Evaluation of recombinant influenza virus-simian immunodeficiency virus vaccines in macaques

There is an urgent need for human immunodeficiency virus (HIV) vaccines that induce robust mucosal immunity. Influenza A viruses (both H1N1 and H3N2) were engineered to express simian immunodeficiency virus (SIV) CD8 T-cell epitopes and evaluated following administration to the respiratory tracts of 11 pigtail macaques. Influenza virus was readily detected from respiratory tract secretions, although the infections were asymptomatic. Animals seroconverted to influenza virus and generated CD8 and CD4 T-cell responses to influenza virus proteins. SIV-specific CD8 T-cell responses bearing the mucosal homing marker 7 integrin were induced by vaccination of naïve animals. Further, SIV-specific CD8 T-cell responses could be boosted by recombinant influenza virus-SIV vaccination of animals with already-established SIV infection. Sequential vaccination with influenza virus-SIV recombinants of different subtypes (H1N1 followed by H3N2 or vice versa) produced only a limited boost in immunity, probably reflecting T-cell immunity to conserved internal proteins of influenza A virus. SIV challenge of macaques vaccinated with an influenza virus expressing a single SIV CD8 T cell resulted in a large anamnestic recall CD8 T-cell response, but immune escape rapidly ensued and there was no impact on chronic SIV viremia. Although our results suggest that influenza virus-HIV vaccines hold promise for the induction of mucosal immunity to HIV, broader antigen cover will be needed to limit cytotoxic T-lymphocyte escape

Controlling the Biological Fate of Micellar Nanoparticles: Balancing Stealth and Targeting

Copyright © 2020 American Chemical Society. Integrating nanomaterials with biological entities has led to the development of diagnostic tools and biotechnology-derived therapeutic products. However, to optimize the design of these hybrid bionanomaterials, it is essential to understand how controlling the biological interactions will influence desired outcomes. Ultimately, this knowledge will allow more rapid translation from the bench to the clinic. In this paper, we developed a micellar system that was assembled using modular antibody-polymer amphiphilic materials. The amphiphilic nature was established using either poly(ethylene glycol) (PEG) or a single-chain variable fragment (scFv) from an antibody as the hydrophile and a thermoresponsive polymer (poly(oligoethylene glycol) methyl ether methacrylate) as the hydrophobe. By varying the ratios of these components, a series of nanoparticles with different antibody content was self-assembled, where the surface presentation of targeting ligand was carefully controlled. In vitro and in vivo analysis of these systems identified a mismatch between the optimal targeting ligand density to achieve maximum cell association in vitro compared to tumor accumulation in vivo. For this system, we determined an optimum antibody density for both longer circulation and enhanced targeting to tumors that balanced stealthiness of the particle (to evade immune recognition as determined in both mouse models and in whole human blood) with enhanced accumulation achieved through receptor binding on tumor cells in solid tumors. This approach provides fundamental insights into how different antibody densities affect the interaction of designed nanoparticles with both target cells and immune cells, thereby offering a method to probe the intricate interplay between increased targeting efficiency and the subsequent immune response to nanoparticles.

Timing of immune escape linked to success or failure of vaccination

Successful vaccination against HIV should limit viral replication sufficiently to prevent the emergence of viral immune escape mutations. Broadly directed immunity is likely to be required to limit opportunities for immune escape variants to flourish. We studied the emergence of an SIV Gag cytotoxic T cell immune escape variant in pigtail macaques expressing the Mane-A*10 MHC I allele using a quantitative RT-PCR to measure viral loads of escape and wild type variants. Animals receiving whole Gag expressing vaccines completely controlled an SIVmac251 challenge, had broader CTL responses and exhibited minimal CTL escape. In contrast, animals vaccinated with only a single CTL epitope and challenged with the same SIVmac251 stock had high levels of viral replication and rapid CTL escape. Unvaccinated na&iuml;ve animals exhibited a slower emergence of immune escape variants. Thus narrowly directed vaccination against a single epitope resulted in rapid immune escape and viral levels equivalent to that of na&iuml;ve unvaccinated animals. These results emphasize the importance of inducing broadly directed HIV-specific immunity that effectively quashes early viral replication and limits the generation of immune escape variants. This has important implications for the selection of HIV vaccines for expanded human trials.<br /

