Results and Discussion Table 1

Characterization of HIV antigen-specific human CD8+ T cell responses evoked by in vitro priming of cultured Dendritic Cells with peptide-containing bi...
Author: Egbert Leonard
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Characterization of HIV antigen-specific human CD8+ T cell responses evoked by in vitro priming of cultured Dendritic Cells with peptide-containing biodegradable microsphere vaccine Rouskey C.J.a, Harris P.E.b, Paranjpe V.a, Patel R.a, Hoban H.a, McLoughlin S.a, Herst C.V.a, Killingbeck S.c, Zompi S.d, Rubsamen R.M.a,c a

Flow Pharma Inc., 1900 University Avenue, Suite 200, East Palo Alto, CA 94303, United States Columbia Department of Medicine at the College of Physicians and Surgeons c UC Berkeley School of Public Health d University of California San Francisco School of Medicine e Massachusetts General Hospital b

Abstract We describe an in vitro assay that uses poly-L-lactide-co-glycolide (PLGA) microparticles containing conserved HIV-1 CTL epitopes to pulse monocyte derived dendritic cells (MDDC) and subsequently prime autologous CD8+ T cell effector function in vitro. We demonstrate that microparticle-primed MDDC are capable of activating, priming and expanding epitope-specific CD8+ T cells that have the capacity to kill autologous HIV-1 -infected CD4+ Target cells and suppress p24 antigen in effector:target co-cultures. These studies reveal the potential of such a delivery mechanism as a vaccination method that targets dendritic cells in vivo, suggesting that immunization using this method is more efficacious than therapeutic DC vaccines primed with peptide only ex vivo. Introduction Emerging data strongly indicate that CD8+ T lymphocytes play an integral role in exerting significant immune pressure in HIV-1 infection [1, 2]. Newly infected hosts mount a potent natural cytotoxic T cell (CTL) response towards the Gag, Nef, Vif and Rev proteins of HIV [3-5]. In the case of elite controllers and slow progressors, this response prevents rapid advancement towards disease and is directed toward highly conserved epitopes [3, 6]. In normal progressors, the Th1mediated response is circumvented, at least in part, by the induction of tolerance through regulatory pathways, and blockading signaling through these pathways may prevent the inhibition of natural CTL responses [7-9]. Patients with access to highly active antiretroviral therapy (HAART) experience a significant reduction in plasma viral load (pVL) and recover CD4+ T cell counts within weeks of beginning treatment [10, 11]. Though HAART manages many of the comorbidities associated with HIV infection, these treatment regimens fail to induce antiviral CD8-mediated CTL responses to conserved epitopes, preventing the clearance of virus from natural reservoirs [12-15]. HAART prevents the maintenance of HIV-specific CD8+ T cells by reducing viral antigens to a non-priming level, suggesting the need for therapeutic vaccines that prime these responses with or without HAART [16-18]. To date, therapeutic vaccination candidates against HIV-1 utilize autologous dendritic cells (DC) pulsed ex vivo with HLA class-specific epitopes, HIV proteins, or autologous HIV [19-24]. Dendritic cells play a pivotal role in the initiation and maintenance of immune responses against viruses and are impaired in individuals with progressive HIV-1 infection [25-27]. In the case of DC-based therapeutic vaccines, MDDC act as adjuvants that deliver conserved HIV-1 CTL epitopes. These methods, though safe and well-tolerated, have yet to elicit the breadth of immune response needed to completely control the virus in vivo [20-22]. We previously described a vaccination method in which conserved CTL epitopes delivered with TLR9 agonist, cytosine-phosphate-guanine (CpG), elicit functional CD8+ T cell responses in mice when delivered in poly-L-lactide-co-glycolide (PLGA) microparticles [28]. We also demonstrated that co-administration of PLGA microparticles with monophosphoryl lipid A (MPLA) greatly

