Michael D. Macklin

Antibody responses to DNA vaccination of horses using the influenza virus hemagglutinin gene

Equine influenza virus infection remains one of the most important infectious diseases of the horse, yet current vaccines offer only limited protection. The equine immune response to natural influenza virus infection results in long-term protective immunity, and is characterized by mucosal IgA and serum IgGa and IgGb antibody responses. DNA vaccination offers a radical alternative to conventional vaccines, with the potential to generate the same protective immune responses seen following viral infection. Antigen-specific antibody isotype responses in serum and mucosal secretions were studied in ponies following particle-mediated delivery of hemagglutinin (HA)-DNA vaccination on three occasions at approximately 63-day intervals. One group of four ponies were vaccinated at skin and mucosal sites and the another group were vaccinated at skin sites only. All ponies were subjected to a challenge infection 30 days after the third vaccination. Skin and mucosal vaccination provided complete protection from clinical signs of infection, while skin vaccination provided partial protection; DNA vaccination provided partial protection from viral shedding. DNA vaccination generated only IgGa and IgGb antibody responses, which occurred with a higher frequency in the skin and mucosa vaccinated ponies. No mucosal IgA response was generated prior to challenge infection and IgA responses were only detected in those ponies which shed virus postchallenge. These results demonstrate that HA-DNA vaccination induces IgG(a) and IgG(b) antibody responses which are associated with protection in the absence of mucosal IgA responses. In addition, additional DNA vaccinations of mucosal sites increased protection and the frequency of seroconversion in ponies.

A Phase I Study of Immunization Using Particle-Mediated Epidermal Delivery of Genes for gp100 and GM-CSF into Uninvolved Skin of Melanoma Patients

Purpose: We examined in vivo particle-mediated epidermal delivery (PMED) of cDNAs for gp100 and granulocyte macrophage colony-stimulating factor (GM-CSF) into uninvolved skin of melanoma patients. The aims of this phase I study were to assess the safety and immunologic effects of PMED of these genes in melanoma patients. Experimental Design: Two treatment groups of six patients each were evaluated. Group I received PMED with cDNA for gp100, and group II received PMED with cDNA for GM-CSF followed by PMED for gp100 at the same site. One vaccine site per treatment cycle was biopsied and divided for protein extraction and sectioning to assess transgene expression, gold-bead penetration, and dendritic cell infiltration. Exploratory immunologic monitoring of HLA-A2+ patients included flow cytometric analyses of peripheral blood lymphocytes and evaluation of delayed-type hypersensitivity to gp100 peptide. Results: Local toxicity in both groups was mild and resolved within 2 weeks. No systemic toxicity could be attributed to the vaccines. Monitoring for autoimmunity showed no induction of pathologic autoantibodies. GM-CSF transgene expression in vaccinated skin sites was detected. GM-CSF and gp100 PMED yielded a greater infiltration of dendritic cells into vaccine sites than did gp100 PMED only. Exploratory immunologic monitoring suggested modest activation of an antimelanoma response. Conclusions: PMED with cDNAs for gp100 alone or in combination with GM-CSF is well tolerated by patients with melanoma. Moreover, pathologic autoimmunity was not shown. This technique yields biologically active transgene expression in normal human skin. Although modest immune responses were observed, additional investigation is needed to determine how to best utilize PMED to induce antimelanoma immune responses.