Complexity of the Inoculum Determines the Rate of Reversion of SIV Gag CD8 T Cell Mutant Virus and Outcome of Infection

Escape mutant (EM) virus that evades CD8+ T cell recognition is frequently observed following infection with HIV-1 or SIV. This EM virus is often less replicatively “fit” compared to wild-type (WT) virus, as demonstrated by reversion to WT upon transmission of HIV to a naïve host and the association of EM virus with lower viral load in vivo in HIV-1 infection. The rate and timing of reversion is, however, highly variable. We quantified reversion to WT of a series of SIV and SHIV viruses containing minor amounts of WT virus in pigtail macaques using a sensitive PCR assay. Infection with mixes of EM and WT virus containing ≥10% WT virus results in immediate and rapid outgrowth of WT virus at SIV Gag CD8 T cell epitopes within 7 days of infection of pigtail macaques with SHIV or SIV. In contrast, infection with biologically passaged SHIVmn229 viruses with much smaller proportions of WT sequence, or a molecular clone of pure EM SIVmac239, demonstrated a delayed or slow pattern of reversion. WT virus was not detectable until ≥8 days after inoculation and took ≥8 weeks to become the dominant quasispecies. A delayed pattern of reversion was associated with significantly lower viral loads. The diversity of the infecting inoculum determines the timing of reversion to WT virus, which in turn predicts the outcome of infection. The delay in reversion of fitness-reducing CD8 T cell escape mutations in some scenarios suggests opportunities to reduce the pathogenicity of HIV during very early infection

Charge has a marked influence on hyperbranched polymer nanoparticle association in whole human blood

In this study, we synthesize charge-varied hyperbranched polymers (HBPs) and demonstrate surface charge as a key parameter directing their association with specific human blood cell types. Using fresh human blood, we investigate the association of 5 nm HBPs with six white blood cell populations in their natural milieu by flow cytometry. While most cell types associate with cationic HBPs at 4 °C, at 37 °C phagocytic cells display similar (monocyte, dendritic cell) or greater (granulocyte) association with anionic HBPs compared to cationic HBPs. Neutral HBPs display remarkable stealth properties. Notably, these charge-association patterns are not solely defined by the plasma protein corona and are material and/or size dependent. As HBPs progress toward clinical use as imaging and drug delivery agents, the ability to engineer HBPs with defined biological properties is increasingly important. This knowledge can be used in the rational design of HBPs for more effective delivery to desired cell targets

Inactivated Simian Immunodeficiency Virus-Pulsed Autologous Fresh Blood Cells as an Immunotherapy Strategy▿ †

Practical immunotherapies for human immunodeficiency virus infection are needed. We evaluated inactivated simian immunodeficiency virus (SIV) pulsed onto fresh peripheral blood mononuclear cells in 12 pigtail macaques with chronic SIVmac251 infection for T-cell immunogenicity in a randomized cross-over design study. The immunotherapy was safe and convincingly induced high levels of SIV-specific CD4+ T-cell responses (mean, 5.9% ± 1.3% of all CD4+ T cells) and to a lesser extent SIV-specific CD8+ T-cell responses (mean, 0.7% ± 0.4%). Responses were primarily directed toward Gag and less frequently toward Env but not Pol or regulatory/accessory SIV proteins. T-cell responses against Gag were generally broad and polyfunctional, with a mean of 2.7 CD4+ T-cell epitopes mapped per animal and more than half of the SIV Gag-specific CD4+ T cells expressing three or more effector molecules. The immunogenicity was comparable to that found in previous studies of peptide-pulsed blood cells. Despite the high-level immunogenicity, no reduction in viral load was observed in the chronically viremic macaques. This contrasts with our studies of immunization with peptide-pulsed blood cells during early SIV infection in macaques. Future studies of inactivated virus-pulsed blood cell immunotherapy during early infection of patients receiving antiretroviral therapy are warranted

