Progress Toward Development of Photodynamic Vaccination Against Infectious/Malignant Diseases and Photodynamic Mosquitocides

Invited Paper

Progress toward development of photodynamic vaccination against infectious/malignant diseases and photodynamic mosquitocides

Kwang Poo Chang (張光樸), Bala K Kolli Department of Microbiology/Immunology, Chicago Medical School/RFUMS, N Chicago, IL 60064, USA

Chia-Kwung Fan (范家堃) Department of Molecular Parasitology and Tropical Diseases, College of Medicine Taipei Medical University, Taipei, Taiwan

Dennis KP Ng (吳基培), Clarence TT Wong (王搢珽) Department of Chemistry, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong

Laura Manna, Raffaele Corso School of Veterinary Medicine/Animal Productions, University of Naples, Federico II, Naples, Italy

Neng-Yao Shih, (施能耀) National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan

Robert Elliott, XP Jiang (江贤朋) Elliott Mastology Center, 541 Shadows Lane, Baton Rouge, LA 70806, USA

Shin-Hong Shiao (蕭信宏) Department of Parasitology, College of Medicine, National Taiwan University, Taipei, Taiwan

Guo-Liang Fu (傅国樑) GeneFirst Ltd, E5 Culham Science Centre, Abingdon, Oxfordshire OX14 3DB, United Kingdom

ABSTRACT

Photodynamic therapy (PDT) uses photosensitizers (PS) that are excited with light to generate ROS in the presence of oxygen for treating various diseases. PS also has the potential use as photodynamic insecticides (PDI) and for light-inactivation of Leishmania for photodynamic vaccination (PDV). PDT-inactivated Leishmania are non-viable, but remain immunologically competent as whole-cell vaccines against leishmaniasis, and as a universal carrier for delivery of add-on vaccines against other infectious and malignant diseases. We have screened novel PS, including Zn- and Si-phthalocyanines (PC) for differential PDT activities against Leishmania, insect and mammalian cells in vitro to assess their PDI and PDV potential. Here, Zn-PC were conjugated with various functional groups. The conjugates were examined for uptake by cells as a prerequisite for their susceptibility to light-inactivation. PDT sensitivity was found to vary with cell types and PS used. PDI potential of several PS was demonstrated by their mosquito larvicidal PDT activities

Proc. of SPIE Vol. 10479 1047912-1 in vitro. PDT-inactivated Leishmania were stored frozen for PDV in several ongoing studies: [1] Open label trial with 20 sick dogs for immunotherapy of canine leishmaniasis after chemotherapy in Naples, Italy. Clinical follow-up for >3 years indicate that the PDV prolongs their survival; [2] PDV of murine models with a human lung cancer vaccine showed dramatic tumor suppression; [3] Open label trial of multiple PDV via compassionate access to 4 advanced cancer patients showed no clinically adverse effects. Two subjects remain alive. Genetic modifications of Leishmania are underway to further enhance their safety and efficacy for PDV by installation of activable mechanisms for self-destruction and spontaneous light-emission.

Keyword list: Photosensitizer, phthalocyanine, porphyrin, photo-inactivation, singlet oxygen, Leishmania, vaccine carrier, vaccination, immunotherapy, canine leishmaniasis, lung cancer, mouse tumor model, compassionate trial, vaccine safety.

Proc. of SPIE Vol. 10479 1047912-2 1. Photodynamic vaccination and photodynamic insecticides (KP Chang, CK Fan, Bala K. Kolli)

Photodynamic vaccination (PDV) and photodynamic insecticides (PDI) have been recently described and reviewed3.

Photodynamic vaccination (PDV) is referred to inactivation of microorganisms, i.e. Leishmania via the principle of photodynamic therapy (PDT) with photosensitizer (PS) for use as vaccines and vaccine carriers. Leishmania are naturally endowed with several unique advantages to serve in these capacities2,4: [1] Leishmania are expected to target their natural and add-on vaccines to antigen-presenting cells (APC) – a desirable destination for vaccination to elicit lasting cell-mediated immunity. This is the case because Leishmania are known to infect dendritic cells (DC) and live in the endosome-lysosome vacuolar systems of macrophages as their exclusive host cells in natural infection; [2] The surface of Leishmania is coated with lipid- and protein-glyconjugates, responsible not only for homing vaccines to APC but also for protection of the vaccine loads they carry from degradation by the lytic factors abundant in the mammalian body fluids; [3] Effective adjuvanticity in addition to the presence natural vaccines is a hallmark of Leishmania, judging from the outcome of its natural infection. Life-long immunity invariably develops after spontaneous or chemotherapeutic cure of all Old World leishmaniasis. This is especially true for simple cutaneous leishmaniasis, which is mostly a rather innocuous and self-healing skin infection. Cutaneous Leishmania are thus expected to offer a very favorable immunological environment as carrier for transgenically add-on vaccines against other infectious and malignant diseases. PS-sensitization of such Leishmania for photo-inactivation has been under investigation by us to render them non-viable and thus non-disease-causing, but retain their favorable immunological properties for immuno-prophylaxis and –therapy as whole-cell vaccines against leishmaniasis and as carrier for add-on vaccines against other diseases.