Immunogenicity and efficacy of baculovirus-expressed and DNA-based equine influenza virus hemagglutinin vaccines in mice

Two fundamentally different approaches to vaccination of BALB/c mice with the hemagglutinin (HA) of A/Equine/Kentucky/1/81 (H3N8) (Eq/KY) were evaluated, that is, administration of HA protein vs administration of HA-encoding DNA. Each vaccine was tested for its immunogenicity and ability to provide protection from homologous virus challenge. HA protein was synthesized in vitro by infection of Sf21 insect cells with a recombinant baculovirus. Intranasal administration of this vaccine induced virus-specific antibodies, as measured by enzyme-linked immunosorbent assay (ELISA), but did not induce virus neutralizing (VN) antibodies. This route of administration provided partial protection from virus challenge, but interestingly, this protection was completely abrogated, rather than enhanced, by co-administration of 10 micrograms of cholera holotoxin. As a second approach, mice were directly vaccinated in vivo by Accell gene gun delivery of plasmid DNA encoding the Eq/KY HA gene. This approach induced VN antibodies as well as virus-specific ELISA antibodies. When two doses of DNA vaccine were administered 3 weeks apart, mice were not protected from challenge, although they cleared the infection more rapidly than control mice. However, when the second DNA vaccination was delayed until 9 weeks after the first, 9 out of 10 vaccinated mice were completely protected. These results indicate that the time between initial and booster DNA vaccinations may be an important variable in determining DNA vaccination efficacy.

Mucosal co-administration of cholera toxin and influenza virus hemagglutinin-DNA in ponies generates a local IgA response

We have previously demonstrated that equine influenza virus hemagglutinin (HA) DNA vaccination protects ponies from challenge infection, and induces protective IgGa and IgGb responses. However, this approach does not induce a nasal IgA response. The objective of this study was to examine the value of cholera toxin (CT) administration as an adjuvant for intranasal HA DNA vaccination, and to measure protection 3 months after DNA vaccination. After an immunogenic dose of CT was determined, ponies were immunized on two occasions by intranasal administration of HA DNA and cholera toxin, or HA DNA alone. Ponies in both groups received two additional HA DNA particle mediated vaccinations at skin and mucosal sites. Antibody responses, and protection from challenge infection 3 months after the last vaccination were studied and compared to an influenza virus naive control group. Ponies in both vaccination groups produced virus-specific IgG antibodies in serum following vaccination and showed clinical protection from challenge infection 3 months after the last vaccination. Co-administration of CT plus HA DNA vaccination induced a nasal IgA response. In addition, analysis of antibody titers in nasal secretions indicated local production of nasal IgGb, which was amplified by CT administration.

Melanoma-induced suppression of tumor antigen-specific T cell expansion is comparable to suppression of global T cell expansion

We have observed that in vivo interaction between melanoma and resting T cells promotes suppression of antigen-driven proliferative T cell expansion. We hypothesized that this suppression would affect tumor antigen-specific T cell populations more potently than tumor-unrelated T cell populations. A B16F10 cell line was stably transfected to express low levels of the lymphocytic choriomeningitis virus (LCMV) glycoprotein GP33 (B16GP33). Mice bearing B16F10 or B16GP33 tumors were infected with LCMV, and proliferative expansion of LCMV epitope-specific T cell populations was quantified. In vitro and in vivo assays confirmed low levels of antigenic GP33 expression by B16GP33 tumors. Suppressed expansion of GP33-specific T cells was equivalent between mice bearing B16F10 and B16GP33 tumors. These observations suggest that the ability of growing melanoma tumors to impair antigen-driven proliferative expansion of activated T cells is global and not antigen-specific, and provide further insight into the influence of cancer on activated T cell homeostasis.

Clonal expansions of 6-thioguanine resistant T lymphocytes in the blood and tumor of melanoma patients.

