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Genetically Modified Dendritic Cell Vaccine Is a Much More Potent Activator of CD4 and CD8 T Cells Than Peptide

Development of Cell Based Tuberculosis Vaccines: Genetically Modified Dendritic Cell Vaccine Is a Much More Potent Activator of CD4 and CD8 T Cells Than Peptide or Protein Loaded Counterparts

Janet I. Malowany1, Sarah McCormick1, Michael Santosuosso1, Xizhong Zhang1, Naoko Aoki1, Patricia Ngai1,
cards against humanity in stores, Jun Wang1, Jaina Leitch1, Jonathan Bramson1, Yonghong Wan1 and Zhou Xing1

1Department of Pathology and Molecular Medicine and Division of Infectious Diseases, Centre for Gene Therapeutics, McMaster University, Hamilton, ON, Canada L8N 3Z5

Correspondence: Zhou Xing, Department of Pathology and Molecular Medicine, Room 4012 MDCL, McMaster University, 1200 Main Street West, Hamilton, ON, Canada L8N 3Z5. Fax: +1 905 522 6750.

Top of pageAbstractGenetically modified dendritic cell (DC) based vaccines have not been explored for immunization against tuberculosis. AdAg85A transduced DC vaccine (AdAg85/DC) expressed higher levels of IL 12 and was much more immunogenic than Ag85 protein loaded (pro/DC) or CD4/CD8 T cell peptide loaded (pep/DC) DC vaccines. Compared to pro/DC or pep/DC, AdAg85/DC elicited a remarkably higher level of ex vivo IFN production by CD4 and CD8 T cells at weeks 2, 6, and 12 postimmunization, which was coupled with higher frequencies of antigen specific T cells. By an in vivo CD8 or CD4 T cell cytotoxicity (CTL) assay, AdAg85/DC was shown to provoke much higher and more sustained levels of CD8 and CD4 CTL activity up to 12 weeks postimmunization. Intramuscular (im) AdAg85/DC immunization was more potent than the iv route of AdAg85/DC immunization. Our results thus suggest that genetically modified DC based TB vaccine is superior to subunit DC vaccines and has the potential for therapeutic applications.

Top of pageIntroductionTuberculosis (TB) is one of the leading infectious causes of death worldwide1. The TB epidemic has been worsened by HIV infection2. Bacille Calmette Gu (BCG) is currently the only TB vaccine available and is administered to humans shortly after birth. While BCG is effective in protecting from childhood TB,
best cards against humanity, it is ineffective in protecting from adult TB3,4. Thus, one of the challenges to TB vaccinologists is to develop effective prophylactic TB vaccines able to confer long term protection against pulmonary TB3,4. In this regard, since BCG may not be safe for immune compromised hosts and has proven ineffective in boosting immune anti TB responses in BCG vaccinees5,6, a therapeutic TB vaccine will likely be a recombinant genetic vaccine that is not only effective but also safe and repeatable. To date, recombinant DNA TB vaccines have been evaluated for their therapeutic effects7 but recent results indicate that this form of vaccine is ineffective for therapeutic purposes8,9.

Dendritic cells (DC) are the most potent professional antigen presenting cells. At the interface of innate and adaptive immunity, these cells are critical to antigen sampling and activation of na T cells, having the power to polarize the immune response toward the type 1 or type 2 phenotype10. Thus, exploiting their potent immune activating properties, DC have been differentiated in vitro and manipulated as vaccines to deliver antigens for cancer immunotherapy11,12 or prevention of infectious diseases13. While the most commonly used method to prepare DC vaccines is to load DC with immunogenic peptide or protein (“subunit DC vaccines”)14, recent studies, particularly those carried out in cancer models, have suggested that the DC vaccines virally transduced to express immunogenic peptides or proteins are more potent than subunit DC vaccines. Of several viral vector systems, recombinant adenoviral vector has been used effectively to transduce tumor antigen coding genes into DC, and such adenovirally transduced DC cancer vaccines have been found to be not only more effective than subunit DC vaccine but also safe and repeatable15,16. While relatively few studies thus far have explored such viral gene modified DC vaccines and compared their potency to subunit DC vaccines for generating immune protection against infectious disease, emerging evidence supports their potential to be used as effective, safe, and repeatable cell based vaccines to prevent a variety of diseases that are significant causes of morbidity and mortality. However, gene modified DC TB vaccines remain to be explored and/or compared with peptide/protein DC counterparts. In the past several years, we have been using adenoviral gene transfer vectors to transduce cancer antigen coding genes into DC and found that such genetically modified DC cancer vaccines are potent in eliciting anti cancer immune responses19,20,21,22. We found that AdAg85A transduced DC vaccine was much more potent than peptide or protein loaded DC counterparts in eliciting long lasting immune activation of both CD4 and CD8 T cells. We believe that our findings hold implications in the future design of DC based vaccines for TB as well as other intracellular infectious diseases.

