Purpose: GEN-003 is a candidate therapeutic HSV-2 vaccine containing a fragment of infected cell protein 4 (ICP4.2), a deletion mutant of glycoprotein D2 (gD2 Delta TMR), and Matrix-M2 adjuvant. In a dose-ranging phase 1/2a clinical trial, immunization with GEN-003 reduced viral shedding and the percentage of reported herpetic lesion days. Here we examine the immune responses in the same trial, to characterize vaccine-related changes in antibody and cell-mediated immunity.Methods: Participants with genital HSV-2 infection were randomized to 1 of 3 doses of GEN-003, antigens without adjuvant, or placebo. Subjects received 3 intramuscular doses, three weeks apart, and were monitored for viral shedding, lesions and immunogenicity. Antibody titers were measured by ELISA and neutralization assay in serum samples collected at baseline and 3 weeks post each dose. T cell responses were assessed pre-immunization and 1 week post each dose by IFN-gamma ELISpot and intracellular cytoldne staining. Blood was also collected at 6 and 12 months to monitor durability of immune responses.Results: Antibody and T cell responses increased with vaccination and were potentiated by adjuvant. Among the doses tested, the rank order of reduction in viral shedding follows the ranking of fold change from baseline in T cell responses. Some immune responses persisted up to 12 months.Conclusion: All measures of immunity are increased by vaccination with GEN-003; however, a correlate of protection is yet to be defined. (C) 2016 Elsevier Ltd. All rights reserved.

Measurement of neutralizing antibodies against herpes simplex virus (HSV) is important for evaluation of candidate vaccines. The established plaque-reduction neutralization assay is time consuming, labor intensive, and difficult to validate and transfer. Here, we describe the characterization of a HSV-neutralization assay based on the expression of a reporter gene, β-galactosidase (β-Gal). Using previously constructed HSV-β-Gal recombinant viruses, HSV-2/Gal and HSV-1/tk12, we developed a colorimetric β-Gal-based neutralization assay that is sensitive and highly reproducible, and performed in less than 48 h. HSV-1 and HSV-2 neutralizing titers measured by the β-Gal-based neutralization assay were equivalent to those obtained by a plaque reduction neutralization assay. Intra- and inter-assay precision studies demonstrated that the β-Gal-based assay was repeatable and yielded low and acceptable variation. In addition, comparison of HSV-2 neutralizing antibody (NAb) titers measured in two independent laboratories by two unique β-Gal-based assays showed a highly significant correlation (r = 0.9499, p < 0.0001) between the two assays. The new assay will serve as an important tool both for preclinical and clinical trials of new HSV vaccines.

Infections with Streptococcus pneumoniae cause substantial morbidity and mortality, particularly in children in developing nations. Polysaccharide-conjugate vaccines provide protection against both invasive disease and colonization, but their use in developing countries is limited by restricted serotype coverage and expense of manufacture. Using proteomic screens, we recently identified several antigens that protected mice from pneumococcal colonization in a CD4(+) T cell- and interleukin-17A (IL-17A)-dependent manner. Since several of these proteins are lipidated, we hypothesized that their immunogenicity and impact on colonization are in part due to activation of Toll-like receptor 2 (TLR2), a receptor for lipoproteins. Here we show that lipidated versions of the antigens elicited significantly higher activation of both human embryonic kidney cells engineered to express TLR2 (HEK-TLR2) and wild-type (WT) murine macrophages than nonlipidated mutant antigens. Lipoprotein-stimulated secretion of proinflammatory cytokines was ∼10× to ∼100× lower in murine TLR2-deficient macrophages than in WT macrophages. Subcutaneous immunization of C57BL/6 mice with protein subunit vaccines containing one or two of these lipoproteins or protein fusion constructs bearing N-terminal lipid adducts elicited a robust IL-17A response and a significant reduction in colonization compared with immunization with alum alone. In contrast, immunization of Tlr2(-/-) mice elicited no detectable IL-17A response and no protection against pneumococcal colonization. These experiments suggest that the lipid moieties enhance the immunogenicity and protective efficacy of pneumococcal TH17 antigens through activation of TLR2. Thus, triggering TLR2 with an antigen-specific protein subunit formulation is a possible strategy for the development of a serotype-independent pneumococcal vaccine that would reduce pneumococcal carriage.