There are two ways to accomplish PS-sensitization of Leishmania for photo-inactivation. One way is to exploit the genetic defect of such trypanosomatid protozoon parasites in heme biosynthesis. Leishmania spp. were genetically transfected with the mammalian cDNAs to express the 2nd and 3rd enzymes in this pathway7,12. The transfectants produce endogenous PS in the form of uroporphyrin I (URO) when exposed to delta-aminolevulinate (ALA), the product of the 1st enzyme in the classic pathway of heme biosynthesis. The absence of the downstream URO utilizing enzyme in Leishmania results in the cytosolic accumulation of URO. These uroporphyic cells are thus extremely light-sensitive. The photo-inactivation of these cells is manifested by a rapid loss of flagellar motility, but not their structural integrity before eventual disintegration. These events are consistent with light excitation of URO for generation of singlet oxygen 1 5 ( O2) , which apparently oxidatively inactivate cytosolic macromolecules in the vicinity, but are too short-lived to cross the plasma membrane to damage the cell surface glycol-conjugates. Another way to PS-sensitize Leishmania is to expose them to exogenous PS for uptake for cellular accumulation - a prerequisite for effective photo-inactivation. Ineffective or less effective by this approach are thus uncharged, water-soluble URO, which is not taken up by cells, including Leishmania. Cationic phthalocyanines (PC) were seen to PS-sensitize Leishmania via membrane association, e.g. Al-PC HCl9, mitochondrion-targeting, e.g. pyridyloxy Si-PC8 and endocytic uptake into endosomes, e.g. Si-PC with axial rods8 1 1 or Amino-PC . All PC are excitable by red light to generate O2, producing similar cellular events of photo-inactivation, as seen after exposure of uroporphyric Leishmania to longwave UV.

PDT-inactivation of Leishmania proved safe and effective for prophylaxis against leishmaniasis and for delivery of surrogate vaccines to APC for T-cell activation in experimental models. Single PS-sensitization and photo-inactivation of L. amazonensis via uroporphyrinogenesis in vivo was shown to elicit T-cell transferable immunity against visceral leishmaniasis in Syrian hamster model11. Double PC/URO sensitization followed by photo-inactivation in vitro proved to be safe, as these PDT-treated Leishmania produced no growth in culture, no infection of macrophages in vitro and, above all, no lesion in mice10. Similar approach followed by in vivo photo-inactivation significantly delayed the onset of lesion development and markedly reduced the parasite loads in a highly susceptible BALB/c mouse model for cutaneous leishmaniasis (Submitted). In addition, Leishmania transgenically modified to express OVA was shown, after PDT- inactivation, to retain the capacity of delivering this T-cell antigen to both DC and macrophages for processing and presentation to activate OVA-epitope-specific CD8 T cells in vitro8.

Proc. of SPIE Vol. 10479 1047912-3 For developing photodynamic insecticides (PDI), we have begun to screen both novel PC and porphyrin derivatives for PDT activities in vitro against cell lines from different insects (generously provided by Dr. Cindy Goodman, USDA, RES, Columbia, MO). Preliminary observations showed differential PDT-sensitivities of cells from different insects, raising the hope of finding PS to discriminate harmful from beneficial insects for developing sun-light activated PDI. Much attention has been devoted to the cells from Aedes mosquito on account of its role as a vector for transmission of serious epidemic viral diseases of Dengue fever and Zika fever. Some novel PC examined showed effective in vitro PDT against these cells in nM concentrations, being more effective than the classic PS, like Rose bengal.

On account of the promising outcome from the aforementioned laboratory work, collaborative studies have been undertaken with interested colleagues who have the expertise with PS or the subjects and/or laboratory models to initiate small scale clinical or experimental trials, viz. PC chemical synthesis in relation to biological activities, PDV immunotherapy of canine leishmaniasis and human lung cancer in experimental murine models, PDV safety evaluation in advanced cancer patients and Aedes larvicidal activities and molecular mechanisms. The work to be summarized in the subsequent sections has been accomplished by collaborators on their own in their respective field of expertise and in their laboratories or clinics. In that sense, each contribution is independent in its own right.

References

[1] Al-Qahtani A, Alkahtani S, Kolli B, Tripathi P, Dutta S, Al-Kahtane AA, Jiang XJ, Ng DK, Chang KP. “Aminophthalocyanine-Mediated Photodynamic Inactivation of Leishmania tropica,” Antimicrob Agents Chemother. 60(4):2003-2011 (2016).

[2] Chang, KP. In: Encyclopedia of Life Sciences, Leishmaniases. John Wiley & Sons, Ltd. http://www.els.net/WileyCDA/ElsArticle/refId-a0001954.html (2012); In: Neglected Tropical Diseases - South Asia, Overview of leishmaniasis with special emphasis on Indian kala-azar. Springer International Publ. AG. https://doi.org/10.1007/978-3-319-68493-2_1 (2017).

[3] Chang KP, Kolli BK; New Light Group. “New "light" for one-world approach toward safe and effective control of animal diseases and insect vectors from leishmaniac perspectives,” Parasit Vectors. 9(1):396 (2016).

[4] Chang KP, Reed SG, McGwire BS, Soong L. “Leishmania model for microbial virulence: the relevance of parasite multiplication and pathoantigenicity,” Acta Trop. 85(3):375-390 (2003).

[5] Dutta S, Kolli BK, Tang A, Sassa S, Chang KP. “Transgenic Leishmania model for delta-aminolevulinate-inducible monospecific uroporphyria: cytolytic phototoxicity initiated by singlet oxygen-mediated inactivation of proteins and its ablation by endosomal mobilization of cytosolic uroporphyrin,” Eukaryot Cell. 7(7):1146-1157 (2008).

