Selective FcγR Co-engagement on APCs Modulates the Activity of Therapeutic Antibodies Targeting T Cell Antigens.

The co-engagement of fragment crystallizable (Fc) gamma receptors (FcγRs) with the Fc region of recombinant immunoglobulin monoclonal antibodies (mAbs) and its contribution to therapeutic activity has been extensively studied. For example, Fc-FcγR interactions have been shown to be important for mAb-directed effector cell activities, as well as mAb-dependent forward signaling into target cells via receptor clustering. Here we identify a function of mAbs targeting T cell-expressed antigens that involves FcγR co-engagement on antigen-presenting cells (APCs). In the case of mAbs targeting CTLA-4 and TIGIT, the interaction with FcγR on APCs enhanced antigen-specific T cell responses and tumoricidal activity. This mechanism extended to an anti-CD45RB mAb, which led to FcγR-dependent regulatory T cell expansion in mice.

Toxicological and pharmacological assessment of AGEN1884, a novel human IgG1 anti-CTLA-4 antibody.

CTLA-4 and CD28 exemplify a co-inhibitory and co-stimulatory signaling axis that dynamically sculpts the interaction of antigen-specific T cells with antigen-presenting cells. Anti-CTLA-4 antibodies enhance tumor-specific immunity through a variety of mechanisms including: blockade of CD80 or CD86 binding to CTLA-4, repressing regulatory T cell function and selective elimination of intratumoral regulatory T cells via an Fcγ receptor-dependent mechanism. AGEN1884 is a novel IgG1 antibody targeting CTLA-4. It potently enhanced antigen-specific T cell responsiveness that could be potentiated in combination with other immunomodulatory antibodies. AGEN1884 was well-tolerated in non-human primates and enhanced vaccine-mediated antigen-specific immunity. AGEN1884 combined effectively with PD-1 blockade to elicit a T cell proliferative response in the periphery. Interestingly, an IgG2 variant of AGEN1884 revealed distinct functional differences that may have implications for optimal dosing regimens in patients. Taken together, the pharmacological properties of AGEN1884 support its clinical investigation as a single therapeutic and combination agent.

HIV-1 Tat second exon limits the extent of Tat-mediated modulation of interferon-stimulated genes in antigen presenting cells.

BACKGROUND: We have shown that HIV-1 Tat interaction with MAP2K3, MAP2K6, and IRF7 promoters is key to IFN-stimulated genes (ISG) activation in immature dendritic cells and macrophages. RESULTS: We evaluated how Tat alleles and mutants differ in cellular gene modulation of immature dendritic cells and monocyte-derived macrophages and what similarities this modulation has with that induced by interferons. The tested alleles and mutants modulated to different degrees ISG, without concomitant induction of interferons. The first exon TatSF21-72 and the minimal transactivator TatSF21-58, all modulated genes to a significantly greater extent than full-length wild type, two-exon Tat, indicating that Tat second exon is critical in reducing the innate response triggered by HIV-1 in these cells. Mutants with reduced LTR transactivation had a substantially reduced effect on host gene expression modulation than wild type TatSF2. However, the more potent LTR transactivator TatSF2A58T modulated ISG expression to a lower degree compared to TatSF2. A cellular gene modulation similar to that induced by Tat and Tat mutants in immature dendritic cells could be observed in monocyte-derived macrophages, with the most significant pathways affected by Tat being the same in both cell types. Tat expression in cells deleted of the type I IFN locus or receptor resulted in a gene modulation pattern similar to that induced in primary immature dendritic cells and monocyte-derived macrophages, excluding the involvement of type I IFNs in Tat-mediated gene modulation. ISG activation depends on Tat interaction with MAP2K3, MAP2K6, and IRF7 promoters and a single exon Tat protein more strongly modulated the luciferase activity mediated by MAP2K3, MAP2K6, and IRF7 promoter sequences located 5' of the RNA start site than the wild type two-exon Tat, while a cysteine and lysine Tat mutants, reduced in LTR transactivation, had negligible effects on these promoters. Chemical inhibition of CDK9 or Sp1 decreased Tat activation of MAP2K3-, MAP2K6-, and IRF7-mediated luciferase transcription. CONCLUSIONS: Taken together, these data indicate that the second exon of Tat is critical to the containment of the innate response stimulated by Tat in antigen presenting cells and support a role for Tat in stimulating cellular transcription via its interaction with transcription factors present at promoters.

Resistance to infection, early and persistent suppression of simian immunodeficiency virus SIVmac251 viremia, and significant reduction of tissue viral burden after mucosal vaccination in female rhesus macaques.