enhanced the immunogenicity of our vaccine. This approach delivers CTL epitopes to DC in vivo and eliminates the need to expand DC ex vivo, making it a simple and efficient alternative to current DC vaccination methods (reference the failures). To further evaluate our vaccination technology, we constructed microparticles with A*02- or B*57restricted HIV-1 p24 epitopes (SL09, SLYNTVATL; and KF11, KAFSPEVIPMF) and evaluated their ability to prime CD8+ T cells for in vitro killing. We demonstrate that these epitopes, when coupled with adjuvants, are effectively presented by MDDC to CD8+ T cells and prime CTL responses against HIV-1. We finally demonstrate that CD8+ T cells primed with class-restricted microparticles suppress HIV in vitro. Methods Microparticles and peptides Poly-L-lactide-co-glycolide powder (PLGA, chemically equivalent to Resomer 502H), acetone (>99.99% pure), ammonium hydroxide (NH4OH; >99.99% pure), D-mannose powder (>99.9% pure), and sterile endotoxin-free water were purchased from Sigma-Aldrich Corporation (St. Louis, MO). The TLR-9 agonist CpG oligodeoxynucleotide (ODN) 2006-B5 (specific for human cells) was purchased from InVivoGen Corporation (Carlsbad, CA). HIV SL-09 peptide (SLYNTVATL) and HIV KF-11 peptide (KAFSPEVIPMF) were synthesized by f-moc chemistry and purified by reverse phase HPLC (American Peptide, Sunnyvale, CA). Peptides were supplied as lyophilized powder. All solutions were prepared fresh on the day of microsphere preparation. PLGA was prepared as a 4% (w/v) solution in acetone. The peptide of choice was prepared as a 10mg/mL solution; SL09 was prepared in 5% NH4OH, whereas KF-11 was prepared in water. Mannose was prepared as a 100mg/ml solution in water. The CpG ODN was prepared as a 10mg/mL solution in water. The solution of PLGA in acetone was next placed in a sonicator. Under constant sonication, the following components were added in order: One hundred microliters of mannose was added to a final concentration of 0.25% (w/w) of PLGA; fifty microliters of CpG ODN was added to a final concentration of 0.025% (w/w) of PLGA; and ten microliters of peptide was added to a final concentration of 0.0025% (w/w) of PLGA. The complete formulation was then sonicated for an additional five minutes. Complete formulations were processed through the Flow Focusing device within one hour of their preparation. Once collected, the resultant microspheres were allowed to air-dry overnight before harvesting. Isolation of PBMC PBMC were isolated using a Histopaque gradient (Sigma, Saint Louis, MO) and were resuspended in RPMI (Life Technologies, Inc.) supplemented with 10% human AB serum, and Penicillin (100U/mL) and Streptomycin (100ug/mL). All PBMCs were incubated for 4 hours at 37C, 5% CO2 to allow monocytes to adhere to the bottom of culture flasks. Adherent monocytes were washed in RPMI and subsequently treated with differentiation media to derive MDDC. Both CD4+ and CD8+ T cell populations were isolated from non-adherent populations using magnetic negative selection (Miltenyi Biotech). Differentiation of monocyte-derived dendritic cells (MDDC) Adherent monocytes were washed with complete RPMI and incubated for six days in the presence of GM-CSF (50ng/mL) and IL-4 (100ng/mL). On day 6, MDDC were treated with LPS (100ug/ml) or poly(I:C) (100ug/mL) and IL-12 (100ng/mL) to drive complete maturation. Prior to vaccination, dendritic cells were evaluated for their expression of maturation markers. In vitro vaccination On day 7, MDDC were washed in complete RPMI, plated in 96-well round bottom plates at the indicated concentration and volumes, and pulsed with either SL09 or KF11 peptide (10uM), or with microparticles containing 0.0025% peptide (50ug/mL). Pulsed dendritic cells were incubated

overnight at 37oC, in 5% CO2. Also on day 7, freshly purified CD4+ and CD8+ T cells were isolated from healthy donors and incubated overnight in 10ng/mL IL-15. The following day, CD4+T cells (18:1), CD8+ T cells (2:1) and dendritic cells were co-cultured for 7 days. Activation was assessed over the time course using flow cytometry. One week following co-culture, aliquots of CD8+ T cells were expanded using autologous MDDC and peptide or microparticles. To expand peptide-specific CTL for use in cell-killing assays, primed CD8+ T cells were fed with fresh IL-2 (50IU/mL) every two days starting on day 18. These cells were expanded for up to two weeks before being used in cell-mediated kill assays.