[6] Dutta S, Chang C, Kolli BK, Sassa S, Yousef M, Showe M, Showe L, Chang KP. “Delta-aminolevulinate-induced host-parasite porphyric disparity for selective photolysis of transgenic Leishmania in the phagolysosomes of mononuclear phagocytes: a potential novel platform for vaccine delivery,” Eukaryot Cell. 11(4):430-441 (2012).

[7] Dutta S, Furuyama K, Sassa S, Chang KP. “Leishmania spp.: delta-aminolevulinate-inducible neogenesis of porphyria by genetic complementation of incomplete heme biosynthesis pathway,” Exp Parasitol. 118(4):629-636 (2008).

[8] Dutta S, Ongarora BG, Li H, Vicente Mda G, Kolli BK, Chang KP. “Intracellular targeting specificity of novel phthalocyanines assessed in a host-parasite model for developing potential photodynamic medicine,” PLoS One. 6(6):e20786 (2011).

[9] Dutta S, Ray D, Kolli BK, Chang KP. “Photodynamic sensitization of Leishmania amazonensis in both extracellular and intracellular stages with aluminum phthalocyanine chloride for photolysis in vitro”. Antimicrob Agents Chemother. 49(11):4474-4484 (2005).

Proc. of SPIE Vol. 10479 1047912-4

[10] Dutta S, Waki K, Chang KP. “Combinational sensitization of Leishmania with uroporphyrin and aluminum phthalocyanine synergistically enhances their photodynamic inactivation in vitro and in vivo,” Photochem Photobiol. 88(3):620-625 (2012).

[11] Kumari S, Samant M, Khare P, Misra P, Dutta S, Kolli BK, Sharma S, Chang KP, Dube A. “Photodynamic vaccination of hamsters with inducible suicidal mutants of Leishmania amazonensis elicits immunity against visceral leishmaniasis,” Eur J Immunol. 39(1):178-191 (2009).

[12] Sah JF, Ito H, Kolli BK, Peterson DA, Sassa S, Chang KP. “Genetic rescue of Leishmania deficiency in porphyrin biosynthesis creates mutants suitable for analysis of cellular events in uroporphyria and for photodynamic therapy,” J Biol Chem. 277(17):14902-14909 (2002).

Proc. of SPIE Vol. 10479 1047912-5 2. Synthesis and properties of novel phthalocyanines for photodynamic therapy (Dennis KP Ng, Clarence TT Wong)

Photodynamic therapy (PDT) is a clinically approved treatment modality against different types of diseases including cancers1. Compared to standard chemotherapy and surgery, PDT has recently gained considerable attention because of its high success rate, cost-effectiveness, less-invasive procedures, and less side effects. Besides cancers, PDT has also been explored for the treatment of localized infection2 and neurological disorders3. The key component in PDT is the photosensitizer, which can be activated by light and converts neighboring oxygen to various reactive oxygen species that cause the therapeutic effects. Among the various classes of photosensitizers being studied, phthalocyanines are of particular interest owing to their desirable characteristics4. Firstly, they are generally more physically and chemically stable compared to other photosensitizers. They can resist high temperature and strong acid and base treatments, which allow them to tolerate a wide range of conditions. In terms of photophysical properties, phthalocyanines exhibit high photostability with strong absorption in the near-infrared region ranging from 600–800 nm that allows deeper tissue penetration. Furthermore, phthalocyanines have a well-balanced fluorescence emission and singlet oxygen generation, which enable them to serve as good imaging agents as well as efficient photosensitizers. As a result, a number of phthalocyanine analogues, including CGP55847, Photosens®, and Photocyanine are currently in different stages of clinical trials for oncological indications5. In addition, the fluorescence emission and singlet oxygen generation can be controlled by various mechanisms such as self-quenching, photoinduced electron transfer, and excitation energy transfer. These properties facilitate the design of activatable phthalocyanine-based theranostic agents for multilevel controlled PDT6. Under specific stimulations such as mRNA, thiols, and acid, these phthalocyanines can be activated to perform PDT at a specific location of the body.

Over the past few decades, the advance in the synthetic chemistry of phthalocyanines has allowed versatile functionalization of the dyes. The synthesis usually involves a base-promoted cyclotetramerization of phthalonitriles, though post-modification after the formation of the phthalocyanine ring has also been reported. In this paper, the cellular uptake and PDT efficacy of a series of phthalocyanine-based photosensitizers against a range of mammalian, insert, and Leishmania cells are reported. Figure 1 shows the molecular structures of these compounds. Compounds 1-3 were prepared by copper-promoted azide-alkyne cycloaddition of our previously reported alkynyl phthalocyanine7 with the corresponding azido peptides. The first two peptides (VHLGYAT and GPIEDRPMG) have been reported to exhibit tumor-targeting property8,9 , while the last peptide (PKKKRKV) is a typical nuclear localization sequence10, of which the lysine-rich nature may promote the cellular uptake by microbial cells. With a view to enhancing the interactions between the peptides and the cell membranes, two additional analogues (4 and 5) were prepared which contain two peptide chains. The synthesis was also involved click chemistry as the key step. Apart from these peptide-conjugated phthalocyanines, we also conjugated phthalocyanines with monosaccharides, which are hydrophilic and biocompatible, and can bind to specific receptors. The mannose analogues 7 and 8 were prepared again by click reaction of the alkynyl phthalocyanine 6 with the corresponding sugars. All these compounds were purified by HPLC and characterized by high-resolution mass spectrometry.

Figure 1. Molecular structures phthalocyanine-based photosensitizers 1-8.