Reactivation of latent herpes simplex virus 2 (HSV-2) infections can be characterized by episodic recurrent genital lesions and/or viral shedding. We hypothesize that infected (HSV-2(pos)) asymptomatic individuals have acquired T cell responses to specific HSV-2 antigen(s) that may be an important factor in controlling their recurrent disease symptoms. Our proteomic screening technology, ATLAS, was used to characterize the antigenic repertoire of T cell responses in infected (HSV-2(pos)) and virus-exposed seronegative (HSV-2(neg)) subjects. T cell responses, determined by IFN-γ secretion, were generated to gL, UL2, UL11, UL21, ICP4, ICP0, ICP47 and UL40 with greater magnitude and/or frequency among cohorts of exposed HSV-2(neg) or asymptomatic HSV-2(pos) individuals, compared to symptomatic recurrent HSV-2(pos) subjects. T cell antigens recognized preferentially among individuals who are resistant to infection or who are infected and have mild or no clinical disease may provide new targets for the design of vaccines aimed at treating and/or preventing HSV-2 infection.

Immunotherapeutic herpes simplex virus 2 (HSV-2) vaccine efficacy depends upon the promotion of antigen-specific immune responses that inhibit reactivation or reactivated virus, thus controlling both recurrent lesions and viral shedding. In the present study, a candidate subunit vaccine, GEN-003/MM-2, was evaluated for its ability to induce a broad-spectrum immune response in mice and therapeutic efficacy in HSV-2-infected guinea pigs. GEN-003 is comprised of HSV-2 glycoprotein D2 (gD2ΔTMR340-363) and a truncated form of infected cell polypeptide 4 (ICP4383-766), formulated with Matrix M-2 (MM-2) adjuvant (GEN-003/MM-2). In addition to eliciting humoral immune responses, CD4(+) and CD8(+) T cells characterized by the secretion of multiple cytokines and cytolytic antigen-specific T cell responses that were able to be recalled at least 44 days after the last immunization were induced in immunized mice. Furthermore, vaccination with either GEN-003 or GEN-003/MM-2 led to significant reductions in both the prevalence and severity of lesions in HSV-2-infected guinea pigs compared to those of phosphate-buffered saline (PBS) control-vaccinated animals. While vaccination with MM-2 adjuvant alone decreased recurrent disease symptoms compared to the PBS control group, the difference was not statistically significant. Importantly, the frequency of recurrent viral shedding was considerably reduced in GEN-003/MM-2-vaccinated animals but not in GEN-003- or MM-2-vaccinated animals. These findings suggest a possible role for immunotherapeutic GEN-003/MM-2 vaccination as a viable alternative to chronic antiviral drugs in the treatment and control of genital herpes disease.

Abstract An effective HSV-2 vaccine is still needed despite the prevalence of antiviral drugs. Recent HSV-2 vaccine clinical trials highlighted the need to elicit potent T cell responses since neutralizing antibodies alone were incapable of providing complete protection. Through proteomic screens using T cells of HSV-2-exposed donors several antigens were identified as promising vaccine targets. Two of these antigens, GB208 and GB217 (GEN-003), were formulated with a saponin-derived adjuvant Matrix M-2, which in recent avian influenza human clinical trials induced cellular and humoral immune responses. In preclinical studies, C57BL/6 mice were used to evaluate the ability of GEN-003 combined with Matrix M-2 to induce antigen-specific T cell and antibody responses. T cell effector functions were evaluated by IFNγ ELISPOT, intracellular cytokine staining, and in vivo cytolysis of fluorescently labeled peptide-pulsed targets. Neutralizing antibody responses as well as total and subclass IgG titers were also assessed. Cellular and humoral immune responses were primed in mice after administering a wide range of protein and adjuvant doses. GB208- and GB217- specific T cell responses were polyfunctional, cytolytic, and persisted for more than 3 months. Antibody responses were neutralizing, and represented balanced Th1/Th2 phenotypes. In conclusion, GEN-003 combined with Matrix M-2 form a promising vaccine candidate that activates both arms of the immune system necessary for viral clearance.