Cell-mediated killing assay Three days before performing the killing assay, CD4+ T cells were isolated from PBMC of autologous healthy donors. The cells were stimulated for two days in complete RPMI and infected on the third day with an R5 strain of HIV obtained from Advanced Biotechnologies, Inc. (Ba-L, Columbia, MD) at an MOI of 0.1. Infection occurred overnight following which HIV-infected CD4+ target cells were washed three times to remove unbound HIV. CD8+ T cells were purified from priming co-cultures and incubated with target cells at an effector:target ratio of 5:1. Assay conditions were established in triplicate and cell killing was assessed via Annexin/PI staining. CD8+ T cells from each well were assessed for the production of IFNg using intracellular cytokine staining. Supernatants were harvested and p24 ELISA was used to confirm viral suppression. Flow Cytometry Flow cytometry was performed as previously described [29] using a BD Facs Accuri C6, and all reagents were purchased from BD Biosciences (San Diego, CA). Briefly, dendritic cell maturation was assessed by staining with an antibody cocktail containing CD86-APC and CD11c-PE. T cell activation was assessed using an antibody cocktail containing CD8-FITC, CD25-PE, and CD38Percp-Cy5.5. Isotype control IgG1-FITC, IgG2a-PE antibodies were used as negative staining controls. Unstained and single color compensation controls were derived from anti-CD3-stimulated (1ug/mL) T cells from each donor. All samples were washed in 1x PBS and stained in FACS Buffer (1x PBS, 2%FBS, 0.2% Na-Azide), according to the manufacturer’s protocol. SL09-specific T cell enumeration Peptide antigen specific T cells from HLA A*02 donors in co-cultures were enumerated using HLA A*02-SL09 pentamer purchased from ProImmune (London, UK). Staining was performed according to the manufacturer’s instructions. Positive control HLA-A*02-restricted, gag(p24)specific CD8+ T cell line was purchased from Isis Innovations (London, UK) and used to validate our TCR staining protocol. Annexin V/PI staining Annexin-V staining was performed to the protocol provided by BD (San Diego, CA). Briefly, 5x10^5 T cells were washed two times in 1x PBS and pelleted at 1600rpm, for 5min. Cell pellets were resuspended in 100uL of Annexin binding buffer to which 1uL of anti-CD8-APC, 5uL of Annexin V and 5uL of PI were added. The cells were incubated in the dark at room temperature for 15 minutes. Lastly 400uL of 1x Annexin binding buffer was added to each sample and dead cells enumerated on the BD FACS Accuri. CD8 negative, HIV+ populations were gated and dead cells within those populations were enumerated. Intracellular Cytokine Staining Aliquots of CD8+ T cells from effector:target co-cultures were stained with a cocktail of CD3, CD8, CD69, and IFNg purchased from BD Biosciences (San Diego, CA). Briefly, cells were harvested and washed in twice in 1x Dulbecco’s PBS (D-PBS), permeabilized at room temperature for 10 minutes, stained, and ultimately fixed in FACS fixative (1xPBS, 1% paraformaldehyde). p24 ELISA

Tissue culture supernatants from cytotoxic T cell assays were harvested and diluted 1:3 in complete RPMI and assayed using a p24 ELISA kit purchased from Abnova (Taipei City, Taiwan). Microsoft Excel was used to generate standard curves, and data were analyzed in GraphPad Prism.