O O O O OO N N N N N N N N N N Zn N N Zn N N N N N N N O O N N O O N O O N N N

= - a - s- eu- - r- a- r- - CONH V l Hi L Gly Ty Al Th Gly COOH (1) = ------CONH Val His Leu Gly Tyr Ala Thr Gly COOH (4) = ------CONH Gly Pro Ile Glu Asp Arg Pro Met Gly COOH (2) ------= CH NHCO Val Lys Arg Lys Lys Lys Pro N Fmoc 5 2 ( ) = - a - s- r - s- s- s- ro- - moc CH2NHCO V l Ly A g Ly Ly Ly P N F (3)

Proc. of SPIE Vol. 10479 1047912-6 OH OH OH OH O O HO HO O N N N HO O O O NH N Zn N 2 O O R O O N = 7 8 N N N R (6) ( ) N N ( ) N N N

References

[1] Moghissi, K., Dixon, K. and Gibbins, S., “A surgical view of photodynamic therapy in oncology: A review,” Surg. J. 1(1): e1-e15 (2015).

[2] Morley, S., et al., “Phase IIa randomized, placebo-controlled study of antimicrobial photodynamic therapy in bacterially colonized, chronic leg ulcers and diabetic food ulcers: a new approach to antimicrobial therapy,” Br. J. Dermatol. 168(3): 617-624 (2013).

[3] Valiente-Gabioud, A. A., et al., “Phthalocyanine as molecular scaffolds to block disease-associated protein aggregation,” Acc. Chem. Res. 49(5): 801-808 (2016).

[4] Ng, D. K. P., “Phthalocyanine-based photosensitizers: more effective photodynamic therapy?,” Future Med. Chem. 6(18): 1991-1993 (2014).

[5] Jiang, Z., Shao, J., Yang, T., Wang, J. and Jia, L., “Pharmaceutical development, composition and quantitative analysis of phthalocyanine as the photosensitizer for cancer photodynamic therapy,” J. Pharm. Biomed. Anal. 87: 98-104 (2014).

[6] Wong, R. C. H., Lo, P. C. and Ng, D. K. P., “Stimuli responsive phthalocyanine-based fluorescent probes and photosensitizers,” Coord. Chem. Rev. In press (2017).

[7] Ke, M. R., Yeung, S. L., Fong, W. P., Ng, D. K. P. and Lo, P. C., “A phthalocyanine-peptide conjugate with high in vitro photodynamic activity and enhanced in vivo tumor-retention property,” Chem. Eur. J. 18(14): 4225-4233 (2012).

[8] Zhang, Y., et al., “Panning and identification of a colon tumor binding peptide from a phage display peptide library,” J. Biomol. Screen. 12(3): 429-435 (2007).

[9] Lee, Y. M., Lee, D., Kim, J., Park, H. and Kim, W. J., “RPM peptide conjugated bioreducible polyethylenimine targeting invasive colon cancer,” J. Control Release, 10(205): 172-180 (2015).

[10] Hodel, M, R., Corbett, A. H. and Hodel, A. E., “Dissection of a nuclear localization signal,” J. Biol. Chem. 276(2): 1317-1325 (2001).

Proc. of SPIE Vol. 10479 1047912-7 3. Immunotherapy of canine leishmaniasis by photodynamic vaccination (Laura Manna/Raffaele Corso)

Background. Visceral Leishmania spp. are protozoa belonging phylogenetically to the family of Trypanosomatidae in the order of Kinetoplastida. Visceral leishmaniasis (VL) is a sand fly-borne, often fatal disease that currently affects 12 million individuals. VL caused by opportunistic infection is of special concern among immunocompromised sub- population, including human immunodeficiency virus-positive patients1. Canine leishmaniasis (CanL) is similarly transmitted as a severe parasitic disease of stray and domestic dog populations with a wide distribution in all continents except Oceania. The etiological agent of CanL is Leishmania infantum (= L. chagasi). The clinical manifestations of the disease range from unapparent subclinical infections to a systemic disease, including weight loss, lymphadenopathy, hemorrhagic diarrhea, ocular lesions, and hyperthermia, frequently associated also with dermatological lesions. The infected dogs are the primary reservoir and play a central role in the transmission of human infantile VL, for example, in the Mediterranean basin. The prevalence and the incidence of CanL are expanding and there is a need for research to improve diagnosis, treatment, and prevention of this complex disease2.

A novel immunotherapy is being developed according to the Leishmania strategy of vaccine delivery3 against difficult- to-cure diseases, like CanVL in Italy. Several drugs used for chemotherapy of the disease are able to improve clinical signs temporarily or seemingly cure dogs clinically, but none of the available treatments reliably eliminates the infection4. The current clinical management of this disease there entails prolonged treatments of sick dogs for 30 days with a heavy daily dosage of toxic drugs (antimonials/miltefosine) followed by a daily maintenance dose of allopurinol for life. Studies have demonstrated that these drugs, alone or in combination, appear to cure most animals clinically but do not lead to a parasitological clearance5,6. Relapses of the disease frequently occur within the first year after chemotherapy4. Given below is the progress of the first clinical trial of immunotherapy to assess its safety and efficacy in symptomatic dogs naturally infected with L. infantum.

Methods. With institutional IRB-approval and dog owner’s consent, a direct observational open label trial was initiated for immunotherapy of 20 diseased dogs hospitalized in the Department of Veterinary Medicine and Animal Productions, University of Naples, Federico II. Diagnosis was based on clinical manifestations, seropositivity of anti-Leishmania antibodies by Immunofluorescence-antibody-test (IFAT test) and detection of Leishmania DNA for parasite loads in infected tissues by real-time PCR. Before treatment, dogs were subjected to a battery of laboratory tests to assess their clinical status based on previously established parameters7. Leishmania were also isolated in culture from lymph-node aspirates of CanL-positive dogs. Collection of samples for these and subsequent studies has been approved by IEC with dog owners’ consent.