Influenza infection enhances antibody-mediated NK cell functions via Type I interferon dependent pathways

NK cells are an important component in the control of influenza infection, acting to both clear virus-infected cells and release antiviral cytokines. Engagement of CD16 on NK cells by antibody-coated influenza-infected cells results in antibody-dependent cellular cytotoxicity (ADCC). Increasing the potency of antibody-mediated NK cell activity could ultimately lead to improved control of influenza infection. To understand if NK cells can be functionally enhanced following exposure to influenza virus-infected cells, we co-cultured human PBMCs with influenza-infected human alveolar epithelial (A549) cells and evaluated the capacity of NK cells to mediate antibody-dependent functions. Pre-incubation of PBMCs with influenza-infected cells markedly enhanced the ability of NK cells to respond to immune complexes containing HA and anti-HA antibodies or transformed allogenic cells in the presence or absence of a therapeutic monoclonal antibody. Cytokine multiplex, RNA sequencing, supernatant transfer, trans-well and cytokine blocking/supplementation experiments showed that type I interferons released from PBMCs were primarily responsible for the influenza-induced enhancement of antibody-mediated NK cell functions. Importantly, the influenza-mediated increase in antibody-dependent NK cell functionality was mimicked by the type I interferon agonist poly(I:C). We conclude that type I interferon secretion induced by influenza virus infection enhances the capacity of NK cells to mediate ADCC, and this pathway could be manipulated to alter the potency of anti-influenza therapies and vaccines

Balancing Reversion of Cytotoxic T-Lymphocyte and Neutralizing Antibody Escape Mutations within Human Immunodeficiency Virus Type 1 Env upon Transmission▿ †

Human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is subject to both neutralizing antibody (NAb) and CD8 T-cell (cytotoxic T-lymphocyte [CTL]) immune pressure. We studied the reversion of the Env CTL escape mutant virus to the wild type and the relationship between the reversion of CTL mutations with N-linked glycosylation site (NLGS)-driven NAb escape in pigtailed macaques. Env CTL mutations either did not revert to the wild type or only transiently reverted 5 to 7 weeks after infection. The CTL escape mutant reversion was coincident, for the same viral clones, with the loss of NLGS mutations. At one site studied, both CTL and NLGS mutations were needed to confer NAb escape. We conclude that CTL and NAb escape within Env can be tightly linked, suggesting opportunities to induce effective multicomponent anti-Env immunity

Templated Polymer Replica Nanoparticles to Facilitate Assessment of Material-Dependent Pharmacokinetics and Biodistribution

Surface modification is frequently used to tailor the interactions of nanoparticles with biological systems. In many cases, the chemical nature of the treatments employed to modify the biological interface (for example attachment of hydrophilic polymers or targeting groups) is the focus of attention. However, isolation of the fundamental effects of the materials employed to modify the interface are often confounded by secondary effects imparted by the underlying substrate. Herein, we demonstrate that polymer replica particles templated from degradable mesoporous silica provide a facile means to evaluate the impact of surface modification on the biological interactions of nanomaterials, independent of the substrate. Poly­(ethylene glycol) (PEG), poly­(<i>N</i>-(2 hydroxypropyl)­methacrylamide) (PHPMA), and poly­(methacrylic acid) (PMA) were templated onto mesoporous silica and cross-linked and the residual particles were removed. The resulting nanoparticles, comprising interfacial polymer alone, were then investigated using a range of in vitro and in vivo tests. As expected, the PEG particles showed the best stealth properties, and these trends were consistent in both in vitro and in vivo studies. PMA particles showed the highest cell association in cell lines in vitro and were rapidly taken up by monocytes in ex vivo whole blood, properties consistent with the very high in vivo clearance subsequently seen in rats. In contrast, PHPMA particles showed rapid association with both granulocytes and monocytes in ex vivo whole blood, even though in vivo clearance was less rapid than the PMA particles. Rat studies confirmed better systemic exposure for PEG and PHPMA particles when compared to PMA particles. This study provides a new avenue for investigating material-dependent biological behaviors of polymer particles, irrespective of the properties of the underlying core, and provides insights for the selection of polymer particles for future biological applications