Shailbala Singh

HIV-1 Vpr protein inhibits telomerase activity via the EDD-DDB1-VPRBP E3 ligase complex.

Background: Telomerase is an essential enzyme for chromosome stability. Results: The HIV-1 accessory protein Vpr targets TERT, a catalytic subunit of telomerase, via ubiquitin-mediated degradation. Conclusion: Vpr inhibits telomerase activity by TERT down-regulation. Significance: Learning how telomerase is deregulated by HIV-1 Vpr is crucial for understanding HIV-1-associated pathogenesis. Viral pathogens utilize host cell machinery for their benefits. Herein, we identify that HIV-1 Vpr (viral protein R) negatively modulates telomerase activity. Telomerase enables stem and cancer cells to evade cell senescence by adding telomeric sequences to the ends of chromosomes. We found that Vpr inhibited telomerase activity by down-regulating TERT protein, a catalytic subunit of telomerase. As a molecular adaptor, Vpr enhanced the interaction between TERT and the VPRBP substrate receptor of the DYRK2-associated EDD-DDB1-VPRBP E3 ligase complex, resulting in increased ubiquitination of TERT. In contrast, the Vpr mutant identified in HIV-1-infected long-term nonprogressors failed to promote TERT destabilization. Our results suggest that Vpr inhibits telomerase activity by hijacking the host E3 ligase complex, and we propose the novel molecular mechanism of telomerase deregulation in possibly HIV-1 pathogenesis.

Unique potential of 4-1BB agonist antibody to promote durable regression of HPV + tumors when combined with an E6/E7 peptide vaccine

Significance Nearly all cervical, anal, vulvar, and penile cancer and up to half of oropharyngeal cancers are driven by the E6 and E7 oncoproteins of human papilloma virus (HPV). Therapeutic vaccination against these HPV proteins can slow disease progression in animal models and in patients, but is rarely curative. We demonstrate that coadministration of agonist antibodies targeting the T-cell costimulatory receptor 4-1BB and an intranasal HPV E6/E7 peptide vaccine promoted durable regression in 100% of animals bearing HPV+ TC-1 tumors established in the female reproductive tract. The efficacy of 4-1BB in this system was unique among immune checkpoint antibodies and provides a paradigm for enhancement of therapeutic cancer vaccines with costimulatory agonist antibodies. Antibody modulation of T-cell coinhibitory (e.g., CTLA-4) or costimulatory (e.g., 4-1BB) receptors promotes clinical responses to a variety of cancers. Therapeutic cancer vaccination, in contrast, has produced limited clinical benefit and no curative therapies. The E6 and E7 oncoproteins of human papilloma virus (HPV) drive the majority of genital cancers, and many oropharyngeal tumors. We discovered 15–19 amino acid peptides from HPV-16 E6/E7 for which induction of T-cell immunity correlates with disease-free survival in patients treated for high-grade cervical neoplasia. We report here that intranasal vaccination with these peptides and the adjuvant alpha-galactosylceramide elicits systemic and mucosal T-cell responses leading to reduced HPV+ TC-1 tumor growth and prolonged survival in mice. We hypothesized that the inability of these T cells to fully reject established tumors resulted from suppression in the tumor microenvironment which could be ameliorated through checkpoint modulation. Combining this E6/E7 peptide vaccine with checkpoint blockade produced only modest benefit; however, coadministration with a 4-1BB agonist antibody promoted durable regression of established genital TC-1 tumors. Relative to other therapies tested, this combination of vaccine and α4-1BB promoted the highest CD8+ versus regulatory FoxP3+ T-cell ratios, elicited 2- to 5-fold higher infiltration by E7-specific CTL, and evoked higher densities of highly cytotoxic TcEO (T cytotoxic Eomesodermin) CD8 (>70-fold) and ThEO (T helper Eomesodermin) CD4 (>17-fold) T cells. These findings have immediate clinical relevance both in terms of the direct clinical utility of the vaccine studied and in illustrating the potential of 4-1BB antibody to convert therapeutic E6/E7 vaccines already in clinical trials into curative therapies.

Intranasal but not intravenous delivery of the adjuvant α-galactosylceramide permits repeated stimulation of natural killer T cells in the lung.

