Elsevier

Vaccine

Volume 31, Issue 49, 2 December 2013, Pages 5806-5813
Vaccine

Candida skin test reagent as a novel adjuvant for a human papillomavirus peptide-based therapeutic vaccine

https://doi.org/10.1016/j.vaccine.2013.10.014Get rights and content

Highlights

  • The vaccine antigens are 4 human papillomavirus type 16 E6 peptides.

  • Candida skin test reagent is the vaccine adjuvant.

  • The partial Langerhans cell maturation effects are due to the peptides.

  • Candida has the T-cell proliferative effect.

  • IL-12 is the most commonly expressed cytokine by the Langerhans cells.

Abstract

A vaccine adjuvant that can effectively promote cell-mediated immunity is currently not available. Because of the ability of a Candida skin test reagent injection to induce common wart regression, our group is using it as a novel adjuvant in a clinical trial of a peptide-based human papillomavirus therapeutic vaccine. The goal of this current study was to investigate the mechanisms of how Candida enhances the vaccine immune responses. Maturation effects on Langerhans cells, capacity to proliferate T-cells, expression of cytokines and pattern recognition receptors by Langerhans cells, and ability to induce Th1, Th2, and Th17 responses were investigated in healthy subjects. The vaccine, human papillomavirus peptides with Candida, demonstrated partial maturation effects on Langerhans cells indicated by significantly up-regulated CD40 (p = 0.00007) and CD80 (p < 0.00001) levels, and showed T-cell proliferative capacity (p < 0.00001) when presented by Langerhans cells in vitro. Interestingly, the maturation effects were due to the peptides while Candida was responsible for the T-cell proliferation. The cytokine profile (IL-1β, IL-6, IL-8, IL-10, IL-12p40, IL-23Ap19, IFN-γ and TNF-α) of Langerhans cells treated with the vaccine or Candida alone showed that IL-12p40 mRNA was most frequently induced, and IL-12p70 protein was detected in the supernatants. The presence of pattern recognition receptors known to associate with Candida albicans (DC-SIGN, dectin-1, dectin-2, galectin-3, mincle, mannose receptor, Toll-like receptors-1, 2, 4, 6 and 9) were demonstrated in all subjects. On the other hand, the induction of Th1 response demonstrated by IFN-γ secretion by CD4 cells stimulated with the vaccine or Candida pulsed Langerhans cells was demonstrated only in one subject. In summary, the Langerhans cell maturation effects of the vaccine were due to the peptides while the T-cell proliferative capacity was derived from Candida, and the most frequently induced cytokine was IL-12.

Introduction

The most widely used adjuvant in approved human vaccines is an alum-based adjuvant that has been shown to elicit a predominantly Th2 immune response [1]. Therefore, the alum-based adjuvant would be useful in a vaccine designed to boost antibody responses, but not for a vaccine designed to stimulate cellular immune responses. Since successful clearance of human papillomavirus (HPV) infection is believed to be induced by cell-mediated immunity [2], [3], an adjuvant that would promote such an immunity is necessary, but not available.

Our group and others have shown that serial intra-lesional injections of common warts with skin testing reagents such as Candida, mumps, and/or Trichophyton can induce regression not only of treated warts but also of distant untreated warts [4], [5], [6], [7], [8], [9]. In a Phase I clinical trial (NCT00569231), our group used Candin® (Allermed, San Diego, CA), a colorless extract of Candida albicans, to treat common warts. Resolution of treated warts occurred in 82% of the subjects, and anti-HPV T-cell responses were demonstrated [8]. Given that Candin is derived from C. albicans, it should contain numerous pathogen-associated molecular patterns (PAMPs). We hypothesized that Candin would be an effective vaccine adjuvant which would stimulate multiple pattern recognition receptors (PRRs) and induce innate as well as adaptive immunity.

Cervical cancer is almost always caused by high-risk HPV infection, and is the 2nd most common cancer among women in the world. Two very effective prophylactic HPV vaccines, Gardasil® (Merck, NJ, USA) and Cervarix® (GlaxoSmithKline, Middlesex, UK), are available, and they work by inducing high titers of neutralizing antibody [10], [11], [12]. However, they are not effective for women with pre-existing HPV infection [10], [12], [13]. Therefore, a therapeutic HPV vaccine that can be used for those already infected with HPV and/or have developed HPV-associated neoplasia is not available. Our group studied naturally induced immunity in women with HPV infection and/or cervical lesions, and have found that the ability to induce T-cell responses against E6, one of the oncoproteins of high-risk HPVs, is associated with HPV clearance and regression of cervical lesions [3], [14], [15]. Therefore, we designed an HPV therapeutic vaccine which consists of four HPV type 16 E6 peptides and Candin, and are conducting a Phase I clinical trial (NCT01653249).

In the current study, we examined the immune enhancing effects of Candin as a vaccine adjuvant. Surprisingly, the E6 peptides were responsible for the partial maturation of Langerhans cells (LCs) while Candin was responsible for the T-cell proliferative effects. The most commonly induced cytokine by the LCs was IL-12.

Section snippets

Generation of monocytes-derived LCs

Mononuclear cells were collected from healthy blood donors (n = 10) by apheresis (Key Biologics, LLC, Memphis, TN). The subjects were numbered in a chronological order. Peripheral blood mononuclear cells (PBMCs) were purified using the ficoll gradient centrifugation method. Monocytes were negatively isolated from PBMC using Monocyte Isolation Kit II (Miltenyi Biotec, Auburn, CA), and were converted to LCs using granulocyte-macrophage colony-stimulating factor, IL-4, and transforming growth factor

Phenotypic maturation of LCs

We evaluated the maturation effects of Candin, and/or “peptides” on LCs (Fig. 1, Fig. 2). For CD40, statistically significant increases in mean fluorescence intensity (MFI) were observed with LCs treated with zymosan (p < 0.00001), “peptides” (p = 0.00003) and Candin/“peptides” (p = 0.00007) compared to untreated LCs. In addition, MFIs of LCs treated with “peptides” and Candin/“peptides” were significantly higher than the MFI of LCs treated with Candin alone (p = 0.001 and 0.003 respectively). For

Discussion

“Adjuvant” is derived from a Latin word, adjuvare, and means to help or to enhance. An effective vaccine adjuvant should be able to promote a strong immune response against the vaccine antigen in terms of size and durability. Antigen presenting cells (APCs) play a critical role in the initiation of immune responses. One of the desired features of an adjuvant is the ability to enhance maturation of APCs and the consequent priming of effective T-cell responses. CD40 and CD80 have been

Acknowledgements

This study was supported by grants from NIH (R01 CA143130, UL1TR000039, and P20GM103625), and the National Natural Science Foundation of China (No. 81101989). Author contributions: X.W.: Designed the study; acquired, analyzed and interpreted data; wrote and approved the manuscript. H.N.C: Trained X.W. in experimental methods; acquired, and analyzed data; edited and approved the manuscript. U.N.: Conceived and designed the study; analyzed and interpreted data; edited and approved the manuscript.

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    Present address: Department of Pediatrics, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.

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