Results and Discussion To generate efficient antigen presenting cells (APCs), monocytes were harvested from PBMC of donors (Table 1) and incubated for six days with differentiating cytokines and maturation agents. Following differentiation and maturation, we assayed MDDC cultures for the expression of maturity markers. Data indicate that our process was successful at producing mature dendritic cells. Though a single donor “KT” produced significantly lower numbers of MDDC (Figure 1). Overall, cell cultures were >55% mature as judged by the expression of CD86 and CD11c. Following assessment and prior to in vitro vaccination percentages of positive cells (designated CD86+CD11c+) were used to calculate the number of available, mature APC. A total of 2e3 mature MDDC were plated and pulsed with epitope-loaded microparticles, or peptide alone. Pulsed (“vaccinated”) MDDC were co-cultured with mixed T cell populations.

Table 1. Donors, haplotypes and inclusion in data analysis. The following donors were studied in two separate experiments. Not all donors were evaluated in CTL kill, IFNg or p24 assays due to a lack of assay or cell availability at the time of testing. If samples are included in analysis, they contain a “yes” in the indicated experimental column. “a”= homozygous at this locus. Haplotypes in bold are haplotypes to which our epitopes potentially bind.

MDDC pulsed with SL09 and KF11 microparticles induce the upregulation of CD25 in a HLA-Class I restricted fashion. To determine if matured MDDC pulsed with SL09 or KF11-microparticles effectively activated CD8+ T cells, we pulsed MDDC with SL09- or KF11-microparticles and co-cultured them with mixed T cell cultures. Each co-culture was evaluated for activation over a 7-day time course (days 9, 11, 13, and 15). We evaluated CD69 (data not shown), CD25 (Figure 2) and CD38 (Figure 3)

expression and considered these markers as key indicators in predicting CTL activation. CD8+ T cells co-cultured with microparticle-pulsed MDDC were activated at greater levels than those cocultured with non-vaccinated MDDC (Figure 2b, 2d; day 15 data). These effects were epitopespecific with HLA-A*02 donors responding specifically to SL09 microparticles and HLA-B*44 donors responding to KF11-microparticles.

Figure 1. Monocyte-derived dendritic cells were activated following differentiation and maturation with IL-12 and poly(I:C). Monocytes were isolated via attachment to culture flasks and differentiated in the presence of GM-CSF and IL-4. Following differentiation, MDDC were matured in the presence of IL-12 and poly(I:C). Following maturation, MDDC were assessed for presentation capacity using a two color flow cytometric assay evaluating expression of CD11c and CD86. MDDC from all donors showed maturation in >65% of the total population. These percentages were used to determine the final number of viable APC in MDDC:T cell co-cultures.

Interestingly, KF11-pulsed MDDC were capable of activating HLA-B*44+ CD8+ T cells - an effect that is not yet fully understood. KF11 is known to be an HLA-B*57-restricted epitope, though evidence indicates that this peptide contains a binding motif that overlaps with the expected B*44 epitope within the Gag protein (p24; EEKAFSPEVIPMF) [30]. A SYFPEITHI database analysis of KF11 indicates that this peptide binds to B*44 with an overall score of 20. When compared with a positive control HHV4-derived peptide (score = 22) this data suggests that presentation of KF11 epitope to B*44 positive donors is likely.

The effects of mature, MDDC on CD38 upregulation were not specific as revealed by flow immunohistochemical measurements in both CD4+ and CD8+ T cell analyses (Figure 3) suggesting that the adjuvant effects of MDDC are capable of inducing non-specific immune

responses in vitro - a finding that has been previously described [31]. Figure 2. Monocyte-derived dendritic cells (MDDC) pulsed with PLGA microparticles with conserved HLA A*02 (SL09; SLYNTVATL) or HLA B*57 (KF11; KAFSPEVIPMF) HIV-1 epitopes activate autologous CD8+ T cells. MDDC were derived from PBMC and pulsed with PLGA-microparticles loaded with CpG and. Dendritic cells were then co-cultured with naive, primed CD8+ and CD4+ T cells at a ratio of 2:1 and 18:1, respectively. Following co-culture, cells were assayed for expression of CD25. Co-culture of autologous MDDC pulsed with SL09 microparticles did not significantly stimulate CD4+ T cells from HLA A*02 (a) or HLA B*44 (c) donors to a level greater than MDDC background (a). However, CD8+ T cells from HLA A*02 (b) and HLA B*44 (d) were stimulated as expected by their respective, class I-restricted epitopes. All samples were gated on CD8+ cells and assessed for CD25 expression. Paired t-tests were performed and p-values are documented (NS; not significant).

