Joshua O. Marshak

Merkel Cell Polyomavirus-Specific CD8+ and CD4+ T-cell Responses Identified in Merkel Cell Carcinomas and Blood

Purpose: Merkel cell polyomavirus (MCPyV) is prevalent in the general population, integrates into most Merkel cell carcinomas (MCC), and encodes oncoproteins required for MCC tumor growth. We sought to characterize T-cell responses directed against viral proteins that drive this cancer as a step toward immunotherapy. Experimental Design: Intracellular cytokine cytometry, IFN-γ enzyme-linked immunospot (ELISPOT) assay, and a novel HLA-A*2402–restricted MCPyV tetramer were used to identify and characterize T-cell responses against MCPyV oncoproteins in tumors and blood of MCC patients and control subjects. Results: We isolated virus-reactive CD8 or CD4 T cells from MCPyV-positive MCC tumors (2 of 6) but not from virus-negative tumors (0 of 4). MCPyV-specific T-cell responses were also detected in the blood of MCC patients (14 of 27) and control subjects (5 of 13). These T cells recognized a broad range of peptides derived from capsid proteins (2 epitopes) and oncoproteins (24 epitopes). HLA-A*2402–restricted MCPyV oncoprotein processing and presentation by mammalian cells led to CD8-mediated cytotoxicity. Virus-specific CD8 T cells were markedly enriched among tumor infiltrating lymphocytes as compared with blood, implying intact T-cell trafficking into the tumor. Although tetramer-positive CD8 T cells were detected in the blood of 2 of 5 HLA-matched MCC patients, these cells failed to produce IFN-γ when challenged ex vivo with peptide. Conclusions: Our findings suggest that MCC tumors often develop despite the presence of T cells specific for MCPyV T-Ag oncoproteins. The identified epitopes may be candidates for peptide-specific vaccines and tumor- or virus-specific adoptive immunotherapies to overcome immune evasion mechanisms in MCC patients. Clin Cancer Res; 17(21); 6671–80. ©2011 AACR.

Improved DNA vaccination by skin-targeted delivery using dry-coated densely-packed microprojection arrays

HSV-2-gD2 DNA vaccine was precisely delivered to immunologically sensitive regions of the skin epithelia using dry-coated microprojection arrays. These arrays delivered a vaccine payload to the epidermis and the upper dermis of mouse skin. Immunomicroscopy results showed that, in 43 ± 5% of microprojection delivery sites, the DNA vaccine was delivered to contact with professional antigen presenting cells in the epidermal layer. Associated with this efficient delivery of the vaccine into the vicinity of the professional antigen presenting cells, we achieved superior antibody responses and statistically equal protection rate against an HSV-2 virus challenge, when compared with the mice immunized with intramuscular injection using needle and syringe, but with less than 1/10th of the delivered antigen.

Cross-presentation and genome-wide screening reveal candidate T cells antigens for a herpes simplex virus type 1 vaccine

Herpes simplex virus type 1 (HSV-1) not only causes painful recurrent oral-labial infections, it can also cause permanent brain damage and blindness. There is currently no HSV-1 vaccine. An effective vaccine must stimulate coordinated T cell responses, but the large size of the genome and the low frequency of HSV-1-specific T cells have hampered the search for the most effective T cell antigens for inclusion in a candidate vaccine. We have now developed what we believe to be novel methods to efficiently generate a genome-wide map of the responsiveness of HSV-1-specific T cells, and demonstrate the applicability of these methods to a second complex microbe, vaccinia virus. We used cross-presentation and CD137 activation-based FACS to enrich for polyclonal CD8+ T effector T cells. The HSV-1 proteome was prepared in a flexible format for analyzing both CD8+ and CD4+ T cells from study participants. Scans with participant-specific panels of artificial APCs identified an oligospecific response in each individual. Parallel CD137-based CD4+ T cell research showed discrete oligospecific recognition of HSV-1 antigens. Unexpectedly, the two HSV-1 proteins not previously considered as vaccine candidates elicited both CD8+ and CD4+ T cell responses in most HSV-1-infected individuals. In this era of microbial genomics, our methods - also demonstrated in principle for vaccinia virus for both CD8+ and CD4+ T cells - should be broadly applicable to the selection of T cell antigens for inclusion in candidate vaccines for many pathogens.

