 hemophilic patients in each of five age-matched groups [uninfected; chronic HCV with or without human immunodeficiency virus (HIV); and cleared HCV with or without HIV]. RESULTS: As expected, patients with HIV had significantly reduced CD4+ and increased CD8+ T cells. Irrespective of HIV, patients with chronic HCV infection had approximately 25% fewer CD19+ B cells than those without chronic HCV infection. CONCLUSIONS: These data support the hypothesis that asymptomatic patients with chronic HCV infection have an altered B-lymphocyte population.

Vitamin D-binding protein and pancreatic cancer: a nested case-control study.

BACKGROUND: Vitamin D-binding protein (DBP) is the primary carrier of 25-hydroxyvitamin D [25(OH)D] in the circulation. One prospective study in male smokers found a protective association between DBP and pancreatic cancer, particularly among men with higher 25(OH)D concentrations. OBJECTIVE: The objective was to examine the association between DBP and pancreatic cancer risk in an American population. DESIGN: We conducted a nested case-control study in the Prostate, Lung, Colorectal, and Ovarian Cancer screening trial cohort of men and women aged 55-74 y at baseline. Between 1993 and 2010, 295 incident pancreatic adenocarcinoma cases were reported (follow-up to 15.1 y). Two controls (n = 590) were matched to each case by age, race, sex, and month of blood draw. We calculated smoking- and diabetes-adjusted ORs and 95% CIs with the use of conditional logistic regression. RESULTS: DBP concentration was not significantly associated with pancreatic cancer overall [highest (≥7149.4 nmol/L) vs. lowest (<3670.4 nmol/L) quintile; OR: 1.75; 95% CI: 0.91, 3.37; P-trend = 0.25]. For serum 25(OH)D compared with the referent (50 to <75 nmol/L), individuals in the highest group had a significantly higher risk (≥100 nmol/L; OR: 3.23; 95% CI: 1.24, 8.44), whereas those in the lowest group had no significant association (<25 nmol/L; OR: 2.50; 95% CI: 0.92, 6.81). Further adjustment for DBP did not alter this association. CONCLUSION: Our results do not support the hypothesis that serum DBP or 25(OH)D plays a protective role in pancreatic cancer. This trial was registered at clinicaltrials.gov as NCT00339495.

Whole blood cryopreservation in epidemiological studies.

Standardized and cost-effective biological sample collection, processing, and storage procedures are needed in large-scale epidemiological studies to provide material for testing a broad range of etiological hypotheses. One component of sample collection in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial involves shipment of blood in acid-citrate-dextrose anticoagulant to a central processing laboratory, where 10% DMSO is added, and whole blood aliquots are cryopreserved. A single technician is able to routinely process 50-60 samples/day. Tests conducted to evaluate potential uses of cryopreserved whole blood showed successful EBV transformation (>90%, up to 20 months of storage). In addition, lymphocytes maintained good viability and stable T-cell:B-cell ratios, and T cells maintained the capacity to proliferate in response to solid phase anti-CD3/CD28 plus interleukin 2. Whole blood cryopreservation is a cost-effective approach to large-scale storage of viable cells in epidemiological studies.

Simultaneous measurement of several cytokines using small volumes of biospecimens.

The role of host immunity in the development of virus-induced cancers has been difficult to elucidate, in part because of our inability to effectively measure multiple immune parameters using available amounts of biological material. The objective of the present study was to validate the use of a newly developed multiplex assay, the LINCOplex assay, for the simultaneous measurement of multiple cytokines [interleukin/(IL)-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IFN-gamma, and tumor necrosis factor-alpha]. Supernatants obtained from peripheral blood mononuclear cell cultures stimulated with various different mitogens and antigens (including phytohemagglutinin, influenza, tetanus, HPV16 E6 and E7 peptides, and media alone) were selected for study. In total, 750 specimens obtained from 26 participants were tested in replicate using the 8-plex LINCOplex assay (25 micro l of specimen required per well). Every specimen was included in duplicate in a blinded fashion. For some specimens, multiple 2-fold dilutions of the same specimen were included to evaluate the linearity of results. The availability of independently obtained IL-2 and IFN-gamma results from standard single cytokine (simplex) assays allowed for a direct comparison between the LINCOplex results and those obtained from the simplex assays. Spearman correlation coefficients for continuous results, and exact agreement rates and weighted kappa statistics for quartiled variables, were computed to evaluate intra- and interassay agreement. IL-4 levels were consistently below the detectable level of the assay (3 pg/ml) whereas IL-6 and IL-8 levels were consistently above the highest detectable level of the assay (10,000 pg/ml), and these three cytokines were, therefore, not evaluated further. For the remaining five cytokines, excellent intra-assay reproducibility was observed, with Spearman correlation coefficients consistently above 0.90 for all five cytokines. Exact agreement rates ranging from 77.6-90.3% and weighted kappas ranging from 0.81-0.92 were observed. Similar results were observed when analysis was restricted to supernatants obtained from cultures that had been stimulated with HPV16 peptides and when analysis was restricted to supernatants obtained from cultures containing no antigen or mitogen, suggesting that the LINCOplex assay is applicable under conditions where moderate or weak cytokine responses/levels are expected. Linearity of results was observed when dilutions of a single specimen were blindly tested, with the exception of IL-2 and IL-10, where deviations from linearity were sometimes observed. For IL-2 and IFN-gamma, where results from simplex assays were available for comparison, the LINCOplex assay and the simplex assay results agreed well. Spearman correlation coefficients were 0.86 and 0.93 for IL-2 and IFN-gamma, respectively. Exact agreement and weighted kappa values were 68.5% and 0.72 (95% confidence interval, 0.65-0.79), respectively, for IL-2 and 67.3% and 0.73 (95% confidence interval, 0.67-0.80), respectively, for IFN-gamma. These results indicate the applicability of the LINCOplex assay for the simultaneous measurement of multiple cytokines using small amounts of biological material.

