Jagannadha K Sastry

Protection against chronic infection and AIDS by an HIV envelope peptide-cocktail vaccine in a pathogenic SHIV-rhesus model

Based on our prior studies in mouse, monkey, chimpanzee, and human experimental systems, we identified six peptides encoded by highly conserved regions of the human immunodeficiency virus type 1 (HIV-1) envelope gene that selectively induce cellular immune responses in the absence of anti-viral antibody production. We tested a cocktail of the six peptides as a prototype vaccine for protection from simian human immunodeficiency virus (SHIV) infection and acquired immunodeficiency syndrome (AIDS) in a rhesus monkey model. Three monkeys were vaccinated with the peptide cocktail in Freunds adjuvant followed by autologous dendritic cells (DC) pulsed with these peptides. All the vaccinated animals exhibited significant induction of T-cell proliferation and cytotoxic T lymphocytes (CTL) responses, but no neutralizing antibodies. Two control mock-vaccinated monkeys showed no specific immune responses. Upon challenge with the pathogenic SHIV(KU-2), both the control and vaccinated monkeys were infected, but efficient clearance of virus-infected cells was observed in all the three vaccinated animals within 14 weeks. These animals also experienced a boosting of antiviral cellular immune responses after infection, and maintained antigen-specific IFN-gamma-producing cells in circulation beyond 42 weeks post-challenge. In contrast, the two mock-vaccinated monkeys had low to undetectable cellular immune responses and maintained significant levels of viral-infected cells and infectious virus in circulation. Further, in both the control monkeys plasma viremia was detectable beyond 38 weeks post-challenge indicating chronic phase infection. In one control monkey, the CD4+ cells dropped to very low levels by 2 weeks post-challenge and became undetectable by week 39 coinciding with high plasma viremia and AIDS, which included cachexia and ataxia. These results serve as proof of principle for the effectiveness of the HIV envelope peptide cocktail vaccine against chronic infection and AIDS, and support the development of multivalent peptide-based vaccine as a viable strategy to induce cell-mediated immunity (CMI) for protection against HIV and AIDS in humans.

Differences in Functional Immune Responses of High vs. Low Hardy Healthy Individuals

An association between the personality trait of hardiness and immune responses was explored. Blood samples were collected from 21 healthy individuals under nonstressful conditions, who had either high or low levels of hardiness. Functional immune assays tested for natural killer (NK) cell activity and proliferation responses to Candida albicans (Candida), purified protein derivative from Mycobacterium tuberculosis (PPD), lipopolysaccharide (LPS), concanavalin A (Con A), and Staphylococcus enterotoxin A (Staph A). Differences between high and low hardy groups on these immune responses were examined using Bonferroni adjusted independent t-tests. Results revealed significant differences between the groups for Candida, PPD, Con A, and Staph A. In all instances, the high hardy group had larger mean proliferative responses than the low hardy group. Implications of the study as well as avenues for future research are discussed.

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.

Dendritic cells enhance detection of antigen-specific cellular immune responses by lymphocytes from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine

Abstract: Detection and enumeration of functional antigen‐specific T cells is important for understanding the breadth of cell‐mediated immunity to infections and experimental vaccines. We tested the utility of dendritic cells (DC), the professional antigen presenting cells, in the enzyme‐linked immunosorbent spot‐forming cell assay (ELISPOT) for efficient monitoring of antigen‐specific immunity in rhesus macaques vaccinated with an HIV envelope peptide‐cocktail. Compared with direct antigen‐specific stimulation of peripheral blood mononuclear cells, the DC‐ELISPOT protocol involving co‐culturing of macaque T cells with autologous DC pulsed with the various peptides from the vaccine cocktail yielded up to 18‐fold higher numbers of interferon‐γ producing cells without increasing the background. Importantly, use of DC in the analyses revealed immune responses in vaccinated macaques that were otherwise undetectable. Similar data were obtained when recall responses to purified protein derivative were analyzed by the DC‐ELISPOT method using blood samples from human volunteers. These data establish the importance of DC in improving detection sensitivity and eliminating false negative results, both essential for efficient monitoring of antigen‐specific cellular immune responses.