Non-viable, but immunologically active whole-cell vaccines were prepared under sterile conditions in standard biosafety hood as follows. Cultured L. infantum promastigotes were harvested and resuspended in Hank’s Balanced Salt solution at 108 cells/ml. The cell suspension was amino-PC-sensitized in the dark overnight, washed once and photo-inactivated with red light (~600 nm wavelength) for 20-30 minutes until their flagellum became immobilized (~1-2 J/cm2). These whole-cell vaccine samples were stored in 1-2 ml Aliquots at -20 C before use for auto-immunotherapy.

The studied dogs were divided in two groups for immunotherapy: Group 1 consisted of 9 dogs, each immunized with thawed photo- inactivated Leishmania at 107 cells/0.1 ml; Group 2 included the remaining 11 dogs, each similarly immunized, but after chemotherapy by daily subcutaneous injections of meglumine antimoniate at 100 mg/kg/day and allopurinol at 10 mg/kg/day for 30 days. All dogs of both groups were subsequently treated with allopurinol alone at the indicated dosage thereafter. Efficacy of immunotherapy was assessed on the basis of parasite loads in all dogs by real- time PCR of lymph node aspirates for Leishmania-specific DNA, starting from day 0 and every three months thereafter for >3 years8. At the same time, all dogs were also assessed clinically for disease signs, hemato-biochemical profiles and anti-Leishmania antibodies by IFAT. A clinical score was thus obtained for each dog for the record.

Results. At the time of diagnosis before immunotherapy, all 20 dogs had clinical signs and clinico-pathological abnormalities characteristic of CanL with Leishmania loads, ranging from 103 to 104 and from 102 to 103 for group 1 and group 2, respectively. Our results based on clinical scores and parasite loads of the dogs indicated that the immunotherapy improved the prognosis of both groups. After the initial 30 day-chemotherapy in group 2, immunotherapy was found to work better than using allopurinol alone by preventing relapse of the disease completely. One month after immunotherapy, 4/9 dogs in Group1 and 8/11 dogs in Group 2 showed a dramatic decrease of parasite

Proc. of SPIE Vol. 10479 1047912-8 loads in the lymph nodes. The parasite loads continued to decrease, dropping to 102/ml lymph node aspirates in 5/9 dogs in Group 1 by month 2 and to 10/ml in 10/11 dogs in group 2 by month 4. Both groups were also improved clinically during the first 4 months after immunotherapy: Clinical scores progressively reduced to 0 reading after 2 month in 6/9 and 7/11 of Group1 and Group 2, respectively. During the period from 4 to 8 months after starting the immunotherapy, 3 dogs in Group 1 and 2 dogs in Group 2 relapsed with progressive increases in clinical scores and parasite loads. They were excluded from further study beyond the stated periods. Among the dogs responsive to immunotherapy, parasite loads remained significantly lower than those of the starting point and zero reading of clinical scores for the subsequent 36 months.

Conclusions/Significance: When applied appropriately, the immunotherapy appears to boost the feeble immunity expected to develop after chemotherapy. Work is on-going to see if it is robust enough to clear the infection completely from immunized dogs, and to enroll additional dogs for both prophylactic and therapeutic trials.

[1] Desjeux P, Alvar J:. Leishmania/HIV co-infections: epidemiology in Europe. Ann. Tro.p Med. Parasitol. 97: 3-15 (2003).

[2] Dantas-Torres F, Solano-Gallego L, Baneth G, RibeiroV.M, de Paiva-Cavalcanti M, and Otranto D: Canine leishmaniosis in the old and New World: unveiled similarities and differences. Trends Parasitol. 2: 531-538 (1012).

[4] Manna L, Corso R, Galiero G, Cerrone A, Muzj P, Gravino AE. Long-term follow-up of dogs with leishmaniosis treated with meglumine antimoniate plus allopurinol versus miltefosine plus allopurinol. Parasit Vectors. 28;8:289 (2015).

[5] Mirò G, Cardoso L, Pennisi MG, Oliva G, Baneth G: Canine leishmaniosis--new concepts and insights on an expanding zoonosis: part two. Trends Parasitol 2008, 24: 371-377.

[6] Manna L, Vitale F, Reale S, Caracappa S, Pavone LM, Della Morte R, Cringoli G, Staiano N, Gravino AE: Comparison of different tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis. Vet. Parasitol. 125: 251-262 (2004).

[7] Manna L, Reale S, Vitale F, Picillo E, Pavone LM, Gravino AE. Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and allopurinol. Vet. J. 177(2):279-82 (2008).

[8] Manna L, Reale S, Viola E, Vitale F, Foglia ManzilloV, Pavone LM, Caracappa S, Gravino AE: Leishmania DNA load and cytokine expression levels in asymptomatic naturally infected dogs. Vet. Parasitol. 142:271-280 (2006).

Proc. of SPIE Vol. 10479 1047912-9 4. Immunotherapy of human and murine lung cancer by photodynamic vaccination in mouse models (Neng-Yao Shih)

Immunogenic tumor-associated antigens (TAAs) are antigens recognized by the immune system. They have been classified into numerous categories, depending on the nature of the antigens (protein or mucin) and their origin (tissue- specific, oncofetus- or virus-derived). There is however another large and diverse group of overexpressed self-antigens that is defined as any protein found in tumors at an increased level compared with normal healthy cells and tissues. Alpha-enolase (ENO1) is a member of this group, which was originally identified and subsequently found frequently by serological screening of malignant non-small cell lung cancer (NSCLC) patients1. Interestingly, upregulation of ENO1 expression has been reported as a common scenario during the progression of not only NSCLC but also many other cancers, e.g. pancreatic duct adenocarcinoma (PDAC), breast and hepatitis virus-related liver cancers.