SL09-specific T cells are expanded in the presence of MDDC pulsed with SL09 microparticles. To determine if the activated CD8+ T cells were able to recognize peptide antigen in the context of autologous MHC Class I, we assayed the the T cell cultures for reactivity with HLA A*02-SL09 pentamer. In mixed T cell cultures, SL09 peptide was less effective than SL09 microparticles at priming these responses (Figure 4), though both free peptide and SL09-microparticles were able to expand epitope specific T cells. Specific T cell enumeration was expected to be in the 1-3% range when compared to other studies that induce Gag-specific CD8+ T cells [32]. Our data are in line

with these studies and suggest that our technology may more efficiently prime these responses in vitro.

Figure 3. T cells co-cultured with MDDC upregulate CD38 in vitro. Mixed T cell cultures co-cultured with autologous microparticle-pulsed dendritic cells, or dendritic cells alone were cultured for 7 days and assessed for the upregulation of activation marker CD38 using flow cytometry. HLA-A*02 and HLA-B*44restricted CD4+ (a,c) and CD8+ (b, d) T cells were activated in the presence of non-pulsed or pulsed MDDC detailing the adjuvant effect of matured, activated MDDC.

CD8+ T cells primed with microparticle-pulsed MDDC effectively kill HIV+ targets. We used Annexin V/PI staining to determine if CD8+ T cells primed with vaccinated MDDC were able to kill autologous HIV+ CD4+ target cells. The kill assay was established at an E:T ratio of 5:1, and co-cultures were established on day 25. On days 26, 28, (data not shown) and day 30 (Figure 5), co-cultures were harvested and the number of dead HIV+CD4+ T cell targets were enumerated. In five out of six donors, treated with MDDC pulsed with SL09-microparticles, specific killing was greater when compared to background (HIV only) and non-pulsed dendritic cell controls (DC+T). These data indicate that our vaccine candidates are taken up by dendritic cells, processed through class I pathways, and effectively prime CD8+ T cells for killing. To further elucidate the mechanism by which HIV+CD4+ target cells were programmed for death, we examined the activation of CD8+ effectors using 4-color flow cytometry. Gating on CD3+CD8+ T cells from co-cultures, we enumerated the percentage of CD3+CD8+ T cells in the population that expressed increased levels of CD69 and IFNg. The early activation marker CD69 indicates that effector cells are receiving a stimulation signal, and expression of IFNg indicates that the

effectors have have acquired a phenotype typical of CTLs. On day 30, significant differences in IFNg production were not observed. Significant differences were observed however, at day 28 in the assay. We document a significant decrease in effector function from D28 to D30 as evidenced through a loss in IFNg production indicating that there may be a period of specific CTL activation prior to D30 in our assay (Figure 6). It is probable that these responses are activated throughout the time course and slowly eradicate HIV+ target cells.

Figure 4. Microparticle-pulsed MDDC prime CD8+ T cells better than SL09 peptide alone. MDDC were pulsed with 10uM peptide or 1uM peptide in SL09 microparticles. Pulsed MDDC were co-cultured with mixed T cell cultures and incubated for 7 days. On the last day of co-culture an CD8+ T cells with the SL09-specific T cell receptor were enumerated using A*02-SL09 pentamer after gating on CD8+ T cell populations. Data represent the results from two experiments. Paired, t-tests were performed to determine statistical significance.

