PLoS One. 2014 Sep 25;9(9):e108383.

Improving Mycobacterium bovis BCG as a vaccine delivery vector for viral antigens by incorporation of glycolipid activators of NKT cells

Manjunatha M. Venkataswamy1,2*, Tony W. Ng1*, Shalu S. Kharkwal1, Leandro J. Carreño1,3, Zheng Liu4, Robert Bittman4, Peter J. Jervis5, Liam R. Cox6, Gurdyal S. Besra5, Xiangshu Wen7, Weiming Yuan7, Moriya Tsuji8, Xiangming Li8, David D. Ho8, John Chan9, Sunhee Lee10, Richard Frothingham10, Barton F. Haynes10, Michael W. Panas11,#, Geoffrey O. Gillard11,##, Jaimie D. Sixsmith11, Birgit Korioth-Schmitz11, Joern E. Schmitz11, Michelle H. Larsen1,12, William R. Jacobs, Jr.1,12, and Steven A. Porcelli1,9

 

1Department of Microbiology and Immunology, 9Department of Medicine, and 12Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, NY 10461, USA

2National Institute of Mental Health and Neuroscience, Bangalore, Karnataka, 560029, India

3Millennium Institute on Immunology and Immunotherapy, Facultad de Medicina, Universidad de Chile, Santiago 8380453, Chile

4Department of Chemistry and Biochemistry, Queens College of CUNY, Flushing, NY 11367, USA

5School of Biosciences and 6School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom

7Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA

8Aaron Diamond AIDS Research Center, Rockefeller University, New York, NY 10016, USA

10Duke University Medical Center, Durham, NC 27710, USA

11Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA

 

*Contributed equally.

#Current Address: Department of Microbiology and Immunology, Stanford Medical School, Palo Alto, CA 94305 USA

## Current Address: Biogen Idec Inc., Cambridge, MA 02142 USA

 

Abstract

Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8+ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8+ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.

PMID: 25255287

 

Supplement

The safety and immunogenicity of the BCG vaccine in human makes it an attractive vector for the development of vaccines against other pathogens including HIV. In our studies, we developed a recombinant BCG (rBCG) that expresses an SIV Gag antigen as a model to induce anti-lentiviral immunity. To further enhance the immune response against SIV Gag, we used synthetic glycolipids that activate natural killer T (NKT). In previous studies, we showed that these glycolipids can be incorporated directly into live BCG to recruit the natural adjuvant activities of NKT cells and increase vaccine efficacy (1). Directly incorporating these glycolipids into the cell wall of the rBCG-SIV Gag vaccine allows presentation of Gag by the dendritic cell while simultaneously activating CD1d-restricted NKT cells (Fig.1), resulting in an enhanced Gag-specific immune response (Fig.2). Using this approach for vaccination of mice, a significant improvement in Gag-specific immune responses was detected following subcutaneous immunization, a clinically relevant route for vaccination.

 

 TN fig1

Figure 1. The glycolipid adjuvant (α-C-GC or 7DW8-5) is stably incorporated into the cell wall or membrane of rBCG expressing SIV Gag. Uptake of the rBCG-SIV Gag by dendritic cells enables the activation of CD1d-restricted NKT cells to be tightly coupled with the presentation of bacterial antigens during immunization to achieve a strong adjuvant effect.

 

 

TN fig2Figure 2. Increasing amounts of the glycolipid adjuvant (7DW8-5) were incorporated into rBCG expressing SIV Gag, and these glycolipid-modified live bacilli were used to immunize mice. The incorporation of the glycolipid adjuvant enhanced the immune response to SIV Gag as shown by staining with the AL11 tetramer, which labels CD8+ T cells specific for the immunodominant SIV Gag epitope.

