A standard three-dose vaccination for HBV was delivered by intramuscular injection at day 0, 1 month, and 6 months. Marijuana smokers were admitted overnight to the UCLA General Clinical Research Center for vaccination and given 3 standardized MJ cigarettes to be smoked during the first day and 1 MJ cigarette on the following morning . HBV vaccination occurred just prior to the third MJ cigarette. Blood samples were collected prior to and approximately 15 minutes following completion of the first MJ cigarette to determine the concentration of THC and its metabolites. Marijuana smokers recorded their daily MJ use in a diary. Non-smokers were vaccinated as outpatients without MJ exposure. Blood samples for immune monitoring werecollected from all subjects at baseline and 3–4 weeks after the second and third vaccine dose. HBsAg-specific proliferation assays were performed using the subject’s fresh blood as noted for the in vitro effects of THC above, except that no exogenous THC was added. Culture medium contained RPMI 1640 supplemented with glutamine, 10% heat-inactivated human AB serum , 10 mM Hepes buffer, and antibioticantimycotic mixture . For intracellular cytokine analysis, fresh purified CD3+ T cells were stimulated in vitro for 7 days with autologous HBsAg-pulsed DC . T cells were recovered and re-stimulated for 5 hours with either HBsAg-pulsed DC or non-antigen-pulsed DC in combination with anti-CD28 mAb . Brefeldin A was added during the last 4 hours. T cells stimulated with Phorbol 12-myristate 13- acetate and Calcium Ionophore A23187 served as positive controls. After stimulation, cells were recovered, fixed, cryo preserved,greenhouse racking and on the day of FACS analysis were treated with BD Bioscience lysing and permeabilizing reagents followed by staining with mAb .
A minimum of 100,000 gated T cells were acquired to determine the frequency of responder subsets. Student’s t-tests evaluated differences between the responses stimulated by control DC and THC-DC. A Fisher’s exact test was used to analyze differences between the frequency of non-responders, responders and good responders with respect to HBsAb titers. A Wilcoxon rank-sum test was employed to compare the distributions of HBV-specific cellular immune assay results for the antibody non-responder vs. responder or good responder groups and for the NS vs. MS subjects. p<0.05 was considered significant. Sample size estimates for the clinical protocol were informed by human epidemiologic data regarding vaccine responses in the general population , mouse model data regarding the effects of systemic THC dosing on vaccination and the results of human in vitro studies assessing T cell activation by THC-DC and control DC . When the NS response to HBV vaccination was assumed to be 80–90% and the responder frequency in MS to be as low as 45%, then ~18 subjects per group were calculated to achieve a power of 0.8 . For a responder rate of ≤30% in the MS group, only 8 subjects per group would be required. Similarly, 10 subjects per cohort were calculated to yield a power of >0.90 if habitual marijuana use resulted in a 50% reduction in T cell proliferation or cytokine production compared to the response in NS. A recruitment goal of 10–20 subjects per group was established prior to the study.Control DC, when loaded with HBsAg, stimulate the proliferation of autologous T cells obtained from healthy HBV-immunized NS . No proliferation was observed in the absence of HBsAg, confirming antigen-specificity . The down-regulation of CFSE expression in proliferating cells was associated with the upregulation of CD25 and the conversion from CD45RA to CD45RO, consistent with the generation of effector/effectormemory cells.
However, the stimulation of HBsAg-specific responder cells was dramatically curtailed when THC-DC were substituted for control DC. Similarly, culture supernatants demonstrated diminished Th1 cytokine production when T cells were stimulated with antigen-loaded THC-DC as compared to control DC . These findings confirmed a profound impact of THC exposure on the generation of HBsAg-specific T cell responses . Healthy MS and NS underwent a standard regimen for HBV vaccination . Forty seven subjects were screened, 21 enrolled, and 18 completed the study . The most common issues preventing enrollment were substance abuse not identified during the preliminary verbal screening, evidence of prior vaccination to HBV by serum HBsAb titer, and difficulty in committing to all of the study visits over the 8–9 month clinical protocol. The 3 subjects that were enrolled but failed to complete the protocol did so for personal reasons unrelated to the study. Table 1 summarizes demographics of the enrolled subjects, MJ and tobacco use, and measured serum levels of THC and THC metabolites. Subjects were predominantly male and matched across groups for age . The average duration of MJ use was 21.7 years and average lifetime exposure was 113.2 joint-years , consistent with moderate to heavy MJ use. Consistent with expectations , all MS had measurable THC metabolites in the blood at baseline. As measured 15–20 minutes following completion of the first marijuana cigarette, the peak serum THC level averaged 42.3 ng/ml and THC metabolites increased from 322.1 to 370.9 ng/ml. Based on pharmacokinetic studies by Schwope DM et al., , this suggests peak serum THC exposures that averaged ~100 ng/ml following each MJ smoking exposure. HBsAb titers represent an established measure of protective immunity with responders defined by the development of a titer >10 IU/L and the expected frequency of responders dependent upon age, gender, and HLA genotype .