Markers of microbial translocation and risk of AIDS-related lymphoma.

BACKGROUND: Depletion of gut-associated lymphocytes by HIV infection facilitates microbial translocation, which may contribute to non-Hodgkin lymphoma (NHL) risk via chronic immune activation and B-cell hyperstimulation. METHOD: We therefore examined associations of four microbial translocation markers with subsequent NHL risk in a case-control study nested within four prospective cohort studies of HIV-infected individuals. Prediagnostic blood specimens for 56 NHL cases and 190 controls matched for age, sex, race, specimen type, cohort, and CD4 T-cell count were tested for the endotoxin lipopolysaccharide (LPS), antiendotoxin core antibody (EndoCab), LPS-binding protein (LBP), and soluble CD14 (sCD14). RESULTS: Elevated levels of sCD14 were associated with significantly increased NHL risk [odds ratio (OR) 2.72 (95% confidence interval [95% CI] 1.29-5.76)]. In subgroup analyses, elevated LPS levels were also associated with significantly increased NHL risk [OR 3.24 (95% CI 1.10-9.53)]. EndoCab and LBP levels were not associated with NHL risk. CONCLUSION: The association of sCD14 and LPS with NHL risk supports an etiologic role for gut microbial translocation in lymphomagenesis among HIV-infected individuals. Additional studies with larger sample sizes are needed to confirm these observations.

Characterization of effect of repeated freeze and thaw cycles on stability of genomic DNA using pulsed field gel electrophoresis.

In this study, we systematically investigated the effects of repeated cycles of freeze/thaw on stability of genomic Deoxyribonucleic acid (DNA) samples as evaluated by changes in DNA size, concentration, and freeze/thaw protocols. The DNA was isolated using standard extraction procedures including phenol/chloroform and commercially available Gentra Puregene and Qiagen QIAmp kits. Changes in DNA were monitored over 18 cycles of freezing and thawing utilizing several freeze/thaw protocols. DNA samples from multiple subjects prepared from whole blood samples were examined by pulsed field gel electrophoresis (PFGE), and shown to have different average molecular sizes and size distribution patterns depending on the extraction method. Results of freeze/thaw experiments, analyzed by PFGE, showed progressive DNA degradation of the samples, with DNA sizes larger than 100 kb most sensitive to freeze/thaw degradation. Increasing the DNA concentration of stored samples from 10 µg/mL to 100 µg/mL had a somewhat protective effect on DNA stability. Variations in freeze/thaw protocols did not have a significant impact on DNA stability during repeated freeze/thaw cycles. At freeze/thaw cycle 18, average molecular size and size distribution of all DNA samples tested approached 25 kb, regardless of their initial average size and size distributions. This study provides insight on DNA degradation during freeze/thaw cycles and offers guidance to storage and handling of DNA samples.

Impact of circulating vitamin D binding protein levels on the association between 25-hydroxyvitamin D and pancreatic cancer risk: a nested case-control study.

High concentrations of circulating 25-hydroxyvitamin D [25(OH)D] have been associated with elevated pancreatic cancer risk. As this is contrary to an expected inverse association between vitamin D status and cancer, we examined whether vitamin D binding protein (DBP), the primary carrier of vitamin D compounds in circulation, plays a role in this relationship. Prediagnostic serum DBP and 25(OH)D were studied in relation to risk of pancreatic cancer in a nested case-control study of 234 cases and 234 controls in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study of Finnish men. ORs and 95% CIs were estimated using logistic regression, and statistical tests were two-sided. We found that DBP and 25(OH)D were correlated (r = 0.27, P < 0.0001), and DBP was inversely associated with pancreatic cancer risk (OR = 0.66, 95% CI = 0.39-1.12, for the highest vs. lowest quartile; P(trend) = 0.02). Importantly, this association seemed to have a threshold between quartiles 2 to 4 and quartile 1, and was primarily evident among men with concurrent high 25(OH)D concentrations (OR = 0.33, 95% CI = 0.16-0.70 for highest vs. lowest quartile; P(trend) = 0.002), with no association in men with lower serum 25(OH)D (OR = 1.28, 95% CI = 0.62-2.61 for highest vs. lowest quartile, P(trend) 0.63, P(interaction) = 0.01). Men with higher 25(OH)D concentrations and serum DBP below the median showed greatly elevated risk of pancreatic cancer (OR = 5.01, 95% CI 2.33-10.78, for highest vs. lowest quartile; P(trend) < 0.0001), while risk was weakly inversely associated with serum 25(OH)D when DBP concentrations were higher (P(interaction) = 0.001). Taken together, our findings indicate that higher DBP concentrations may sequester more 25(OH)D and reduce free 25(OH)D bioavailability. Simultaneous examination of DBP and 25(OH)D may be important in determining the association of vitamin D with cancer risk.