DNA vaccine delivery by densely-packed and short microprojection arrays to skin protects against vaginal HSV-2 challenge

There is an unmet medical need for a prophylactic vaccine against herpes simplex virus (HSV). DNA vaccines and cutaneous vaccination have been tried for many applications, but few reports combine this vaccine composition and administration route. We compared DNA administration using the Nanopatch™, a solid microprojection device coated with vaccine comprised of thousands of short (110 μm) densly-packed projections (70 μm spacing), to standard intramuscular DNA vaccination in a mouse model of vaginal HSV-2 infection. A dose-response relationship was established for immunogenicity and survival in both vaccination routes. Appropriate doses administered by Nanopatch™ were highly immunogenic and enabled mouse survival. Vaginal HSV-2 DNA copy number day 1 post challenge correlated with survival, indicating that vaccine-elicited acquired immune responses can act quickly and locally. Solid, short, densely-packed arrays of microprojections applied to the skin are thus a promising route of administration for DNA vaccines.

A novel DNA vaccine technology conveying protection against a lethal herpes simplex viral challenge in mice.

While there are a number of licensed veterinary DNA vaccines, to date, none have been licensed for use in humans. Here, we demonstrate that a novel technology designed to enhance the immunogenicity of DNA vaccines protects against lethal herpes simplex virus 2 (HSV-2) challenge in a murine model. Polynucleotides were modified by use of a codon optimization algorithm designed to enhance immune responses, and the addition of an ubiquitin-encoding sequence to target the antigen to the proteasome for processing and to enhance cytotoxic T cell responses. We show that a mixture of these codon-optimized ubiquitinated and non-ubiquitinated constructs encoding the same viral envelope protein, glycoprotein D, induced both B and T cell responses, and could protect against lethal viral challenge and reduce ganglionic latency. The optimized vaccines, subcloned into a vector suitable for use in humans, also provided a high level of protection against the establishment of ganglionic latency, an important correlate of HSV reactivation and candidate endpoint for vaccines to proceed to clinical trials.

Vaxfectin-adjuvanted plasmid DNA vaccine improves protection and immunogenicity in a murine model of genital herpes infection

The herpes simplex type 2 (HSV-2) envelope glycoprotein (gD2) was evaluated as a potential antigen candidate for a plasmid DNA (pDNA)-based HSV-2 vaccine. The pDNA was formulated with Vaxfectin, a cationic lipid-based adjuvant, and tested in a murine HSV-2 lethal challenge model. gD2 was expressed as full-length (FL) and secreted (S) gD2 forms. A 0.1 µg pDNA dose was tested to distinguish treatment conditions for survival and a 100 µg pDNA dose was tested to distinguish treatment conditions for reduction in vaginal and latent HSV-2 copies. Vaxfectin-formulated gD2 pDNA significantly increased serum IgG titres and survival for both FL gD2 and S gD2 compared with gD2 pDNA alone. Mice immunized with FL gD2 formulated with Vaxfectin showed reduction in vaginal and dorsal root ganglia (DRG) HSV-2 copies. The stringency of this protection was further evaluated by testing Vaxfectin-formulated FL gD2 pDNA at a high 500 LD(50) inoculum. At this high viral challenge, the 0.1 µg dose of FL gD2 Vaxfectin-formulated pDNA yielded 80 % survival compared with no survival for FL gD2 pDNA alone. Vaxfectin-formulated FL gD2 pDNA, administered at a 100 µg pDNA dose, significantly reduced HSV-2 DNA copy number, compared with FL gD2 DNA alone. In addition, 40 % of mice vaccinated with adjuvanted FL pDNA had no detectable HSV-2 viral genomes in the DRG, whereas all mice vaccinated with gD2 pDNA alone were positive for HSV-2 viral genomes. These results show the potential contribution of Vaxfectin-gD2 pDNA to a future multivalent HSV-2 vaccine.