The HBsAb titer can also be used to identify a subgroup of “good responders” defined by a titer of ≥100 IU/L at 4–8 weeks post vaccination. High responders exhibit a higher frequency of responding B cells, superior protection from chronic infection, and evidence of broader T cell based anti-viral responses . While there was a numerical difference in the absolute number of non-responders this was not significant given the number of subjects evaluated and both groups were within the expected frequency of vaccine responders given the age range of our study groups . The number of good responders was similar between groups , and the distribution of HBsAb titers between MS and NS at completion of vaccination was not significantly different . Cellular immunity also plays a role in the host response to HBV vaccination and our in vitro studies suggested that T cell responses are particularly susceptible to the immunosuppressive effects of THC. T cells collected at baseline and 3–4 weeks after each vaccination were analyzed for proliferation, effector/effector-memory phenotype, and production of both Th1 cytokines and a Th2 cytokine when stimulated in vitro with HBsA gloaded DC. Similar to the observed HBsAb titers, a range of T cell responses was generated with some subjects showing HBsAg-specific T cell proliferation and cytokine production and others having no measurable response . Good responders demonstrated HBsAg specific proliferation and phenotypic changes after the 2nd vaccination, which increased further after the 3rd vaccination . Proliferating T cells upregulated CD25 and CD45RO and these responses only occurred in the presence of HBsAg, confirming specificity . Furthermore, good responders demonstrated IFN-γ/TNF-α production by CD69+ T cells . In contrast to the relatively uniform stimulation of IFN-γ/TNF-α-producing CD69+ T cells that was observed in the good responders, only 2 of the NS and 2 of the MS exhibited measurable frequencies of IL-10+ T cells . All of these IL-10 responses occurred in good responders and the Th1/Th2 ratio averaged 27:1 in the NS group and 29:1 in the MS group. Non-responding subjects failed to demonstrate HBsAg-specific proliferation at any time point and produced limited cytokine responses . T cells from all subjects, regardless of smoking group or responder status, produced cytokines at similar levels following stimulation with a mitogen, suggesting that the lack cytokine production in response to HBsAg was not due to an overall T cell deficit. HBsAg-specific cellular responses,indoor cannabis grow system including T cell proliferation and intracellular IFN-γ/ TNF-α, occurred almost exclusively in good HBsAb responders irrespective of smoking status . In fact, a comparison of good HBsAb responders to non-responders identified significant between group differences with respect to the HBsAb titer, T cell proliferation, intracellular IFN-γ/TNF-α, and production of IL-2 during the DC:T cell coculture .
This correlation between individuals who are good HBsAb responders and the generation of cellular responders is consistent with other studies which have shown enhanced HBV-specific T cell proliferation and Th1 cytokine production in HBsAb responders , and a correlation between humoral response and in vitro anti-HBsAg cellular activity . However, when all HBsAb responders are included in the analysis the correlations between humoral and cellular responses are lost, consistent with reports that HBsAg-specific lymphoproliferation does not correlate with “any” sign of seroconversion . In contrast to some reports, we did not see HBsAg-specific proliferation in T cells from the HBsAb non-responders. This may be due to the relatively small number of non-responders in our study, or the use of purified T cells and HBsAg-pulsed DC which maximizes stimulation and minimizes non-specific proliferation. When comparing NS and MS for HBsAg-specific T cell responses and cytokine production, there were no significant differences for any of the measurements . In addition, we identified no baseline differences between the NS and MS cohorts with respect to the distribution of peripheral blood cell subsets or their phenotypic characteristics . While other investigators have reported reduced seroconversion rates , reduced HBsAb titer , or both as effects of substance abuse, the majority of these studies focused on IV drug users. In a recent meta-analysis of HBV vaccine studies in drug-using populations, Kamath et al. determined that the immune response among drug users was suboptimal. When multiple factors were assessed in a study of drug users , the only independent predictor of HBsAb non-responsiveness was alcohol use and seroconversion was not significantly related to other drug use . Our MS subjects were healthy, screened to exclude prior HBV exposure, and were not using other drugs, eliminating these potentially confounding factors. Based on the documented immuno suppressive properties of cannabinoids and the potential implications for public health, we designed a controlled clinical investigation to examine whether habitual exposure to MJ smoke impairs the integrated immune response to a HBV vaccination. The immunoregulatory effects of THC on human T cells, B cells, and DC are dramatic when cells are exposed in vitro and there is clear evidence that THC can disrupt protective responses to vaccination, cancer and infectious challenges in mouse models in vivo . For example, 60% of the control mice immunized against Legionella pneumophila were protected from a subsequent lethal challenge while none of the animals who received an intravenous injection of THC prior to immunization survived the challenge . The failed vaccine response was associated with a down-regulation of IL-12, upregulation of IL-4, and switch in immunoglobulin isotype profile. Both DC and T cell function were impaired, recapitulating human in vitro findings. Similar effects were observed in a cancer model when mice were injected with THC , which promoted more rapid growth of tumors and skewed cytokine responses . The important role that cannabinoids play in regulating adaptive immunity has also been suggested by studies in CB1−/−/CB2−/− double knock-out mice that lack functional cannabinoid receptors. This strain exhibits more mature DC, lower numbers of naive T cells, more robust T cell responses to antigen challenge, enhanced production of IL-17 and IFN-γ following challenge with influenza and evidence of post-inflammatory lung damage following influenza infection . Consistent with these publications, our current results demonstrate that short term exposure of human DC to THC in vitro significantly impaired their capacity to stimulate HBsAg-specific T cell and Th1 cytokine responses. We therefore hypothesized that habitual MJ smoking would significantly decrease the frequency and magnitude of HBsAg immune responses to vaccination. Hepatitis B was chosen as a test due to its clinical importance, especially in individuals with a history of substance abuse, the protective value associated with the development of a positive HBsAb titer and cellular immune responses, and our ability to carefully track and quantitate both humoral and cellular immune responses in vitro. Contrary to our expectations and the underlying hypothesis for this study, no major differences were observed between the MS and NS groups with respect to the frequency of HBsAb responders, the magnitude of the HBsAb titer, the number of “good responders”, or the frequency of HBsAg-specific cellular responses or Th1 cytokine production. Furthermore, we did not observe any baseline differences in the distribution of immune cell subsets, DC marker expression, or control mitogen responses between the two groups.