Efficient induction of antigen‐specific immunity is achieved by delivering multiple doses of vaccine formulated with appropriate adjuvants that can harness the benefits of innate immune mediators. The synthetic glycolipid α‐galactosylceramide (α‐GalCer) is a potent activator of NKT cells, a major innate immune mediator cell type effective in inducing maturation of DCs for efficient presentation of co‐administered antigens. However, systemic administration of α‐GalCer results in NKT cell anergy in which the cells are unresponsive to subsequent doses of α‐GalCer. We show here that α‐GalCer delivered as an adjuvant by the intranasal route, as opposed to the intravenous route, enables repeated activation of NKT cells and DCs, resulting in efficient induction of cellular immune responses to co‐administered antigens. We show evidence that after intranasal delivery,α‐GalCer is selectively presented by DCs for the activation of NKT cells, not B cells. Furthermore, higher levels of PD‐1 expression, a potential marker for functional exhaustion of the NKT cells when α‐GalCer is delivered by the intravenous route, are not observed after intranasal delivery. These results support a mucosal route of delivery for the utility of α‐GalCer as an adjuvant for vaccines, which often requires repeated dosing to achieve durable protective immunity.

Natural killer T cell and TLR9 agonists as mucosal adjuvants for sublingual vaccination with clade C HIV-1 envelope protein.

The vast majority of HIV-1 infections occur at mucosa during sexual contact. It may therefore be advantageous to provide mucosal barrier protection against this entry by mucosal vaccination. While a number of mucosal routes of vaccination are possible, many like enteric oral vaccines or intranasal vaccines have significant impediments that limit vaccine efficacy or pose safety risks. In contrast, immunogens applied to the sublingual region of the mouth could provide a simple route for mucosal vaccination. While sublingual immunization is appealing, this site does not always drive strong immune responses, particularly when using protein antigens. To address this issue, we have tested the ability of two mucosal adjuvants: alpha-galactosylceramide (αGalCer) that is a potent stimulator of natural killer T cells and CpG-oligodeoxynucleotide (CpG-ODN) a TLR9 agonist for their ability to amplify immune responses against clade C gp140 HIV-1 envelope protein antigen. Immunization with envelope protein alone resulted in a weak T cell and antibody responses. In contrast, CD4(+) and CD8(+) T cells responses in systemic and mucosal tissues were significantly higher in mice immunized with gp140 in the presence of either αGalCer or CpG-ODN and these responses were further augmented when the two adjuvants were used together. While both the adjuvants effectively increased gp140-specific serum IgG and vaginal IgA antibody levels, combining both significantly improved these responses. Memory T cell responses 60 days after immunization revealed αGalCer to be more potent than CpG-ODN and the combination of the αGalCer and CpG-ODN adjuvants was more effective than either alone. Serum and vaginal washes collected 60 days after immunization with gp140 with both αGalCer and CpG-ODN adjuvants had significant neutralization activity against Tier 1 and Tier 2 SHIVs. These data support the utility of the sublingual route for mucosal vaccination particularly in combination with αGalCer and CpG-ODN adjuvants.

Nerium oleander derived cardiac glycoside oleandrin is a novel inhibitor of HIV infectivity

We evaluated the effectiveness of Anvirzel™, an aqueous extract of Nerium oleander on HIV infection of human peripheral blood mononuclear cells. Oleandrin, the principle cardiac glycoside (CG) in Anvirzel™ has been shown to exhibit anti-cancer properties but its efficacy against HIV is unknown. Treatment with Anvirzel™ significantly reduced the infectivity of virus produced from infected cells without any change in the total amount of virus produced. This is in contrast to treatment with AZT, a potent inhibitor of HIV replication that has been shown to significantly reduce virus production. Relative to untreated cultures, virus in cultures treated with oleandrin had significantly reduced expression of the envelope protein gp120, the sole determinant of virus infectivity, suggesting a novel mechanism underlying the impaired infectivity. These results support the potential utility of the Nerium oleander aqueous extract, containing the CG oleandrin as a novel candidate anti-HIV therapeutic.

Sublingual Vaccination Induces Mucosal and Systemic Adaptive Immunity for Protection against Lung Tumor Challenge

Sublingual route offers a safer and more practical approach for delivering vaccines relative to other systemic and mucosal immunization strategies. Here we present evidence demonstrating protection against ovalbumin expressing B16 (B16-OVA) metastatic melanoma lung tumor formation by sublingual vaccination with the model tumor antigen OVA plus synthetic glycolipid alpha-galactosylceramide (aGalCer) for harnessing the adjuvant potential of natural killer T (NKT) cells, which effectively bridge innate and adaptive arms of the immune system. The protective efficacy of immunization with OVA plus aGalCer was antigen-specific as immunized mice challenged with parental B16 tumors lacking OVA expression were not protected. Multiple sublingual immunizations in the presence, but not in the absence of aGalCer, resulted in repeated activation of NKT cells in the draining lymph nodes, spleens, and lungs of immunized animals concurrent with progressively increasing OVA-specific CD8+ T cell responses as well as serum IgG and vaginal IgA levels. Furthermore, sublingual administration of the antigen only in the presence of the aGalCer adjuvant effectively boosted the OVA-specific immune responses. These results support potential clinical utility of sublingual route of vaccination with aGalCer-for prevention of pulmonary metastases.