The identification of specific lymphocyte populations that mediate tumor immune responses is required for elucidating the mechanisms underlying these responses and facilitating therapeutic interventions in humans with cancer. To this end, mutant hypoxanthine‐guanine phosphoribosyltransferase (HPRT) deficient (HPRT‐) T‐cells were used as probes to detect T‐cell clonal amplifications and trafficking in vivo in patients with advanced melanoma. Mutant T‐cells from peripheral blood were obtained as clonal isolates or in mass cultures in the presence of 6‐thioguanine (TG) selection and from tumor‐bearing lymph nodes (LNs) or metastatic melanoma tissues by TG‐selected mass cultures. Nonmutant (wild‐type) cells were obtained from all sites by analogous means, but without TG selection. cDNA sequences of the T‐cell receptor (TCR) beta chains (TCR‐β), determined directly (clonal isolates) or following insertion into plasmids (mass cultures), were used as unambiguous biomarkers of in vivo clonality of mature T‐cell clones. Clonal amplifications, identified as repetitive TCR‐β V‐region, complementarity determining region 3 (CDR3), and J‐region gene sequences, were demonstrated at all sites studied, that is, peripheral blood, LNs, and metastatic tumors. Amplifications were significantly enriched among the mutant compared with the wild‐type T‐cell fractions. Importantly, T‐cell trafficking was manifested by identical TCR‐β cDNA sequences, including the hypervariable CDR3 motifs, being found in both blood and tissues in individual patients. The findings described herein indicate that the mutant T‐cell fractions from melanoma patients are enriched for proliferating T‐cells that infiltrate the tumor, making them candidates for investigations of potentially protective immunological responses. Environ. Mol. Mutagen., 2008. Published 2008 Wiley‐Liss, Inc.

Preparations for Particle-Mediated Gene Transfer Using the Accell® Gene Gun

Particle-mediated delivery involves coating materials onto the surface of dense sub-cellular sized (0.5-5 mm) particles and accelerating the particles to sufficient velocity to penetrate target cells. The technique was invented by Sanford and Wolf at Cornell University (1) to transfer DNA into intact plant cells (2), and was further developed into an effective process for producing genetically engineered crop plants by several groups (reviewed in 3). Subsequent work has shown that this method is generally applicable for transferring materials including DNA, RNA, proteins, peptides and pharmacological compounds into a wide variety of tissue and cell types in vivo, ex vivo, or in vitro (reviewed in 4).

In vivo 6-thioguanine-resistant T cells from melanoma patients have public TCR and share TCR beta amino acid sequences with melanoma-reactive T cells

In vivo hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient T cells (MT) from melanoma patients are enriched for T cells with in vivo clonal amplifications that traffic between blood and tumor tissues. Melanoma is thus a model cancer to test the hypothesis that in vivo MT from cancer patients can be used as immunological probes for immunogenic tumor antigens. MT were obtained by 6-thioguanine (TG) selection of lymphocytes from peripheral blood and tumor tissues, and wild-type T cells (WT) were obtained analogously without TG selection. cDNA sequences of the T cell receptor beta chains (TRB) were used as unambiguous biomarkers of in vivo clonality and as indicators of T cell specificity. Public TRB were identified in MT from the blood and tumor of different melanoma patients. Such public TRB were not found in normal control MT or WT. As an indicator of T cell specificity for melanoma, the >2600 MT and WT TRB, including the public TRB from melanoma patients, were compared to a literature-derived empirical database of >1270 TRB from melanoma-reactive T cells. Various degrees of similarity, ranging from 100% conservation to 3-amino acid motifs (3-mer), were found between both melanoma patient MT and WT TRBs and the empirical database. The frequency of 3-mer and 4-mer TRB matching to the empirical database was significantly higher in MT compared with WT in the tumor (p=0.0285 and p=0.006, respectively). In summary, in vivo MT from melanoma patients contain public TRB as well as T cells with specificity for characterized melanoma antigens. We conclude that in vivo MT merit study as novel probes for uncharacterized immunogenic antigens in melanoma and other malignancies.