ENO1 has been depicted increasingly in the literature as a moonlighting protein involved in different ways in the pathogenesis of many different diseases. It is known as a house-keeping glycolytic enzyme catalyzing the interconversion between 2-phosphoglycerate and phosphoenolpyruvate. Only recently was it detected on the cell surface and characterized as a plasminogen receptor, leading to the speculation that it may have a role in tissue invasion and tumor metastasis. An increase in the expression of cell-surface ENO1 was indeed found to markedly enhance the tumor cell invasive capability, thereby promoting tumor metastasis in NSCLC and PDAC.2,3 The invasiveness of these tumor cells is severely impaired by blocking the plasminogen-binding activity of ENO1 with specific antibodies. In NSCLC patients, a significant correlation was also noted between down-regulation of immune response to ENO1and stage- dependent escalation of disease progression4. Moreover, in animals xenografted with ENO1 overexpressing human tumor, the level of circulating anti-ENO1 antibodies was reduced not only via their sequestration by the grafted tumor cells but also by the generation of ENO1-specific regulatory T cells. These observations are highly consistent with the clinical outcomes of the NSCLC patients5. Together, all these data strongly indicate that ENO1 can be a therapeutic target for cancer immunotherapy by eliciting cell-mediated and/or humoral responses against this tumor-associated antigen.

We have begun to target ENO1 by active and adoptive immunotherapy of tumor in mouse models by photodynamic vaccination. Leishmania were episomally transfected with cDNA to express human ENO1 (E) or with empty plasmids to serve as the control (C). Western blot analysis of both showed that only E cells expressed hENO1. All transfectants were grown to stationary phase and resuspended in HBSS to 108 cells/ml. Both E and C cell suspensions were amino- PC1 sensitized overnight in the dark and photo-inactivated with red light for 30-60 min until Leishmania flagellum became immobilized (1-2 J/cm2). All samples were stored frozen until use. In active immunotherapy model, mice were inoculated with ML-1 murine lung cancer cells subcutaneously. After inoculation for 7-14 days before the emergence of tumor, mice in groups of five were immunized and boosted with murine rENO1 or GST controls, both with thawed C Leishmania cells (LS) as adjuvant. In adoptive immunotherapy model, two groups of five BALB/c mice were immunized and boosted with thawed Leishmania E cells and C cells as controls, respectively. Pooled splenic cells from both groups were adoptively transferred intrasplenically into respective groups of five SCID/NOD mice, which were xenograted with human lung cancer cells （CL1-5F4）7 days before the adoptive cell transfer. Tumor development was dramatically suppressed in mice in the experimental groups, but not in the control groups for both schemes of immunotherapy. Although the detailed immune mechanisms behind the effective tumor-suppressive immunity await further investigation, the findings of unprecedented antitumor efficacy induced by photodynamic vaccination support the idea that ENO1 is a promising target when used with Leishmania for future cancer immunotherapy.

References:

[1] Chang GC, Liu KJ, Hsieh CL, Hu TS, Charoenfuprasert S, Liu HK, Luh KT, Hsu LH, 1. Wu CW, Ting CC, Chen CY, Chen KC, Yang TY, Chou TY, Wang WH, Whang-Peng J, Shih NY. “Identification of alpha-enolase as an

Proc. of SPIE Vol. 10479 1047912-10 autoantigen in lung cancer: its overexpression is associated with clinical outcomes.” Clin Cancer Res. 12(19):5746- 54 (2006).

[2] Hsiao KC, Shih NY, Fang HL, Huang TS, Kuo CC, Chu PY, Hung YM, Chou SW, Yang YY, Chang GC, Liu KJ. “Surface α-enolase promotes extracellular matrix degradation and tumor metastasis and represents a new therapeutic target.” PLoS One. 8(7):e69354. (2013).

[3] Principe M, Ceruti P, Shih NY, Chattaragada MS, Rolla S, Conti L, Bestagno M, Zentilin L, Yang SH, Migliorini P, Cappello P, Burrone O, Novelli F. “Targeting of surface alpha-enolase inhibits the invasiveness of pancreatic cancer cells.” Oncotarget. 6(13):11098-113. (2015).

[4] Shih NY, Lai HL, Chang GC, Lin HC, Wu YC, Liu JM, Liu KJ, Tseng SW. “Anti-alpha-enolase autoantibodies are down-regulated in advanced cancer patients.” Jpn J Clin Oncol. 40(7):663-9. (2010).

[5] Hsiao KC, Shih NY, Chu PY, Hung YM, Liao JY, Chou SW, Yang YY, Chang GC, Liu KJ. Anti-α-enolase is a prognostic marker in postoperative lung cancer patients. Oncotarget. 6(33):35073-86. (2015).