Amplified and Persistent Immune Responses Generated by Single Cycle Replicating Adenovirus Vaccines

ABSTRACT Replication-competent adenoviral (RC-Ad) vectors generate exceptionally strong gene-based vaccine responses by amplifying the antigen transgenes they carry. While they are potent, they also risk causing adenovirus infections. More common replication-defective Ad (RD-Ad) vectors with deletions of E1 avoid this risk but do not replicate their transgene and generate markedly weaker vaccine responses. To amplify vaccine transgenes while avoiding production of infectious progeny viruses, we engineered “single-cycle” adenovirus (SC-Ad) vectors by deleting the gene for IIIa capsid cement protein of lower-seroprevalence adenovirus serotype 6. In mouse, human, hamster, and macaque cells, SC-Ad6 still replicated its genome but prevented genome packaging and virion maturation. When used for mucosal intranasal immunization of Syrian hamsters, both SC-Ad and RC-Ad expressed transgenes at levels hundreds of times higher than that of RD-Ad. Surprisingly, SC-Ad, but not RC-Ad, generated higher levels of transgene-specific antibody than RD-Ad, which notably climbed in serum and vaginal wash samples over 12 weeks after single mucosal immunization. When RD-Ad and SC-Ad were tested by single sublingual immunization in rhesus macaques, SC-Ad generated higher gamma interferon (IFN-γ) responses and higher transgene-specific serum antibody levels. These data suggest that SC-Ad vectors may have utility as mucosal vaccines. IMPORTANCE This work illustrates the utility of our recently developed single-cycle adenovirus (SC-Ad6) vector as a new vaccine platform. Replication-defective (RD-Ad6) vectors produce low levels of transgene protein, which leads to minimal antibody responses in vivo. This study shows that replicating SC-Ad6 produces higher levels of luciferase and induces higher levels of green fluorescent protein (GFP)-specific antibodies than RD in a permissive Syrian hamster model. Surprisingly, although a replication-competent (RC-Ad6) vector produces more luciferase than SC-Ad6, it does not elicit comparable levels of anti-GFP antibodies in permissive hamsters. When tested in the larger rhesus macaque model, SC-Ad6 induces higher transgene-specific antibody and T cell responses. Together, these data suggest that SC-Ad6 could be a more effective platform for developing vaccines against more relevant antigens. This could be especially beneficial for developing vaccines for pathogens for which traditional replication-defective adenovirus vectors have not been effective.

Prime-boost vaccination using chemokine-fused gp120 DNA and HIV envelope peptides activates both immediate and long-term memory cellular responses in rhesus macaques

HIV vaccine candidates with improved immunogenicity and induction of mucosal T-cell immunity are needed. A prime-boost strategy using a novel HIV glycoprotein 120 DNA vaccine was employed to immunize rhesus macaques. The DNA vaccine encoded a chimeric gp120 protein in fusion with monocyte chemoattractant protein-3, which was hypothesized to improve the ability of antigen-presenting cells to capture viral antigen through chemokine receptor-mediated endocytosis. DNA vaccination induced virus-reactive T cells in peripheral blood, detectable by T cell proliferation, INFγ ELISPOT and sustained IL-6 production, without humoral responses. With a peptide-cocktail vaccine containing a set of conserved polypeptides of HIV-1 envelope protein, given by nasogastric administration, primed T-cell immunity was significantly boosted. Surprisingly, long-term and peptide-specific mucosal memory T-cell immunity was detected in both vaccinated macaques after one year. Therefore, data from this investigation offer proof-of-principle for potential effectiveness of the prime-boost strategy with a chemokine-fused gp120 DNA and warrant further testing in the nonhuman primate models for developing as a potential HIV vaccine candidate in humans.