HSV-2 infection is characterized by chronic intermittent shedding of virus and episodic clinical outbreaks. T cells and antibodies play a critical role in controlling the infection. While patients possess T cell reactivity to multiple HSV-2 antigens, reactivity to specific antigens may be associated with control of clinical disease. Recurrence of clinical disease is episodic, but little is known about the temporal variation in peripheral T cell responses to HSV-2. T cell and antibody responses to two antigens previously associated with asymptomatic infection were studied among 27 HSV-2 seropositive individuals with a history of HSV outbreaks by IFN-γ ELISPOT and endpoint titer antibody ELISA, respectively. Peripheral blood was collected 4 times, 3 weeks apart and within 48 hours of any genital lesion outbreak. Patients were categorized as high or low responders. None of high and 9 of 23 low T cell responders experienced clinical outbreaks. Immune responses did not vary significantly during the study period or during clinical outbreaks. The correlation of immune response and subclinical disease is currently under evaluation. These results indicate that in chronic HSV-2 infection, levels of peripheral T cells and antibodies remain consistent over time and are not boosted during viral outbreaks. This supports the hypothesis that a vaccination strategy to increase T cell responses to select HSV-2 antigens may provide a therapeutic benefit to the patients.

Vaccine strategies that utilize human DCs to enhance antitumor immunity have yet to realize their full potential. Approaches that optimally target a spectrum of antigens to DCs are urgently needed. Here we report the development of a platform for loading DCs with antigen. It is based on killed but metabolically active (KBMA) recombinant Listeria monocytogenes and facilitates both antigen delivery and maturation of human DCs. Highly attenuated KBMA L. monocytogenes were engineered to express an epitope of the melanoma-associated antigen MelanA/Mart-1 that is recognized by human CD8+ T cells when presented by the MHC class I molecule HLA-A*0201. The engineered KBMA L. monocytogenes induced human DC upregulation of costimulatory molecules and secretion of pro-Th1 cytokines and type I interferons, leading to effective priming of Mart-1-specific human CD8+ T cells and lysis of patient-derived melanoma cells. KBMA L. monocytogenes expressing full-length NY-ESO-1 protein, another melanoma-associated antigen, delivered the antigen for presentation by MHC class I and class II molecules independent of the MHC haplotype of the DC donor. A mouse therapeutic tumor model was used to show that KBMA L. monocytogenes efficiently targeted APCs in vivo to induce protective antitumor responses. Together, our data demonstrate that KBMA L. monocytogenes may be a powerful platform that can both deliver recombinant antigen to DCs for presentation and provide a potent DC-maturation stimulus, making it a potential cancer vaccine candidate.

3044 Background: Melanoma is one of the most rapidly growing cancers worldwide yet there is no satisfactory treatment. Dendritic cells (DC) are attractive cellular adjuvants which can increase host resistance to tumor. To optimally prime naïve T cells, DCs must not only present high levels of MHC-peptide complexes, but must also undergo activation and maturation. Methods: The highly attenuated killed but metabolically active (KBMA) Listeria monocytogenes (Lm) were engineered to express the HLA-A*0201 CD8+ T cell epitopes of melanoma-associated antigen (MAA) MelanA/Mart-1 and of control influenza matrix protein, both encoded within the OVA scaffold protein. These bacteria served as a DC antigen-loading platform. Monocyte-derived DC were generated from PBMCs of healthy donors, infected with recombinant attenuated KBMA Lm (rLm) and evaluated for maturation and presentation of the tumor-specific epitope. These DC were also used to generate tumor-specific CD8+ T cell lines, which were tested for effector potential through lysis of HLA matched patient-derived tumor cell lines. Efficacy of the KBMA rLm-infected DCs was confirmed in a B16-OVA mouse tumor model. Survival and tumor burden was compared between the vaccinated and control group. Tumor survival results were analyzed by one-way ANOVA and two-tailed Student’s t test for statistical significance. Results: Highly attenuated KBMA Lm expressing the MelanA/Mart-1 epitope induced up-regulation of costimulatory molecules and lymph node homing molecule CCR7 on human DC, secretion of proinflammatory cytokines, including IL-12p70 and IFNα. In vitro co-culture with naive T cells from healthy donors significantly induced MelanA/Mart-1 specific CD8+ T cells, which in turn were able to lyse melanoma cells expressing the antigen but not a control tumor cell line. In mice, vaccination with rLm-infected DCs resulted in reduced tumor burden and enhanced survival. Conclusions: Our studies demonstrate that KBMA Lm is a novel and effective ex vivo DC antigen loading platform combining a potent DC-maturation stimulus with the cytosolic delivery of recombinant antigen for presentation. The platform results in priming of tumor antigen-specific and protective CD8+ T cells. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Anza Therapeutics Anza Therapeutics Anza Therapeutics Anza Therapeutics