Proc. of SPIE Vol. 10479 1047912-11 5. Safety and efficacy of photodynamic vaccination in advanced cancer patients (Robert L. Elliott, XP Jiang)

Until recently the role of the cancer patient’s immune system was ignored. In fact, the mainstream cancer therapies of chemotherapy and radiation are immunosuppressive. We realized in the late 1980s, the importance of a healthy immune system in the long term survival of the cancer patient by doing a Lymphocyte Blastogenesis Assay before and after therapy. This led to the development of a patented autologous breast cancer vaccine that was shown to improve disease specific survival of breast cancer patients with depressed lymphocyte immunity1,2. These results inspired us to explore and develop personalized vaccines for other solid tumors. We now use innovative cancer immunotherapy in combination with chemotherapy and radiation. The timing of the vaccine during the treatment cycle is very important3-5. Cancer immunotherapy has recently arrived and there are many new techniques promoting it to a proven treatment modality; we are encouraged with the progress, which in our opinion was late coming, as host immunity in clinical oncology was too long ignored.

We are thus interested in exploring new vaccine formulations, including the PS-sensitized and photo-inactivated Leishmania, which were episomally transfected by electroporation with cDNA to express various known tumor- associated antigens (see Ref. # 3 in the 1st section of this article). Expression of these antigens was verified by Western blot analysis of the transfectants under selective pressures. The transfectants were grown for 1-cycle in drug-free medium and resuspended in sterile HBSS to 108 cells/ml. The cell suspensions were first subjected to PS-sensitization, e.g. 1 uM amino-PC in the dark and then photo-inactivated with red light (1-2 J/cm2). Flagellar immobilization provided evidence for effective inactivation of Leishmania. The inactivated cells were washed and resuspended in sterile physiological buffered saline (~500 ug protein/ml). We referred to these samples as “Leishmania vaccine”, which arrived as frozen samples and stored at -20 C until use. Each patient received variable doses of “Leishmania vaccine” at >100 ul (50 ug proteins) at the Inguinal triangle together with our own cancer vaccine preparations, e.g. CEA: carcinoembryonic antigen; CA15-3: breast cancer antigen CA15-3; CA125: ovarian cancer antigen CA125; TAA: autologous breast cancer cells; HTA: allogeneic breast cancer cell line MCF-7.

Four patients have received the “Leismania vaccine”. Three patients had Stage IV breast cancer. All three had lung metastasis and one had also liver metastasis. The fourth patient had Stage IV lung cancer and was 5 years disease free from Stage IV anorectal cancer when diagnosed with the lung cancer.

The Stage IV breast cancer patients received a vaccine containing proteins CA-15-3 (breast cancer antigen), CEA (carcinoembryonic antigen), CA-125 (ovarian cancer antigen), HTA (allogeneic breast cancer cell line) (MCF-7), plus CpG DNA and the mammoglobin A in Leishmania vaccine. Leishmania volume was 0.3ml in total. The Stage IV lung cancer patient received lung HTA (CRL5883), CpG DNA and Leishmania expressing the following cancer vaccine candidates: mammoglobin A, MAGE-A12, and GAGE-1. All patients had the injection in the left femoral triangle intradermally. The number of times varied with each patient and the time interval varied according to the patient’s other treatment protocol. The same volume of the Leismania vaccine was used in each patient.

Patient one: 73 year old white female with Stage IV lung cancer March 2014. Received her vaccines on 4-7-14, 6-5-14, 11-11-14, 6-16-15 and 6-24-16. She is stable, still on treatment with the PD-1 checkpoint inhibitor OPDIVO. She is doing well and has not had any toxicity to the vaccine.

Patient two: A 65 year old white female that has had Stage III-IV breast cancer for 11 years. She has had lung metastasis for the last 5 years and a liver metastasis for 3 years. She received our vaccine with the Leismania vaccine on 7-25-13 and 11-22-13. Her disease was stable until recently and is still working. She is on Femara and IBRANCE and had another vaccine in late November 2016.

Proc. of SPIE Vol. 10479 1047912-12 Patient three: 66 year old white female with numerous lung metastases. She recurred after being disease-free for nearly 20 years. She could not tolerate standard chemotherapy, but received Cis-PT-TF complex IV and got the vaccine mix 10-22-13, 10-30-13, 2-4-14, 3-18-14, 4-28-14, 5-12-14 and 5-2-14. She expired late 2014 but the vaccine palliated her and made her feel better and there was no toxicity.

Patient four: 69 year old white female with lung and brain METS, no other therapy except some IV Cis-PT-TF complex. She received the vaccine mix on 8-11-14, 10-7-14, and 12-16-14. She experienced great palliation and had no toxicity. She expired sometime in early 2015.

These very preliminary studies revealed no adverse reactions of the patients to any of the vaccine preparations used.

References

[1] Elliott RL, Head JF. “Adjuvant breast cancer vaccine improves disease specific survival of breast cancer patients with depressed lymphocyte immunity.” Surg Oncol. 22(3):172-7 (2016). doi: 10.1016/j.suronc.2013.05.003. PMID: 23791552.

[2] Elliott RL. “Combination cancer immunotherapy "Expanding Paul Ehrlich's Magic Bullet Concept"”. Surg Oncol. 21(1):53-5 (2012). doi: 10.1016/j.suronc.2010.02.002. PMID: 20338747.

[3] Elliott RL, Head JF. “Complete resolution of malignant ascites in stage IV breast cancer by peritoneal drainage and innovative chemoimmunotherapy: a case report”. Cancer Biother Radiopharm. 21(2):138-45 (2006). PMID: 16706634.

[4] Jiang XP, Yang DC, Elliott RL, Head JF. “Vaccination with a mixed vaccine of autogenous and allogeneic breast cancer cells and tumor associated antigens CA15-3, CEA and CA125--results in immune and clinical responses in breast cancer patients”. Cancer Biother Radiopharm. 15(5):495-505 (2000). PMID: 11155821.