Serum and mucosal antibody responses to inactivated polio vaccine after sublingual immunization using a thermoresponsive gel delivery system

Administering vaccines directly to mucosal surfaces can induce both serum and mucosal immune responses. Mucosal responses may prevent establishment of initial infection at the port of entry and subsequent dissemination to other sites. The sublingual route is attractive for mucosal vaccination, but both a safe, potent adjuvant and a novel formulation are needed to achieve an adequate immune response. We report the use of a thermoresponsive gel (TRG) combined with a double mutant of a bacterial heat-labile toxin (dmLT) for sublingual immunization with a trivalent inactivated poliovirus vaccine (IPV) in mice. This TRG delivery system, which changes from aqueous solution to viscous gel upon contact with the mucosa at body temperature, helps to retain the formulation at the site of delivery and has functional adjuvant activity from the inclusion of dmLT. IPV was administered to mice either sublingually in the TRG delivery system or intramuscularly in phosphate-buffered saline. We measured poliovirus type-specific serum neutralizing antibodies as well as polio-specific serum Ig and IgA antibodies in serum, saliva, and fecal samples using enzyme-linked immunosorbent assays. Mice receiving sublingual vaccination via the TRG delivery system produced both mucosal and serum antibodies, including IgA. Intramuscularly immunized animals produced only serum neutralizing and binding Ig but no detectable IgA. This study provides proof of concept for sublingual immunization using the TRG delivery system, comprising a thermoresponsive gel and dmLT adjuvant.

Use of Acyclovir for Suppression of Human Immunodeficiency Virus Infection Is Not Associated with Genotypic Evidence of Herpes Simplex Virus Type 2 Resistance to Acyclovir: Analysis of Specimens from Three Phase III Trials

ABSTRACT Herpes simplex virus type 2 (HSV-2) is the most common cause of genital ulcer disease and is a cofactor for HIV-1 acquisition and transmission. We analyzed specimens from three separate phase III trials of acyclovir (ACV) for prevention of HIV-1 acquisition and transmission to determine if failure of ACV to interrupt HIV acquisition and transmission was associated with genotypic ACV resistance. Acyclovir (400 mg twice daily) or placebo was provided to HSV-2-infected persons at risk of HIV-1 infection in the Mwanza and HPTN 039 trials and to persons dually infected with HSV-2 and HIV-1 who had an HIV-negative partner in the Partners in Prevention study. We extracted HSV DNA from genital ulcer swabs or cervicovaginal lavage fluids from 68 samples obtained from 64 participants randomized to ACV and sequenced the HSV-2 UL23 gene encoding thymidine kinase. The UL23 sequences were compared with published and unpublished data. Variants were observed in 38/1,128 (3.4%) nucleotide positions in the UL23 open reading frame, with 58% of these encoding amino acid changes. No deletions, insertions, or mutations known to be associated with resistance were detected. Thirty-one of the variants (81.5%) are newly reported, 15 of which code for amino acid changes. Overall, UL23 is highly polymorphic compared to other loci in HSV-2, but no drug resistance mutations were detected that could explain the failure to reduce HIV incidence or to prevent HIV-1 transmission in these studies.Herpes simplex virus type 2 (HSV-2) is the most common cause of genital ulcer disease and is a cofactor for HIV-1 acquisition and transmission. We analyzed specimens from three separate phase III trials of acyclovir (ACV) for prevention of HIV-1 acquisition and transmission to determine if failure of ACV to interrupt HIV acquisition and transmission was associated with genotypic ACV resistance. Acyclovir (400 mg twice daily) or placebo was provided to HSV-2-infected persons at risk of HIV-1 infection in the Mwanza and HPTN 039 trials and to persons dually infected with HSV-2 and HIV-1 who had an HIV-negative partner in the Partners in Prevention study. We extracted HSV DNA from genital ulcer swabs or cervicovaginal lavage fluids from 68 samples obtained from 64 participants randomized to ACV and sequenced the HSV-2 UL23 gene encoding thymidine kinase. The UL23 sequences were compared with published and unpublished data. Variants were observed in 38/1,128 (3.4%) nucleotide positions in the UL23 open reading frame, with 58% of these encoding amino acid changes. No deletions, insertions, or mutations known to be associated with resistance were detected. Thirty-one of the variants (81.5%) are newly reported, 15 of which code for amino acid changes. Overall, UL23 is highly polymorphic compared to other loci in HSV-2, but no drug resistance mutations were detected that could explain the failure to reduce HIV incidence or to prevent HIV-1 transmission in these studies.