Transcutaneously refillable nanofluidic implant achieves sustained level of tenofovir diphosphate for HIV pre-exposure prophylaxis

ABSTRACT Pre‐exposure prophylaxis (PrEP) with antiretroviral (ARV) drugs are effective at preventing human immunodeficiency virus (HIV) transmission. However, implementation of PrEP presents significant challenges due to poor user adherence, low accessibility to ARVs and multiple routes of HIV exposure. To address these challenges, we developed the nanochannel delivery implant (NDI), a subcutaneously implantable device for sustained and constant delivery of tenofovir alafenamide (TAF) and emtricitabine (FTC) for HIV PrEP. Unlike existing drug delivery platforms with finite depots, the NDI incorporates ports allowing for transcutaneous refilling upon drug exhaustion. NDI‐mediated drug delivery in rhesus macaques resulted in sustained release of both TAF and FTC for 83 days, as indicated by concentrations of TAF, FTC and their respectively metabolites in plasma, PBMCs, rectal mononuclear cells and tissues associated with HIV transmission. Notably, clinically relevant preventative levels of tenofovir diphosphate were achieved as early as 3 days after NDI implantation. We also demonstrated the feasibility of transcutaneous drug refilling to extend the duration of PrEP drug delivery in NHPs. Overall, the NDI represents an innovative strategy for long‐term HIV PrEP administration in both developed and developing countries. Graphical abstract Figure. No caption available. HighlightsRefillable nanofluidic implant achieves preventive level of tenofovir diphosphate for HIV pre‐exposure prophylaxis.

Kinetics of Intratumoral Immune Cell Activation During Chemoradiation for Cervical Cancer

PURPOSE Radiation therapy has direct cytotoxic effects on tumor-infiltrating lymphocytes, but it also has immune stimulatory effects that increase immune cell infiltration. The dynamics of these competing effects on immune cells at the site of the tumor are poorly characterized during chemoradiation treatment (CRT) because of the difficulty of obtaining consecutive tumor biopsies. We used a minimally invasive cervical cytobrushing method to analyze the kinetics of intratumoral immune cell changes in patients with cervical cancer during CRT. METHODS AND MATERIALS Cervical brushings were obtained from 20 patients with cervical cancer at baseline and during fractionated radiation therapy and cisplatin (weeks 1, 3, and 5). Matching peripheral blood mononuclear cells were obtained from 9 patients at the same time points. Cells were analyzed using multispectral flow cytometry to identify T cell and myeloid cell subsets and their activation status. Changes in immune cell subsets throughout treatment were calculated using matched-pair analysis with Wilcoxon rank sum test. RESULTS We observed a significant decline in CD3+ total T cells, as well as CD8+ and CD4+ T-cell subsets in the first week of treatment from baseline, followed by variable expansion at weeks 3 and 5. This coincided with higher levels of proliferating CD8+ T cells expressing Ki67 at week 3 of treatment. The percentages of activated CD8+ T cells expressing CD69 continuously increased over the course of treatment, whereas the percentage of activated CD11c+CD11b- dendritic cells was highest during the first week. Many of these changes were not observed in the blood. CONCLUSIONS Our results identified immune dynamic changes during CRT, indicating that CRT may be immune activating at the site of the tumor. This study also suggests the importance of sequential analyses of the local tumor microenvironment in addition to peripheral blood.

Efficacy of sustained delivery of GC-1 from a Nanofluidic system in a spontaneously obese non-human primate: a case study

With nearly 40% of U.S. adults obese, and childhood and adolescent rates rising, obesity and associated comorbidities are serious public health concerns with massive societal costs. Often, lifestyle interventions do not offer sufficient weight loss to improve health, requiring surgery and medications as adjunct management strategies. Here, we present a 4-month case study in which the sustained, low-dose, and constant administration of the thyroid receptor β selective agonist GC-1 (sobetirome) from a novel nanochannel membrane implant was assessed in an obese, pre-diabetic rhesus macaque. Dramatic loss of white adipose tissue in the abdomen from 36 to 18% was observed via magnetic resonance imaging in conjunction with normalized serum insulin and glycemia, with no signs of cardiotoxicity shown. The non-human primate study highlights sustained low-dose delivery of GC-1 from our minimally invasive subcutaneous implant as a valuable approach to induce weight loss and manage obesity and comorbidities, including type 2 diabetes.