The efficacy of oral, intestinal, nasal, and vaginal vaccinations with DNA simian immunodeficiency virus (SIV)/interleukin-2 (IL-2)/IL-15, SIV Gag/Pol/Env recombinant modified vaccinia virus Ankara (rMVA), and AT-2 SIVmac239 inactivated particles was compared in rhesus macaques after low-dose vaginal challenge with SIVmac251. Intestinal immunization provided better protection from infection, as a significantly greater median number of challenges was necessary in this group than in the others. Oral and nasal vaccinations provided the most significant control of disease progression. Fifty percent of the orally and nasally vaccinated animals suppressed viremia to undetectable levels, while this occurred to a significantly lower degree in intestinally and vaginally vaccinated animals and in controls. Viremia remained undetectable after CD8(+) T-cell depletion in seven vaccinated animals that had suppressed viremia after infection, and tissue analysis for SIV DNA and RNA was negative, a result consistent with a significant reduction of viral activity. Regardless of the route of vaccination, mucosal vaccinations prevented loss of CD4(+) central memory and CD4(+)/α4ß7(+) T-cell populations and reduced immune activation to different degrees. None of the orally vaccinated animals and only one of the nasally vaccinated animals developed AIDS after 72 to 84 weeks of infection, when the trial was closed. The levels of anti-SIV gamma interferon-positive, CD4(+), and CD8(+) T cells at the time of first challenge inversely correlated with viremia and directly correlated with protection from infection and longer survival.

Immunogenicity of a vaccine regimen composed of simian immunodeficiency virus DNA, rMVA, and viral particles administered to female rhesus macaques via four different mucosal routes.

A comparative evaluation of the immunity stimulated with a vaccine regimen that includes simian immunodeficiency virus (SIV), interleukin 2 (IL-2), and IL-15 DNAs, recombinant modified vaccinia virus Ankara (rMVA), and inactivated SIVmac239 particles administered into the oral and nasal cavities, small intestine, and vagina was carried out in female rhesus macaques to determine the best route to induce diverse anti-SIV immunity that may be critical to protection from SIV infection and disease. All four immunizations generated mucosal SIV-specific IgA. Oral immunization was as effective as vaginal immunization in inducing SIV-specific IgA in vaginal secretions and generated greater IgA responses in rectal secretions and saliva samples compared to the other immunization routes. All four immunizations stimulated systemic T-cell responses against Gag and Env, albeit to a different extent, with oral immunization providing greater magnitude and nasal immunization providing wider functional heterogeneity. SIV-specific T cells producing gamma interferon (IFN-γ) dominated these responses. Limited levels of SIV-specific IgG antibodies were detected in plasma samples, and no SIV-specific IgG antibodies were detected in secretions. Vaccination also induced CD4(+) and CD8(+) T-cell responses in the rectal and vaginal mucosa with greater functional heterogeneity than in blood samples. Rectal T-cell responses were significantly greater in the orally vaccinated animals than in the other animals. The most balanced, diverse, and higher-magnitude vaginal T-cell responses were observed after intestinal vaccination. Significantly higher CD8(+) granzyme B-positive T-cell responses were observed systemically after intestinal vaccination and in rectal cells after oral immunization. The majority of SIV-specific T cells that produced granzyme B did not produce cytokines. Of the immunization routes tested, oral vaccination provided the most diverse and significant response to the vaccine.

Long-term control of simian immunodeficiency virus mac251 viremia to undetectable levels in half of infected female rhesus macaques nasally vaccinated with simian immunodeficiency virus DNA/recombinant modified vaccinia virus Ankara.

The efficacy of two SIV DNA plus recombinant modified vaccinia virus Ankara nasal vaccine regimens, one combined with plasmids expressing IL-2 and IL-15, the other with plasmids expressing GM-CSF, IL-12, and TNF-α, which may better stimulate humoral responses, was evaluated in two female rhesus macaque groups. Vaccination stimulated significant SIV-specific mucosal and systemic cell-mediated immunity in both groups, whereas SIV-specific IgA titers were sporadic and IgG titers negative. All vaccinated animals, except one, became infected after intravaginal SIV(mac251) low-dose challenge. Half of the vaccinated, infected animals (7/13) promptly controlled virus replication to undetectable viremia for the duration of the trial (130 wk) and displayed virological and immunological phenotypes similar to those of exposed, uninfected individuals. When all vaccinated animals were considered, a 3-log viremia reduction was observed, compared with controls. The excellent viral replication containment achieved in vaccinated animals translated into significant preservation of circulating α4ß7(high+)/CD4(+) T cells and of circulating and mucosal CD4(+)/C(M) T cells and in reduced immune activation. A more significant long-term survival was also observed in these animals. Median survival was 72 wk for the control group, whereas >50% of the vaccinated animals were still disease free 130 wk postchallenge, when the trial was closed. There was a statistically significant correlation between levels of CD4(+)/IFN-γ(+) and CD8(+)/IFN-γ(+) T cell percentages on the day of challenge and the control of viremia at week 60 postchallenge or survival. Postchallenge immunological correlates of protection were systemic anti-SIV Gag + Env CD4(+)/IL-2(+), CD4(+)/IFN-γ(+), and CD8(+)/TNF-α(+) T cells and vaginal anti-SIV Gag + Env CD8(+) T cell total monofunctional responses.