Acyclovir suppression for HIV prevention is not associated with genotypic evidence of acyclovir resistance to HSV-2: Analysis of specimens from three phase III trials.

ABSTRACT Herpes simplex virus type 2 (HSV-2) is the most common cause of genital ulcer disease and is a cofactor for HIV-1 acquisition and transmission. We analyzed specimens from three separate phase III trials of acyclovir (ACV) for prevention of HIV-1 acquisition and transmission to determine if failure of ACV to interrupt HIV acquisition and transmission was associated with genotypic ACV resistance. Acyclovir (400 mg twice daily) or placebo was provided to HSV-2-infected persons at risk of HIV-1 infection in the Mwanza and HPTN 039 trials and to persons dually infected with HSV-2 and HIV-1 who had an HIV-negative partner in the Partners in Prevention study. We extracted HSV DNA from genital ulcer swabs or cervicovaginal lavage fluids from 68 samples obtained from 64 participants randomized to ACV and sequenced the HSV-2 UL23 gene encoding thymidine kinase. The UL23 sequences were compared with published and unpublished data. Variants were observed in 38/1,128 (3.4%) nucleotide positions in the UL23 open reading frame, with 58% of these encoding amino acid changes. No deletions, insertions, or mutations known to be associated with resistance were detected. Thirty-one of the variants (81.5%) are newly reported, 15 of which code for amino acid changes. Overall, UL23 is highly polymorphic compared to other loci in HSV-2, but no drug resistance mutations were detected that could explain the failure to reduce HIV incidence or to prevent HIV-1 transmission in these studies.Herpes simplex virus type 2 (HSV-2) is the most common cause of genital ulcer disease and is a cofactor for HIV-1 acquisition and transmission. We analyzed specimens from three separate phase III trials of acyclovir (ACV) for prevention of HIV-1 acquisition and transmission to determine if failure of ACV to interrupt HIV acquisition and transmission was associated with genotypic ACV resistance. Acyclovir (400 mg twice daily) or placebo was provided to HSV-2-infected persons at risk of HIV-1 infection in the Mwanza and HPTN 039 trials and to persons dually infected with HSV-2 and HIV-1 who had an HIV-negative partner in the Partners in Prevention study. We extracted HSV DNA from genital ulcer swabs or cervicovaginal lavage fluids from 68 samples obtained from 64 participants randomized to ACV and sequenced the HSV-2 UL23 gene encoding thymidine kinase. The UL23 sequences were compared with published and unpublished data. Variants were observed in 38/1,128 (3.4%) nucleotide positions in the UL23 open reading frame, with 58% of these encoding amino acid changes. No deletions, insertions, or mutations known to be associated with resistance were detected. Thirty-one of the variants (81.5%) are newly reported, 15 of which code for amino acid changes. Overall, UL23 is highly polymorphic compared to other loci in HSV-2, but no drug resistance mutations were detected that could explain the failure to reduce HIV incidence or to prevent HIV-1 transmission in these studies.

The murine intravaginal HSV-2 challenge model for investigation of DNA vaccines.

DNA vaccines have been licensed in veterinary medicine and have promise for humans. This format is relatively immunogenic in mice and guinea pigs, the two principle HSV-2 animal models, permitting rapid assessment of vectors, antigens, adjuvants, and delivery systems. Limitations include the relatively poor immunogenicity of naked DNA in humans and the profound differences in HSV-2 pathogenesis between host species. Herein, we detail lessons learned over the last few years investigating candidate DNA vaccines in the progesterone-primed female mouse vaginal model of HSV-2 infection as a guide to investigators in the field.