Top of pageResultsImmune characteristics of DC vaccinesTo examine whether different modes of DC antigen loading may differentially alter the property of DC, we prepared DC vaccines as described under Materials and Methods and analyzed them by FACS at 24 h posttreatment. We used lipopolysaccharide (LPS) as a positive control for stimulation. We gated on DC stained positive for MHC class II and subsequently examined them for CD11c and surface activation markers or cytokine expression by FACS analysis software. Untreated DC, compared with AdAg85A infected DC (AdAg85/DC) and Ag85 protein loaded DC (pro/DC), similar to LPS/DC, had strikingly increased cell surface expression of B7.1 (Fig. 1) and CD40 (data not shown), whereas CD4/CD8 T cell peptide loaded DC (pep/DC) did not demonstrate such increased expression. In comparison, untreated DC already expressed similarly high levels of B7.2, and AdAg85/DC, pro/DC, and LPS treated DC demonstrated only mildly increased B7.2 or MHC class II expression (data not shown). By using intracellular cytokine staining (ICCS) we examined cytokine production by the different DC vaccine formulations. We found that compared to untreated DC, AdAg85/DC, pro/DC, or pep/DC showed little increase in TNF production (Fig. 1). While LPS treatment did not increase TNF production above the level seen in other DC at 24 h (Fig. 1), it did markedly increase TNF responses at an earlier time of 5 h (data not shown). In sharp contrast to TNF responses, compared to untreated DC, AdAg85/DC had a remarkably higher level of IL 12 production than pro/DC or pep/DC and such increased IL 12 expression in AdAg85/DC was comparable to that induced by LPS stimulation (Fig. 1). These data suggest that while viral gene modified DC retain other properties of dendritic cells, they assume a much more pronounced type 1 immune activating phenotype than protein or peptide loaded counterparts. In vitro differentiated dendritic cells were washed 5 h after being incubated without (DC) or with AdAg85A (AdAg85/DC), Ag85 complex proteins (Pro/DC), T cell peptides (Pep/DC), or LPS (LPS/DC). The cells were then incubated for an additional 19 h before immunostaining, FACS analysis, and/or ICCS. FACS analysis was carried out by gating on MHC class II and CD11c positive cells.

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Intramuscular AdAg85/DC vaccination leads to potent T cell activationTo compare the potential of immune activation by various DC vaccines, we immunized mice im by AdAg85/DC, pro/DC, or pep/DC and sacrificed them at weeks 2, 6, and 12 postvaccination. By using ICCS and FACS techniques, we first analyzed the number of antigen specific, IFN releasing CD4 and CD8 T cells in the spleen. At all time points compared to pro/DC and pep/DC vaccines, AdAg85/DC vaccination generated a much greater number of splenic IFN producing CD8 or CD4 T cells upon stimulation by respective immune dominant peptides (Figs. 2A and 2B). The frequency of antigen specific CD8 T cells was greater than that of CD4 T cells. The level of increased CD8 T cells appeared to be better sustained than the CD4 counterpart. The control DC vaccine (DC infected only with an empty adenoviral vector) elicited little response (data not shown). Since AdAg85A/DC vaccine was prepared in such a way that DC were infected with an adenoviral vector expressing Ag85A, there was a possibility that the lack of efficiency by pep/DC or pro/DC vaccine was due to the lack of DC activation caused directly by viral infection per se. To rule it out, we prepared pep/DC and pro/DC vaccines that were also infected with a control adenoviral vector (pepAddl/DC and proAddl/DC) and compared the immunogenicity of these two additional control vaccines with that of pep/DC, pro/DC, or AdAg85A/DC. We found that adenoviral infection of pep/DC or pro/DC still failed to activate Ag85A specific CD8 or CD4 T cells effectively (Figs. 3A and 3B). By using different culture conditions and ELISA, we examined the IFN production capacity of T cells and found that overall such increased numbers of antigen specific T cells by AdAg85/DC vaccine were accompanied by proportionately increased levels of IFN protein release into culture supernatants (Figs. 4A and 4B). Of note, at 12 weeks, the IFN production level was lower than at earlier times, suggesting that the majority of antigen specific T cells have entered a memory phase at this time. Overall, these observations indicate that AdAg85/DC vaccine is very potent in activating both CD8 and CD4 T cells,
against humanity game, whereas pro/DC and pep/DC vaccines are weaker stimulators. Mice were im immunized with various DC vaccines and sacrificed at weeks 2, 6, and 12. The whole splenocytes were cultured for 6 h with or without (A) CD8 or (B) CD4 T cell peptide. The cells were then subjected to immunostaining, ICCS, and FACS analysis. The data are expressed as the average % SEM (IFN positive cells of all splenocytes) from three or four mice/DC vaccine/time. Mice were im immunized with various DC vaccines and sacrificed at week 2. The whole splenocytes were cultured for 6 h with or without (A) CD8 or (B) CD4 T cell peptide. The cells were then subjected to immunostaining, ICCS, and FACS analysis. The data are expressed as the average % of two mice/vaccine (IFN positive cells of all splenocytes). Mice were im immunized with various DC vaccines and sacrificed at weeks 2, 6, and 12. The whole splenocytes pooled from three or four mice/vaccine/time were cultured for 24 h with or without (A) CD8 or (B) CD4 T cell peptide stimulation. The amount of IFN protein released into the culture supernatant was quantified by ELISA. Results are expressed as the means SEM of triplicate cultures.