DNA-MVA vaccine protection after X4 SHIV challenge in macaques correlates with day-of-challenge antiviral CD4+ cell-mediated immunity levels and postchallenge preservation of CD4+ T cell memory.

The ability of vaccines to induce immunity both in mucosal and systemic compartments may be required for prevention of HIV infection and AIDS. We compared DNA-MVA vaccination regimens adjuvanted by IL-12 DNA, administered intramuscularly and nasally or only nasally. Most of the vaccinated Rhesus macaques developed mucosal and systemic humoral and cell-mediated SHIV-specific immune responses. Stimulation of mucosal anti-Env IgA responses was limited. After rectal challenge with SHIV 89.6P, all vaccinated and naive animals became infected. However, most of the vaccinated animals showed significant control of viremia and protection from CD4(+) T cell loss and AIDS progression compared to the control animals. The levels of CD4(+) and CD8(+) T cell virus-specific responses measured on the day of challenge correlated with the level of viremia control observed later during the chronic infection. Postchallenge viremia levels inversely correlated with the preservation of SHIV-specific CD4(+)/IL-2(+) and CD8(+)/TNF-alpha(+) T cells but not with CD4(+)/IFN-gamma(+) T cells measured over time after challenge. We also found that during the early chronic infection SHIV vaccination permitted a more significant preservation of both naive and memory CD4(+) T cells compared to controls. In addition, we observed a more significant and prolonged preservation of memory CD4(+) T cells after SHIV vaccination and challenge than that observed after SIV vaccination and challenge. As the antiviral immunity stimulated by vaccination is present in the memory CD4(+) T cell subpopulations, its more limited targeting by SHIV compared to SIV may explain the better control of X4 tropic SHIV than R5 tropic SIVs by vaccination.

Functional relationship between the matrix proteins of feline and simian immunodeficiency viruses.

To investigate the functional relationship between the matrix (MA) proteins of feline and simian immunodeficiency viruses (FIV and SIV, respectively), we generated chimeric proviruses in which the MA-coding region of an SIV infectious molecular clone was partially or fully replaced by its FIV counterpart. Chimeric SIV proviruses containing the amino-terminal 36 residues or the central and carboxy-terminal regions of the FIV MA assembled into virions as efficiently as wild-type SIV. However, the resulting virions were noninfectious in single-cycle infectivity assays. Furthermore, a chimeric SIV provirus containing the entire FIV MA was found to be severely impaired in virion production due to inefficient membrane binding of the chimeric Gag polyprotein. Interestingly, the assembly defective phenotype of this chimeric Gag precursor could be reversed either by introducing the G31K/G33K double amino acid substitution in the FIV-derived MA domain or by coexpression with wild-type SIV Gag. Of note, a chimeric FIV provirus expressing the SIV MA not only assembled into particles as efficiently as wild-type FIV, but also replicated in feline T cells with wild-type kinetics. Our results thus provide novel information about the functional homology between the MA proteins of distantly related lentiviruses.

Functional domains in the feline immunodeficiency virus nucleocapsid protein.

Retroviral nucleocapsid (NC) proteins are small Gag-derived products containing one or two zinc finger motifs that mediate genomic RNA packaging into virions. In this study, we addressed the role of the feline immunodeficiency virus (FIV) NC protein in the late stages of virus replication by analyzing the assembly phenotype of FIV NC mutant viruses and the RNA binding activity of a panel of recombinant FIV NC mutant proteins. Substitution of serine for the first cysteine residue in the NC proximal zinc finger was sufficient to impair both virion assembly and genomic RNA binding. A similar defective phenotype with respect to particle formation and RNA binding was observed when the basic residues Lys28 and Lys29 in the region connecting both zinc fingers were replaced by alanine. In contrast, mutation of the first cysteine residue in the distal zinc finger had no effect on virion production and allowed substantial RNA binding activity of the mutant NC protein. Moreover, this NC mutant virus exhibited wild-type replication kinetics in the feline MYA-1 T-cell line. Interestingly, amino acid substitutions disrupting the highly conserved PSAP and LLDL motifs present in the C-terminus of the FIV NC abrogated virion formation without affecting the NC RNA binding activity. Our results indicate that the proximal zinc finger of the FIV NC is more important for virion production and genomic RNA binding than the distal motif. In addition, this study suggests that assembly domains in the FIV NC C-terminus may be functionally equivalent to those present in the p6 domain of the Gag polyprotein of primate lentiviruses.