Pilot study of safety and feasibility of DNA microseeding for treatment of spontaneous canine melanoma

Abstract Spontaneous canine malignant melanoma provides an excellent pre‐clinical model to study DNA vaccines for melanoma immunotherapy. A USDA‐approved xenogeneic human tyrosinase (huTYR) plasmid DNA vaccine delivered intramuscularly induces detectable immune responses and has clinical activity in some dogs with melanoma. The objective of this pilot study was to evaluate the feasibility, safety and immunogenicity of huTYR plasmid DNA administered to the skin via microseeding in dogs with spontaneous melanoma. DNA microseeding utilizes a modified tattooing device as an alternate and potentially more potent delivery method for DNA immunization. DNA was delivered to shaved inner thigh skin of six companion dogs with melanoma approximately every 14 days for a planned total of four vaccination time points. An anti‐huTYR ELISA was used to test pre‐ and post‐treatment sera. Biopsies of treated skin were obtained for detection of huTYR transgene expression. DNA microseeding was well tolerated with no significant toxicity detected beyond local site irritation, and there were no signs of autoimmunity. huTYR‐expressing cells were observed in biopsies of huTYR DNA microseeding sites. Increased humoral anti‐huTYR antibodies were seen in two of five evaluable dogs following microseeding compared to baseline. DNA microseeding is well tolerated in companion dogs with melanoma. Further investigation is needed to determine if combining DNA microseeding with other immunotherapy regimens potentiates this delivery platform for cancer immunotherapy.

Abstract 2366: Pilot study of DNA microseeding to activate immune rejection of canine melanoma

Background: Canine malignant melanoma provides a model to study DNA vaccine delivery systems. A xenogeneic human tyrosinase (huTYR) DNA vaccine delivered by Biojector2000 received United States Department of Agriculture licensure when it appeared to prolong survival of dogs with melanoma compared to historical, stage-matched controls and to stimulate immune responses in some dogs. The current study evaluates toxicity, transgene expression, and antibody responses to huTYR in companion dogs with spontaneously developing melanoma following delivery of huTYR DNA to the skin via a modified tattoo device, a method termed DNA microseeding. Methods: Five companion dogs with melanoma were scheduled to receive huTYR DNA at two sites (Site A and Site B) on the inner thigh by DNA microseeding every 2 weeks for 4 administrations at a range of huTYR DNA doses; 2 dogs (50 μg [Site A] and 100 μg [Site B]); 2 dogs (200 μg [Site A] and 400 μg [Site B]); and 1 dog (83 μg [Site A] and 83 μg commercial huTYR plasmid [Site B]). Vaccine site biopsies were obtained to determine transgene expression 24 hours after the 1st and 3rd vaccination time-points, and 48 hours after the 2nd and 4th vaccination time-points. Blood samples were obtained at baseline and 2 weeks after the 2nd, 3rd, and 4th vaccinations to quantify TYR-specific antibodies via indirect ELISA. Results: No toxicity, beyond local site irritation, related to the vaccine administration was observed. The 3 dogs with known disease at study entry received 3, 1 and 4 treatments before discontinuation of treatment due to progressive disease. The 2 dogs without evidence of disease at study entry received all 4 planned treatments and remained without evidence for recurrence after treatment for the duration of the study (6 weeks). Only rare huTYR+ cells with macrophage-like morphology were observed in some vaccine site biopsies. A significant increase in anti-huTYR IgG was detected at Day 57 compared to pre-treatment in 2 of the 4 evaluable dogs (p = 0.03 Wilcoxon Mann Whitney), and these were the 2 dogs without evidence of disease at study entry. Baseline anti-huTYR IgG levels were also greater compared to IgG levels against an irrelevant control antigen. Conclusions: While microseeding of huTYR plasmid DNA resulted in only rare transgene expression at DNA doses up to 400 μg, humoral responses against huTYR were substantially boosted in 2 of 4 evaluable dogs. Additional testing is needed to determine if DNA microseeding enhances huTYR DNA vaccine immunogenicity compared to Biojector delivery. Citation Format: Cindy L. Zuleger, Chulhi Kang, Erik A. Ranheim, Ilene Kurzman, Michael D. Macklin, Michael A. Newton, David M. Vail, Jedd D. Wolchok, Elof Eriksson, Mark R. Albertini. Pilot study of DNA microseeding to activate immune rejection of canine melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2366.