Extraction of DNA from serum for high-throughput genotyping: findings from pilot studies within the Prostate Cancer Prevention Trial.

OBJECTIVES: Deoxyribonucleic acid (DNA) extraction from blood and genotyping for candidate single nucleotide polymorphisms (SNP) is now an important part of almost all molecular epidemiologic studies. However, in many studies the amount of blood sample is limited or only serum is available. We conducted several pilot studies to identify methods for DNA extraction and high-throughput SNP genotyping of both white blood cell (WBC) and serum DNA that can be done centrally and reliably for large numbers of samples. METHODS: We used biospecimens from the Prostate Cancer Prevention Trial (PCPT), a phase III, double-blind, placebo-controlled trial that tested the efficacy of finasteride for the primary prevention of prostate cancer. DNA was extracted from WBCs, from serum, and also from serum after organic solvent extraction for analysis of hormones. We also conducted blinded high-throughput genotyping in three laboratories to assess feasibility and reliability of results with differing methodologies using DNA from WBCs and from serum. RESULTS: Genotyping of DNA extracted from WBCs resulted in highly reliable, reproducible results across laboratories using different genotyping platforms. However, genotyping with DNA extracted from serum did not provide reliable data using high-throughput multiplex approaches such as Sequenom (hME and iPLEX) and Applied Biosystems SNPlex, but was successful using Taqman. CONCLUSIONS: Based on the results of these pilot studies, we conclude that DNA obtained from serum must be used judiciously, and that genotyping using multiplex methods is not suitable for serum DNA.

HPV-16 L1 VLP vaccine elicits a broad-spectrum of cytokine responses in whole blood.

Here, we evaluated innate and adaptive immune system cytokine responses induced by HPV-16 L1 VLP in whole blood (WB) cultures from individuals receiving the vaccine (n=20) or placebo (n=4) before and after vaccination. 11 cytokines were measured: IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, and GM-CSF using multiplex bead arrays. Cytokine profiles from WB samples clearly discriminated between vaccine and placebo recipients and between pre and post-vaccination responses. Significant increases in Th1, Th2 and inflammatory cytokines were observed in WB assays following vaccination. Results from WB assays were compared against parallel PBMC-based assays in a subset of patients. Differences between whole blood assay and PBMC were observed, with the highest levels of induction found for WB for several cytokines. Our results indicate that multiplex assays for cytokine profiling in WB are an efficient tool for assessing broad spectrum, innate and adaptive immune responses to vaccines and identifying immunologic correlates of protection in efficacy studies.

Cellular immune responses to human papillomavirus (HPV)-16 L1 in healthy volunteers immunized with recombinant HPV-16 L1 virus-like particles.

The causal association between papillomavirus (HPV) infection and cervical cancer has been demonstrated; the development of a prophylactic vaccine to protect against HPV infection may therefore reduce the incidence of this cancer worldwide. Noninfectious HPV-like particles (VLPs), composed of the L1 major capsid protein, are current candidate vaccines for prevention of HPV infection and cervical neoplasia. Although neutralizing antibodies have a pivotal role in the prevention of initial infection, cellular immune responses to HPV antigens may have an important role in viral clearance. A phase II trial was conducted to further evaluate the immunogenicity of a recombinant HPV-16 L1 VLP vaccine administered intramuscularly, without adjuvant, at 0, 1, and 6 months. Cell-mediated immune responses (lymphoproliferation and cytokine production) to HPV-16 L1 VLPs were evaluated in peripheral blood mononuclear cells (PBMCs) from 43 individuals receiving the L1 VLP vaccine and from 10 individuals receiving placebo. Vaccination resulted, at months 2 and 7 (i.e., 1 month after the second immunization and 1 month after third immunization, respectively), in increases in T cell-proliferative response to HPV-16 L1 VLPs (P<.001). In addition, significant increases in cytokine (interferon-gamma, interleukin [IL]-5 and IL-10) responses to L1 VLPs were observed after vaccination (P<.001). The strongest cytokine responses at month 7 were observed in individuals with high antibody titers at month 2, suggesting that neutralizing antibodies generated by initial vaccination may augment T cell responses to subsequent booster vaccinations. No significant increases in lymphoproliferative or cytokine responses to L1 VLPs were observed in individuals receiving placebo. In summary, the HPV-16 L1 vaccine induces not only robust B cell responses but also L1-specific T cell responses detectable by proliferation of both CD4+ and CD8+ T cells and in vitro production of both Th1- and Th2-type cytokines. Future efficacy studies are needed to evaluate whether and/or how VLP vaccines confer protection against genital HPV infection and associated disease.