Dendritic cells (DCs) that capture apoptotic cells (ACs) in the steady state mediate peripheral tolerance to self-antigens. ACs are recognized by an array of receptors on DCs, the redundancy of which is not completely defined. We made use of an AC surrogate system to address the individual roles of the alpha v beta 5 and complement receptors (CRs) in the phagocytosis and induction of immunity. CR3 and CR4, while substantially less efficient than alpha v beta 5 in internalizing ACs, initiate signals that render DCs tolerogenic. Responding T cells show impaired proliferation and IFN gamma production and subsequently die by apoptosis. While tolerogenic DCs are not induced via alpha v beta 5, coligation of CR3 and alpha v beta 5 maintains the DC's tolerogenic profile. This immunomodulatory role, however, is countered by a significant inflammatory stimulus such as bacterial infection. Overall, our data suggest that under steady-state conditions, signaling via CRs predominates to render DCs tolerogenic.

This chapter contains sections titled: Introduction Acquisition of Antigens for Crosspresentation Mechanisms of Crossprocessing and Crosspresentation Physiological Relevance of Crosspresentation

Presentation of bacteria-derived CD8 T cell epitopes by dendritic cells (DC) requires either their direct infection or that DC acquire and cross-present Ags from other infected cells. We found that cross-presentation of Listeria monocytogenes-derived CD8 T cell epitopes was much stronger than direct Ag presentation by infected murine DC. Cross-presentation of Listeria-derived CD8 T cell epitopes showed unique physiological requirements. It was dependent upon the delivery of unstable bacterial translation products by infected, but still viable, Ag donor cells. Cross-presentation was enhanced both when unstable translation products in infected Ag donor cells were protected from proteasomal degradation and when the production of misfolded bacterial proteins was increased. The requirement of unstable translation products for cross-presentation may represent a novel pathway that functions to focus the CD8 T cell response toward epitopes derived from newly synthesized proteins.