 

Below are three highlights of the manuscript:

  1. Dose sparing effects and improvement of T cell memory with glycolipid-modified rBCG. The glycolipid adjuvant decreased the dosage of the rBCG-SIV Gag vaccine for effective subcutaneous immunization. This dose sparing effect of the glycolipid adjuvant reduced the dosage of vaccine needed for CD8+ T cell priming by 10 to 100 fold.   Immunization with a relatively low dose of the glycolipid adjuvanted vaccine promoted formation of more Gag-specific CD8+ memory T cells, and mice that were immunized with this vaccine responded more rapidly to boosting with SIV-Gag delivered by a heterologous vector.
  2. Absence of NKT cell anergy. When free glycolipids are injected into mice, the strong activation of NKT cells can drive them into a state of anergy, which means they are unresponsive to subsequent stimulation. In this study, the approach of administering the glycolipid in physical association with the rBCG vaccine was shown to circumvent the induction of NKT cell anergy. Thus, NKT cells from mice immunized with the glycolipid adjuvanted vaccine could still response to glycolipid stimulation, whereas NKT cells from mice injected with similar quantities of the free glycolipid were unresponsive to subsequent glycolipid stimulation.
  3. The potent glycolipid adjuvant 7DW8-5 is active in a human CD1d knock-in mouse model. It has been shown that the glycolipid 7DW8-5 is highly active for stimulating human NKT cells, and that it is an effective vaccine adjuvant in a nonhuman primate model. In this study, we tested the efficacy of the 7DW8-5 incorporated into the rBCG-SIV Gag vaccine in a mouse model that has been genetically modified to express the human CD1d molecule. Indeed, 7DW8-5 showed superior activity in this humanized mouse model, supporting the potential clinical relevance of this approach for improving rBCG vaccines in humans.

 

Importance of this study:

The incorporation of synthetic glycolipid activators of NKT cells into live rBCG-SIV Gag improved the immunogenicity of this vaccine. The glycolipid adjuvant also lowered the dose needed for immunization, which can reduce the cost of vaccine production and may also avoid adverse effects. This approach of incorporating glycolipids onto the surface of bacteria can also be easily applied to other live whole cell vaccines (2).

Clinical studies in nonhuman primates and human are ethically challenging, time consuming, resource intensive and expensive. To bridge that gap using the far more tractable laboratory mouse model, we used a human CD1d knock-in mouse to test the impact of glycolipid 7DW8-5 on vaccine efficacy. The potency of the 7DW8-5 glycolipid in this humanized mouse model was encouraging and supports future extension of this work into nonhuman primate models.

 

Reference

  1. Venkataswamy MM, Baena A, Goldberg MF, Bricard G, Im JS, Chan J, Reddington F, Besra GS, Jacobs WR, Jr., Porcelli SA. 2009. Incorporation of NKT cell-activating glycolipids enhances immunogenicity and vaccine efficacy of Mycobacterium bovis bacillus Calmette-Guerin. J Immunol 183: 1644-56
  2. Singh M, Quispe-Tintaya W, Chandra D, Jahangir A, Venkataswamy MM, Ng TW, Sharma-Kharkwal S, Carreno LJ, Porcelli SA, Gravekamp C. 2014. Direct incorporation of the NKT-cell activator alpha-galactosylceramide into a recombinant Listeria monocytogenes improves breast cancer vaccine efficacy. Br J Cancer 111: 1945-54

 

Acknowledgements:

This work was supported by grants OPP38614 and OPP1033104 from the Bill and Melinda Gates Foundation (Collaboration for AIDS Vaccine Discovery – CAVD). Portions of this work were also supported by grants from the NIH/NIAID (RO1 AI45889 and RO1 AI093649 to SAP; RO1 AI070258 to MT; PO1 AI063537 to SAP, WRJ and JC; R01 AI091987 to WY). Flow cytometry resources were supported by the Einstein Cancer Center (P30 CA013330) and the Einstein Center for AIDS Research (P30 AI051519). GSB acknowledges support from a Personal Research Chair from Mr. James Bardrick, a Royal Society Wolfson Research Merit Award, a former Lister Institute-Jenner Research Fellowship, the Medical Research Council and The Wellcome Trust (084923/B/08/Z). LJC is a Pew Latin American Fellow.

 

Contact:

Steven A. Porcelli, MD

Professor and Chair, Department of Microbiology and Immunology

Murray and Evelyne Weinstock Professor

Albert Einstein College of Medicine

Room 416 Forchheimer Building

1300 Morris Park Avenue

Bronx, NY 10461  USA

 

 

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