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Intramuscular AdAg85/DC vaccination leads to the development of potent CD8 and CD4 T cell mediated cytotoxicity in vivoWe next set out to evaluate whether enhanced T cell activation elicited by AdAg85/DC vaccination was accompanied by enhanced antigen specific T cytotoxic activity in vivo. To address this, we employed an in vivo CD8 or CD4 T cell cytotoxicity (CTL) assay24,27. To examine CD8 CTL, we immunized mice im with Addl/DC, AdAg85/DC, pro/DC, or pep/DC vaccine, and at weeks 2, 6, or 12 postvaccination, we injected the mice iv with CD8 T cell Ag85A peptide pulsed, carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled target cells and sacrificed them 6 h post target delivery. We then analyzed the splenocytes by FACS for the presence of CFSE labeled cells and calculated the percentage of lysis of the peptide pulsed targets. At week 2, while there was an appreciable level of target cell lysis in mice elicited by all three forms of DC vaccine, animals vaccinated with AdAg85/DC were 4.5 times more effective (more than 80% target lysis) in killing CD8 peptide pulsed target cells in vivo than pro/DC and pep/DC groups (Figs. 5A and 5B). At both weeks 6 and 12, the level of CD8 T cell mediated killing markedly declined to a minimal level in both pro/DC and pep/DC groups, whereas the level of CD8 CTL was sustained by AdAg85/DC vaccination, maintaining 50% antigen specific target lysis (Fig. 5A). In contrast, the control DC vaccine (Addl/DC) failed to elicit any antigen specific target lysis at any time points (data not shown). Mice were im immunized with pep/DC, pro/DC, or AdAg85/DC vaccine. (A) At weeks 2,
cards against humanity black cards, 6, and 12 postimmunization, CFSE labeled Ag85A specific CD8 T cell peptide pulsed target cells were transferred intravenously into the immunized mice and the splenocytes were isolated 5 h after target cell transfer and examined by FACS analysis for the measurement of CD8 T cell mediated cytotoxicity (CTL), with the representative histogram of FACS analysis presented (B; week 2). (C) In separate experiments Ag85A specific CD4 T cell peptide pulsed target cells were transferred intravenously into the immunized mice and the splenocytes were isolated and examined by FACS analysis for the measurement of CD4 T cell mediated CTL. Results represent % loss of peptide pulsed target cells and are expressed as the means SEM of three or four mice/DC vaccine/time.

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By pulsing the target cells with a CD4 T cell peptide, we also examined the level of CD4 T cell mediated CTL in vivo. CD4 T cell mediated CTL has been found to play a role in host defense against mycobacterial infection28,29. To this end, we carried out an in vivo CD4 CTL assay as described under Materials and Methods and sacrificed the mice 24 h post target cell delivery. Compared to CD8 CTL, the magnitude of CD4 CTL was lower. At both weeks 2 and 6 postvaccination, pro/DC and pep/DC induced a small level of CD4 CTL activity, which decreased to a minimal level by 12 weeks postimmunization (Fig. 5C). In contrast, AdAg85/DC elicited higher levels of CD4 CTL at all time points, with a peak activity detected at week 6 (Fig. 5C), which differed from AdAg85/DC induced CD8 CTL, peaking at week 2 (Fig. 5A). The above CD8 and CD4 CTL data indicate that when given intramuscularly, AdAg85/DC vaccine is a much stronger activator of Ag85A specific CD8 and CD4 effector T cells than pro/DC or pep/DC vaccine.