Figure 5. Microparticle primed T cells kill HIV+ CD4+ Target cells. Following activation in co-culture CD8+ T cells were purified and incubated with CD4+ HIV-infected target cells. Wells were established in triplicate and assessed over a 5 day killing period. Maximum cytotoxic effect was observed on the final day of the assay as determined by Annexin V/PI staining. Data are divided into two figures based on HLA haplotype with HLAA*02 (a) and HLA-A*02/B*44 (b) donors separated for analysis and indicate that SL09-microparticle pulsed MDDC are capable of inducing specific killing in HLA-A*02 donors.

Figure 6. Effector function was determined via intracellular cytokine staining on days 28 (a) and 30 (b). Comparisons between haplotypes are presented. SL09 microparticle-pulsed DC primed CD8+ T cells for specific killing, though these results were not statistically significant (ns). Interferon gamma (IFNg) expression was significantly higher in CD8+ T cells pulsed with SL09 microparticles when compared to

background killing using a paired, two-tailed t-test. Figure 7. CD8+ T cells primed with MDDC pulsed with SL09 or KF11 microparticles suppress HIV in vitro. Microparticle-pulsed MDDC were used to prime CD8+ T cells and generated effector CTL were used in cytotoxic kill assays. Supernatants containing p24 antigen were assayed using p24 ELISA. Statistical significance was observed using a paired, two-tailed t-test, p-values are indicated.

To determine the efficacy of microparticle-primed MDDC to create specific CTL capable of suppressing HIV in vitro we evaluated the presence of p24 in the supernatants of our CTL assay co-cultures. As evidenced in our p24 ELISA, T cells primed by MDDC treated with or without vaccine have the potential to stimulate HIV-suppressive responses (Figure 7). Microparticle-pulsed MDDC primed CD8+ T cells to suppress p24 to levels below untreated CD4+HIV+ controls. The level of p24 present in supernatants from SL09 and KF11-treated co-cultures was significantly lower than the p24 produced in target cells without effector T cells. Interestingly, unpulsed MDDC were also able to confer a level of p24 suppression though it was not significantly different from background suppression. These data, when taken with specific T cell-mediated killing and activation data indicate that our vaccine candidates not only specifically activate CD8+ T cells through class I MHC presentation, but that they also prime these cells to recognize autologous, HIV-infected CD4+ T cells and thus suppress HIV in vitro. Previous studies indicate that dendritic cells are potent initiators of immune responses. These responses do not always occur in an antigen-specific way, and in vitro assessment of vaccination models utilizing DC as adjuvant describe non-specific immune activation in vivo. Our data indicate that ex vivo pulsing of MDDC with epitope-specific PLGA microparticles not only stimulates activation, but also primes specific-killer T cell responses in vitro.

We are aware of the limitations of this in vitro study as measure of vivo efficacy of a possible therapeutic/prophylactic vaccine. Never-the-less, the application of PLGA microparticles as antigen

delivery vehicles with adjuvant activity remains promising in the development of highly effective vaccines against HIV-1. Our findings are further supported in recent research presented by Climent et al., in which PLGA-nanoparticles loaded with full length Gag (p24) were used to prime MDDC and stimulate T cell effector function. Though this work did not enumerate epitope-specific CD8+ T cells, their results demonstrate antigen-specific, CTL-mediated killing of gag-expressing MDDC. Previously, we demonstrated in vivo efficacy of microparticles eliciting T cell responses to OVA (SIINFEKL) and VSV (RGYVYQGL) epitopes. We demonstrated that PLGA microparticles were in fact capable of eliciting T cell responses, and that specific killer T cell responses were greater when co-administered with DC-adjuvant, MPLA (28). Our current studies elucidate a pathway through which antigen-loaded PLGA microparticles are processed by MDDC and presented via HLA class I to elicit downstream effector function. These studies also add further conceptual support to a vaccination strategy where multiple preparations of PLGA microspheres, each carrying one or two peptide antigens, are prepared with the critical MHC Class I restricted conserved HIV epitopes as defined by the strategy of ( INSERT RECENT WALKER PAPER). We hypothesize that injection of multiple, different peptide PLGA microspheres, at different intradermal locations, in HLA typed individuals, would confer a broad range of focused CTL responses avoiding issues of epitope competition associated with the use of full length proteins.

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