Dendritic cells (DCs) are bone marrow-derived professional antigen presenting cells which play a pivotal role in initiating immune responses. To achieve optimal potency for priming of naive T cells, DCs must not only present high levels of MHC peptide complexes, but must also undergo maturation. An ideal DC-targeting vaccine formulation, therefore, should simultaneously deliver multiple antigens and activate DCs to increase expression of surface MHC-peptide complexes, costimulatory molecules, and production of immuno-stimulatory cytokines. Despite the development of a variety of ex vivo methods, no standard efficient and cost effective methods combining antigen loading and activation/maturation of DCs for clinical use currently exist. Here we report the development of a novel DC antigen-loading platform based on killed, but metabolically active (KBMA) recombinant Listeria monocytogenes (Lm). While recombinant Lm has a relatively long history as a cancer vaccine platform, its utility as a DC activation and loading platform has not been fully explored. KBMA Lm were 8-logs more sensitive to photochemical inactivation by the combined treatment of S-59 psoralen (amotosalen HCl) and long-wave ultraviolet light (S-59/UVA), by virtue of abrogation of nucleotide excision repair (NER) capacity, through engineered deletion of the bacterial UvrAB genes. Due to random distribution of infrequent psoralen adducts, KBMA Lm cannot propagate, but are metabolically active and can express their genetic repertoire, and program presentation of expressed heterologous antigens via the MHC class I and class II pathways. Infection of human monocyte-derived or blood myeloid DCs with KBMA Listeria induced their maturation and production of pro-inflammatory and pro- Th1 cytokines. The DCs achieved a fully mature phenotype, comparable to that induced by the standard maturation stimulus MCM-mimic. To determine whether KBMA Lm could program presentation of encoded antigens by the MHC class I pathway, we constructed a recombinant Lm vaccine expressing CD8+ T cell epitopes of influenza matrix protein (MP58–66) and MelanA/Mart-1 protein (Mart126–35). DCs infected with the recombinant KBMA Lm vaccine presented the recombinant class I epitopes and induced IFN-γ production by MP58–66- or Mart126–35-specific human CD8+ T cell clones. These studies demonstrate that KBMA Listeria is a novel and powerful ex vivo DC antigen loading platform combining a potent DC-maturation stimulus with the cytosolic delivery of recombinant antigen for presentation.

Presentation of bacteria-derived CD8 T cell epitopes by dendritic cells (DC) requires either their direct infection or that DC acquire and cross-present Ags from other infected cells. We found that cross-presentation of Listeria monocytogenes-derived CD8 T cell epitopes was much stronger than direct Ag presentation by infected murine DC. Cross-presentation of Listeria-derived CD8 T cell epitopes showed unique physiological requirements. It was dependent upon the delivery of unstable bacterial translation products by infected, but still viable, Ag donor cells. Cross-presentation was enhanced both when unstable translation products in infected Ag donor cells were protected from proteasomal degradation and when the production of misfolded bacterial proteins was increased. The requirement of unstable translation products for cross-presentation may represent a novel pathway that functions to focus the CD8 T cell response toward epitopes derived from newly synthesized proteins.

The identification of T cell epitopes is crucial for the understanding of the host response during infections with pathogenic microorganisms. Generally, the identification of relevant T cell responses is based on the analysis of T cell lines propagated in vitro. We used an ex vivo approach for the analysis of the CD8 T cell response against Listeria monocytogenes that is based upon the fractionation of naturally processed antigenic peptides and subsequent analysis with T cells in an enzyme-linked immunospot (ELISPOT) assay. Our data indicate that the direct ex vivo ELISPOT analysis of peptides extracted from infected tissues represents a versatile and potent test system for the analysis of the CD8 T cell immunome of microorganisms that furthermore requires neither the knowledge of the microbial genome nor of the specificity of responding T cells.

Dendritic cells require a special 'license' to present exogenous antigens to CD8+ T cells. Such permission may be provided by type I interferons during viral infections.

IFN-γ is an essential component of the early Listeria monocytogenes-specific immune response, and is also an important regulator of Ag processing and presentation. Ag presentation is required for the induction and also the effector function of antimicrobial T cells. To evaluate the effect of IFN-γ on bacterial Ag presentation in vivo, macrophages and dendritic cells were separated from L. monocytogenes-infected tissues and analyzed with peptide-specific CD4 and CD8 T cell lines in a sensitive ELISPOT-based ex vivo Ag presentation assay. The comparison of professional APCs isolated from infected IFN-γ-deficient and wild-type mice revealed different peptide presentation patterns of L. monocytogenes-derived CD8 T cell epitopes, while the presentation pattern of CD4 T cell epitopes remained unchanged. The further in vitro analysis of the generation of CD8 T cell epitopes revealed a peptide-specific effect of IFN-γ on MHC class I-restricted Ag presentation. These results show that despite this modulation of the Ag presentation pattern of CD8 T cell epitopes, IFN-γ is not generally required for the MHC class I- and MHC class II-restricted presentation of L. monocytogenes-derived antigenic peptides by professional APCs in vivo.