HIV Antigen Incorporation within Adenovirus Hexon Hypervariable 2 for a Novel HIV Vaccine Approach

Adenoviral (Ad) vectors have been used for a variety of vaccine applications including cancer and infectious diseases. Traditionally, Ad-based vaccines are designed to express antigens through transgene expression of a given antigen. However, in some cases these conventional Ad-based vaccines have had sub-optimal clinical results. These sub-optimal results are attributed in part to pre-existing Ad serotype 5 (Ad5) immunity. In order to circumvent the need for antigen expression via transgene incorporation, the “antigen capsid-incorporation” strategy has been developed and used for Ad-based vaccine development in the context of a few diseases. This strategy embodies the incorporation of antigenic peptides within the capsid structure of viral vectors. The major capsid protein hexon has been utilized for these capsid incorporation strategies due to hexon's natural role in the generation of anti-Ad immune response and its numerical representation within the Ad virion. Using this strategy, we have developed the means to incorporate heterologous peptide epitopes specifically within the major surface-exposed domains of the Ad capsid protein hexon. Our study herein focuses on generation of multivalent vaccine vectors presenting HIV antigens within the Ad capsid protein hexon, as well as expressing an HIV antigen as a transgene. These novel vectors utilize HVR2 as an incorporation site for a twenty-four amino acid region of the HIV membrane proximal ectodomain region (MPER), derived from HIV glycoprotein gp41 (gp41). Our study herein illustrates that our multivalent anti-HIV vectors elicit a cellular anti-HIV response. Furthermore, vaccinations with these vectors, which present HIV antigens at HVR2, elicit a HIV epitope-specific humoral immune response

Expression of inflammasome proteins and inflammasome activation occurs in human, but not in murine keratinocytes

Inflammasomes are multimeric protein complexes that assemble upon sensing of a variety of stress factors. Their formation results in caspase-1-mediated activation and secretion of the pro-inflammatory cytokines pro-interleukin(IL)-1β and -18, which induce an inflammatory response. Inflammation is supported by a lytic form of cell death, termed pyroptosis. Innate immune cells, such as macrophages or dendritic cells, express and activate inflammasomes. However, it has also been demonstrated that human primary keratinocytes activate different types of inflammasomes in vitro, for example, upon UVB irradiation or viral infection. Keratinocytes are the main cell type of the epidermis, the outermost layer of the body, and form a protective barrier consisting of a stratified multi-layered epithelium. In human, gain-of-function mutations of the NLRP1 gene cause syndromes mediated by inflammasome activation in keratinocytes that are characterised by skin inflammation and skin cancer susceptibility. Here we demonstrate that murine keratinocytes do not activate inflammasomes in response to stimuli, which induce IL-1β and -18 secretion by human keratinocytes. Whereas murine keratinocytes produced caspase-1 and proIL-18, expression of the inflammasome proteins Nlrp1, Nlrp3, Aim2, Asc, and proIL-1β was, compared to human keratinocytes or murine dendritic cells, very low or even undetectable. Priming of murine keratinocytes with cytokines commonly used for induction of proIL-1β and inflammasome protein expression did not rescue inflammasome activation. Nevertheless, UVB-induced inflammation and neutrophil recruitment in murine skin was dependent on IL-1β and caspase-1. However, also under these conditions, we did not detect expression of proIL-1β by keratinocytes in murine skin, but by immune cells. These results demonstrate a higher immunological competence of human compared to murine keratinocytes, which is reflected by stress-induced IL-1β secretion that is mediated by inflammasomes. Therefore, keratinocytes in human skin can exert immune functions, which are carried out by professional immune cells in murine skin

Effects of Herpes Simplex Virus Amplicon Transduction on Murine Dendritic Cells

The herpes simplex virus (HSV)-based amplicon is a versatile vaccine platform that has been preclinically vetted as a gene-based immunotherapeutic for cancer, HIV, and neurodegenerative disorders. Although it is well known that injection of dendritic cells (DCs) transduced ex vivo with helper virus-free HSV amplicon vectors expressing disease-relevant antigens induces antigen-specific immune responses, the cellular receptor(s) by which the amplicon virion gains entry into DCs, as well as the effects that viral vector transduction impinges on the physiological status of these cells, is less understood. Herein, we examine the effects of amplicon transduction on mouse bone marrow-derived DCs. We demonstrate that HSV-1 cellular receptors HveC and HveA are expressed on the cell surface of murine DCs, and that HSV amplicons transduce DCs at high efficiency (>90%) with minimal effects on cell viability. Transduction of dendritic cells with amplicons induces a transient DC maturation phenotype as represented by self-limited upregulation of MHCII and CD11c markers. Mature DCs are less sensitive to HSV amplicon transduction than immature DCs regarding DC-related surface marker maintenance. From this and our previous work, we conclude that HSV amplicons transduce DCs efficiently, but impart differential and transient physiological effects on mature and immature DC pools, which will facilitate fine-tuning of this vaccination platform and further exploit its potential in immunotherapy

HIV epitopes incorporated in HVR2 are exposed on the virion surface.

<p>A) In the assay, varying amounts of AdCMVGag, Ad5/HVR2-MPER24-L15(Gag), and Ad5/HVR2-MPER-L15Δ<i>E1</i> were immobilized in the wells of ELISA plates and incubated with anti-gp41 antibody. The binding was detected with an HRP-conjugated secondary antibody. B) In the assay 6×10⁸ VP of either AdCMVGag, Ad5/HVR2-MPER-L15(Gag), and Ad5/HVR2-MPER-L15Δ<i>E1</i> were immobilized on an ELISA plate followed by varying dilutions of gp41 antibody (1;6,000; 1∶3,000;1∶1,500; and 1∶750). The binding was detected with an HRP-conjugated secondary antibody.</p

Capsid-modified vectors can induce a greater number of Gag-specific CD8 T cells and memory T cells than wild type vectors.

<p>Cohorts of BALB/c mice (n = 8) were immunized (prime) by injection of 10¹⁰ vp i.m. with of one of the following Ad vectors: AdCMVGag, Ad5/HVR2-MPER-L15(Gag), and Ad5/HVR2-MPER-L15Δ<i>E1</i>. Gag-specific T cells were detected in the peripheral blood of mice 2 weeks following the initial vaccination, and given a boost immunization in the same manner on day 40. Peripheral blood Gag-specific CD8 T cells were enumerated 26 d.p.p, 69 d.p.p, and 84 d.p.b. A) Flow cytometric analyses bivariate pseudocolor plots are shown for a single mouse from each group for each time point. B) The percent and total number of Gag-specific T cells per million lymphocytes are shown for each mouse and their level of T cells linked for prime and boost time points. C) Paired scatter plots show significant differences in the percent and number of Gag-specific CD8 T cells induced by either MPER-modified or AdCMVGag gene encoding vaccines. Statistical significance was determined by student's t-test, (two-tailed) P<0.03.</p