Intramuscular route of AdAg85/DC vaccination is more potent than intravenous vaccinationSince intravenous (iv) immunization with a DC TB vaccine loaded with CD4/CD8 T cell peptides was previously found to be effective18, we compared the level of immune activation by im and iv vaccination with pro/DC, pep/DC, or AdAg85/DC. Similar to im immunization, iv immunization with pro/DC or pep/DC vaccine induced a minimum level of activation of CD4 and CD8 T cells at both weeks 2 and 6 as assessed by ICCS (data not shown). In contrast, at week 2, iv immunization with AdAg85/DC induced a significantly increased number of CD8 T cells accompanied by a relatively small number of CD4 T cells (Figs. 6A and 6B). By 6 weeks, the number of CD8 T cells markedly increased. In comparison, im AdAg85/DC vaccination triggered peak responses of CD8 or CD4 T cell activation at week 2, which was sustained up to week 6 (Figs. 6A and 6B). Since Elispot immunoassay is more sensitive than ICCS, we also used the IFN Elispot assay to assess the frequency of antigen specific CD4 and CD8 T cells. Similar to im vaccination, iv vaccination with the control DC vaccine (Addl/DC), pep/DC, or pro/DC caused some basal levels of antigen specific IFN release; it elicited little antigen specific response at either week 2 or week 6 (Figs. 7A and 7B). In basic agreement with the results obtained by ICCS, however, iv vaccination with AdAg85/DC elicited enhancement of CD4 and CD8 T cell responses. Intramuscular AdAg85/DC vaccination induced further increased numbers of CD4 and CD8 T cell responses at both time points (Figs. 7A and 7B); this was particularly evident for CD8 T cells. Accompanying greater numbers of CD4 and CD8 T cells by im AdAg85/DC were higher levels of IFN protein in culture supernatant (data not shown). Of interest, while im AdAg85/DC elicited a higher level of in vivo CD8 CTL activity than iv AdAg85/DC at week 2, the CTL activities were similar between iv and im by week 6 (Fig. 7C). These data suggest that, first, regardless of the route of vaccination (im or iv), pro/DC and pep/DC are much weaker DC vaccines than gene modified DC and, second, gene modified DC vaccine, when im administered, elicited stronger immune activation than when it was iv administered. Mice were im or iv immunized with AdAg85/DC and sacrificed at weeks 2 and 6. The whole splenocytes were cultured for 6 h with or without (A) CD8 or (B) CD4 T cell peptide. The cells were then subjected to immunostaining, ICCS, and FACS analysis. The data are expressed as the average % SEM (IFN positive cells of all splenocytes) from three or four mice/vaccination route/time. Mice were immunized iv with pep/DC, pro/DC, AdAg85/DC, or Addl/DC as a control or immunized im with AdAg85/DC. Mice were then sacrificed at (A) week 2 or (B) week 6. The whole splenocytes were pooled from three or four mice/group and cultured for 24 h with or without CD8 or CD4 T cell peptide. The cells were then subjected to Elispot assay. The number of IFN positive spots/million cells was enumerated. The data are expressed as the means SEM from triplicate wells/DC vaccine/time. (C) In separate experiments, mice were immunized iv or im with AdAg85/DC. At weeks 2 and 6 postimmunization, CFSE labeled Ag85A specific CD8 T cell peptide pulsed target cells were transferred intravenously to the immunized mice and the splenocytes were isolated 5 h after the target cell transfer and examined by FACS analysis for the measurement of CD8 T cell mediated CTL. Results represent % loss of peptide pulsed target cells and are expressed as the means SEM of three or four mice/DC vaccine/time. Mice immunized im or iv with pro/DC or pep/DC vaccine had relatively high levels of colony forming units (cfu) in the spleen (Fig. 8). The mice immunized iv with AdAg85/DC also had similarly high levels of cfu. 8). The level of infection in the lung was much lower than that in the spleen and the trend was similar to that in the spleen (data not shown).

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