[5] Jiang XP, Yang DC, Elliott RL, Head JF. “Reduction in serum IL-6 after vacination of breast cancer patients with tumour-associated antigens is related to estrogen receptor status”. Cytokine. 12(5):458-65 (2000). PMID: 10857759.

Proc. of SPIE Vol. 10479 1047912-13 6. Novel mosquito larvicides using light-activated phthalocyanines and porphyrin derivatives (Shin-Hong Shiao)

Mosquitoes are significant insect vectors transmitting many serious infectious diseases, including the recent epidemics of global significance caused by Zika, Dengue and Chikungunya viruses. We have embarked on the investigation of molecular mechanisms crucial for the development of these insects1, 2 and their immunity to pathogens as their vector3-6. Understanding these mechanisms is expected to help us develop effective mosquito control strategies.

The insecticides in use for mosquito control are toxic and ineffective due to development of resistance. The new approach to reduce mosquito population by releasing genetically modified males to cause female infertility is still in experimental trials. Serious objection has been already raised by many against the use of such genetically modified organisms. A safe and effective alternative is photodynamic insecticides, which make use of food, drug, fabric and other dyes as photosensitizers for activation with sun light to generate insecticidal oxygen free radicals.

We have begun such investigation in parallel with the molecular approaches. Preliminary studies of laboratory reared Aedes albopictus led to the discovery of novel phthalocyanines (PC) and porphyrin derivatives (PPD) with light- activable mosquito larvicidal activities at nanomolar concentrations. This was instar- and light-exposure-time dependent. In general, the 2nd instar mosquito larvae were most susceptible. Some photosensitizers (PS) were also found effective for photo-dynamic inactivation of older larvae. By fluorescent microscopy of mosquito cells (C6/36 and other cell lines) exposed to effective PS, PPD appeared to be more diffused in the cytosol and PC more granular, reminiscent of endosomal localization, as seen in other cells. Exposure of effective PC- and PPD-loaded cells to their respective excitation wavelengths resulted in their inactivation and disintegration, consistent with the cytotoxicity known to occur with the expected generation of singlet oxygen and other ROS. To gain insight in that direction, photodynamically inactivated larvae were subjected to proteomic and transcriptomic analyses by mass spectrometry and quantitative Real- Time PCR, respectively. Preliminary results suggest that certain inhibitors of apoptosis were upregulated by the treatment.

The preliminary findings warrant further investigation of the molecular mechanisms in conjunction with insect development and immunity toward the development of safe and effective integrated strategies to control mosquitoes and other harmful insects.

[1] Weng SC, Shiao SH. Frizzled 2 is a key component in the regulation of TOR signaling-mediated egg production in the mosquito Aedes aegypti. Insect Biochem Mol Biol. 61:17-24 (2015).

[2] Shiao SH, Hansen IA, Zhu J, Sieglaff DH, Raikhel AS. Juvenile hormone connects larval nutrition with target of rapamycin signaling in the mosquito Aedes aegypti. J Insect Physiol. 54(1):231-9 (2008).

[3] Shiao SH, Whitten MM, Zachary D, Hoffmann JA, Levashina EA. Fz2 and cdc42 mediate melanization and actin polymerization but are dispensable for Plasmodium killing in the mosquito midgut. PLoS Pathog. 2(12):e133 (2006).

[4] Whitten MM, Shiao SH, Levashina EA. Mosquito midguts and malaria: cell biology, compartmentalization and immunology. Parasite Immunol. 28(4):121-30 (2006).

[5] Blandin S, Shiao SH, Moita LF, Janse CJ, Waters AP, Kafatos FC, Levashina EA. Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae. Cell. 116(5):661-70 (2004).

[6] Shiao SH, Higgs S, Adelman Z, Christensen BM, Liu SH, Chen CC. Effect of prophenoloxidase expression knockout on the melanization of microfilariae in the mosquito Armigeres subalbatus. Insect Mol Biol. 10(4):315-21 (2001).

Proc. of SPIE Vol. 10479 1047912-14 7. Molecular genetic modifications of Leishmania to improve their safety and efficacy for use in photodynamic vaccination (Guoliang Fu, Bala K. Kolli, KP Chang)

Work-in-progress includes two different projects: (1) Transfection of uroporphyrinogenic Leishmania to express luciferases for self-light emission. Both fire fly and Renella luciferases were successfully installed epigenetically with enzyme activities. Lysates of these transfectants emit luminescence on addition of their respective luciferins. In addition, uroporphyric Leishmania are inactivated and undergo cytolysis when transfected with Renella luciferase followed by exposure to cell penetrating luciferins. This is expected, since the wavelength of this luminescence coincides with that to excite uroporphyrin. The resulting emission wavelength is ~600 nm, which is further expected to excite phthalocyanines when Leishmania is further loaded with these photosensitizers. Work is also underway to install bacterial luciferase- luciferin cassette, consisting of five different genes. If successfully expressed, PDV can be simplified by using ALA alone for uroporphrinogensis followed by automated light emission from the gene products of the installed bacterial cassette. Additional PC-sensitization is thus expected to put Leishmania in double jeopardy of URO-PC combination, thereby ensuring their complete inactivation and release of vaccines in APC. (2) Episomal and chromosomal installation of suicidal genes for expression under the teto/tets regulation with oxycyclines for expressing T7 RNA polymerase. If successful, such transfectants alone will be able to deliver add-on vaccines for PDV and can be an additional safety mechanism when used in combination with uroporphyringenic Leishmania.

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