Enhancement of Mucosal Immunogenicity of Viral Vectored Vaccines by the NKT Cell Agonist Alpha-Galactosylceramide as Adjuvant

Gene-based vaccination strategies, specifically viral vectors encoding vaccine immunogens are effective at priming strong immune responses. Mucosal routes offer practical advantages for vaccination by ease of needle-free administration, and immunogen delivery at readily accessible oral/nasal sites to efficiently induce immunity at distant gut and genital tissues. However, since mucosal tissues are inherently tolerant for induction of immune responses, incorporation of adjuvants for optimal mucosal vaccination strategies is important. We report here the effectiveness of alpha-galactosylceramide (α-GalCer), a synthetic glycolipid agonist of natural killer T (NKT) cells, as an adjuvant for enhancing immunogenicity of vaccine antigens delivered using viral vectors by mucosal routes in murine and nonhuman primate models. Significant improvement in adaptive immune responses in systemic and mucosal tissues was observed by including α-GalCer adjuvant for intranasal immunization of mice with vesicular stomatitis virus vector encoding the model antigen ovalbumin and adenoviral vectors expressing HIV env and Gag antigens. Activation of NKT cells in systemic and mucosal tissues along with significant increases in adaptive immune responses were observed in rhesus macaques immunized by intranasal and sublingual routes with protein or adenovirus vectored antigens when combined with α-GalCer adjuvant. These results support the utility of α-GalCer adjuvant for enhancing immunogenicity of mucosal vaccines delivered using viral vectors.

Procedures for Mucosal Immunization and Analyses of Cellular Immune Response to Candidate HIV Vaccines in Murine and Nonhuman Primate Models

Sampling the mucosal tissues and analyses of immune responses are integral to vaccine-development strategies against human immunodeficiency virus (HIV), which is transmitted predominantly across the oro-genital mucosa. While immune assay development and standardization attempts employ mouse models, immunogenicity and protective efficacy that can be extrapolated to humans are realized only from experiments in nonhuman primates. Here, we describe commonly used practices for immunizations in rhesus macaques (Macaca mulatta) along with procedures for obtaining important mucosal tissues samples from macaques and mice. We also describe detailed protocols for two important assays applicable in mouse as well as primate experiments for determining antigen-specific T cells responses induced after vaccination.

Prophylactic sublingual immunization with Mycobacterium tuberculosis subunit vaccine incorporating the natural killer T cell agonist alpha-galactosylceramide enhances protective immunity to limit pulmonary and extra-pulmonary bacterial burden in mice

Infection by Mycobacterium tuberculosis (Mtb) remains a major global concern and the available Bacillus Calmette-Guerin (BCG) vaccine is poorly efficacious in adults. Therefore, alternative vaccines and delivery strategies focusing on Mtb antigens and appropriate immune stimulating adjuvants are needed to induce protective immunity targeted to the lungs, the primary sites of infections and pathology. We present here evidence in support of mucosal vaccination by the sublingual route in mice using the subunit Mtb antigens Ag85B and ESAT-6 adjuvanted with the glycolipid alpha-galactosylceramide (α-GalCer), a potent natural killer T (NKT) cell agonist. Vaccinated animals exhibited strong antigen-specific CD4 and CD8 T cells responses in the spleen, cervical lymph nodes and lungs. In general, inclusion of the α-GalCer adjuvant significantly enhanced these responses that persisted over 50 days. Furthermore, aerosolized Mtb infection of vaccinated mice resulted in a significant reduction of bacterial load of the lungs and spleens as compared to levels seen in naïve controls or those vaccinated with subunit proteins, adjuvant , or BCG alone. The protection induced by the Mtb antigens and-GalCer vaccine through sublingual route correlated with a TH1-type immunity mediated by antigen-specific IFN-γ and IL-2 producing T cells.

Lessons on Non-Progression of HIV Disease from Monkeys

Rhesus macaques infected with simian immunodeficiency virus (SIV) represent the most widely used model for studies related to understanding infection, pathology, immunology, and intervention strategies for HIV infection and AIDS in humans. This model recapitulates the significant impact of host immunogenetics and inter-individual variations on the natural disease course of HIV-AIDS in humans. In addition to the parallel kinetics for fast- and slow-progressing disease course observed in majority of animals, a small subset of animals exhibit long-term non-progression (LTNP) or elite control (EC) status defined as low to undetectable viral loads in the absence of any interventions for prolonged periods. Therefore, the rhesus macaque model has been and continues to be the most extensively adopted experimental system for studies to not only confirm observations from HIV-ADIS in humans but also to validate several mechanistic features of the infection and disease course, which otherwise would be impractical and immoral to be performed in humans.