A Self-Emulsified Adjuvant System Containing the Immune Potentiator Alpha Tocopherol Induces Higher Neutralizing Antibody Responses than a Squalene-Only Emulsion When Evaluated with a Recombinant Cytomegalovirus (CMV) Pentamer Antigen in Mice.

The development of new vaccine adjuvants represents a key approach to improvingi the immune responses to recombinant vaccine antigens. Emulsion adjuvants, such as AS03 and MF59, in combination with influenza vaccines, have allowed antigen dose sparing, greater breadth of responses and fewer immunizations. It has been demonstrated previously that emulsion adjuvants can be prepared using a simple, low-shear process of self-emulsification (SE). The role of alpha tocopherol as an immune potentiator in emulsion adjuvants is clear from the success of AS03 in pandemic responses, both to influenza and COVID-19. Although it was a significant formulation challenge to include alpha tocopherol in an emulsion prepared by a low-shear process, the resultant self-emulsifying adjuvant system (SE-AS) showed a comparable effect to the established AS03 when used with a quadrivalent influenza vaccine (QIV). In this paper, we first optimized the SE-AS with alpha tocopherol to create SE-AS44, which allowed the emulsion to be sterile-filtered. Then, we compared the in vitro cell activation cytokine profile of SE-AS44 with the self-emulsifying adjuvant 160 (SEA160), a squalene-only adjuvant. In addition, we evaluated SE-AS44 and SEA160 competitively, in combination with a recombinant cytomegalovirus (CMV) pentamer antigen mouse.

Rapamycin reduces CCR5 mRNA levels in macaques: potential applications in HIV-1 prevention and treatment.

G1 cytostatic drugs reduce CCR5 co-receptor expression and enhance the antiviral activity of a CCR5 antagonist in vitro. The administration of rapamycin, a G1 cytostatic agent, to three cynomolgous macaques led to decreased CCR5 messenger RNA expression in peripheral blood mononuclear cells and cervicovaginal tissue. These results support further clinical evaluation of G1 cytostatic agents such as rapamycin targeting the downregulation of CCR5 expression as a strategy for both the prevention and treatment of HIV infection.

CCR5 density levels on primary CD4 T cells impact the replication and Enfuvirtide susceptibility of R5 HIV-1.

OBJECTIVE AND DESIGN: Studies in cell lines have demonstrated that CCR5 coreceptor levels influence the replication efficiency and Enfuvirtide (T-20) susceptibility of R5 HIV-1 strains. At present, however, the role that CCR5 levels on primary CD4 T cells--which are markedly lower than in cell lines and vary only approximately fivefold among most donors--may play in virus replication levels or susceptibility to T-20 is not known. In the present study we evaluated the impact of differences in CCR5 levels among donor CD4 T cells on the infection efficiency and T-20 susceptibility of R5 HIV-1. METHODS: CD4 and CCR5 density levels were determined by Quantitative FACS analysis. Virus infectivity assays were conducted in cell lines and primary cells. Associations between coreceptor density, virus replication and T-20 sensitivity were tested using the Spearman's correlation test. RESULTS: We found a positive correlation (r, 0.55; P = 0.011) between CCR5 density levels on primary CD4 T cells and replication of R5 HIV-1. In cell lines expressing physiologically relevant levels of CD4 and CCR5, T-20 was significantly more potent in cells with low CCR5 levels. In addition, T20 50% inhibitory concentrations for R5 HIV-1 replication varied approximately 100-fold among primary cells from different donors and they were positively correlated with CCR5 density values (r, 0.84; P = 0.00004). CONCLUSIONS: These results suggest that CCR5 density levels in HIV-1 patients may impact the activity of T-20 against R5 strains and that therapeutic approaches to alter CCR5 density may potentiate T-20.

In vitro suppression of latent HIV-1 activation by vitamin E: potential clinical implications.

We evaluated the effect of vitamin E in controlling HIV-1 production upon activation of the patients' reservoir of resting CD4 lymphocytes in tissue culture experiments. The addition of vitamin E to patients' cultures resulted in significantly reduced levels of p24 virus production (P = 0.0015). These results suggest that vitamin E supplementation may interfere with the emergence of drug-resistant HIV-1 variants archived in the resting cell reservoir and delay or limit virus rebound upon treatment interruptions.

Indirubin-3'-monoxime, a derivative of a Chinese antileukemia medicine, inhibits P-TEFb function and HIV-1 replication.

OBJECTIVE: To evaluate the effects of the cyclin dependent kinase (CDK) inhibitor Indirubin-3'-monoxime (IM) on Tat-mediated transactivation function, a step of the HIV-1 cycle that is not currently targeted in antiviral therapy. METHODS: The effects of IM on CDK implicated in HIV-1 Tat transactivation function were evaluated by kinase assays, transfection experiments, RNase protection assay and RT-PCR analysis of viral transcripts. The antiviral effect of IM was investigated in cells from HIV-1 infected individuals as well as in cell lines, primary lymphocytes and monocyte-derived macrophages. The antiviral activity of IM was also tested against drug-resistant HIV-1. RESULTS: IM inhibits the kinase activity of CDK9 [50% inhibitory concentration (IC50) of 0.05 microM], the catalytic subunit of Positive transcription elongation factor b (P-TEFb). Inhibition of CDK9 activity by IM results in abrogation of Tat-induced expression of HIV-1 RNA in cell lines. In addition, IM inhibits the replication of HIV-1 in both peripheral blood mononuclear cells (IC50 of 1 microM) and macrophages (IC50 of 0.5 microM). IM is effective against primary and drug-resistant strains of HIV-1. Importantly, the antiviral effects of the drug were seen at concentrations that did not affect cell proliferation. CONCLUSIONS: Non-toxic concentrations of IM inhibit HIV-1 by blocking viral gene expression mediated by the cellular factor P-TEFb. The drug is effective against wild-type and drug-resistant strains of HIV-1. IM may help control replication of HIV-1 in patients by disrupting a step of the HIV-1 cycle that is not being targeted in current antiretroviral treatments.

TSG101 exposure on the surface of HIV-1 infected cells: implications for monoclonal antibody therapy for HIV/AIDS.

HIV infection remains a major global public health problem, in part because of the ability of the virus to elude antiretroviral therapies. Most conventional drugs were designed to directly target virus-encoded mechanisms. However, there is increasing appreciation that certain host-encoded molecules are comparably important for the viral life cycle and could therefore represent potential antiviral targets. Prominent among these is TSG101, a cytoplasmic molecule that is "hijacked" by HIV and used to facilitate viral budding and release. In our present report, we demonstrate thatTSG101 is uniquely exposed on the surface of HIV-infected cells and is available to antibody-based therapies. We also characterize the development of a monoclonal antibody, CB8-2, which reduces virus production from infected cells. These studies demonstrate the potential of TSG101-directed antibodies to combat HIV/AIDS.

The use of Random Homozygous Gene Perturbation to identify novel host-oriented targets for influenza.

Conventional approaches for therapeutic targeting of viral pathogens have consistently faced obstacles arising from the development of resistant strains and a lack of broad-spectrum application. Influenza represents a particularly problematic therapeutic challenge since high viral mutation rates have often confounded many conventional antivirals. Newly emerging or engineered strains of influenza represent an even greater threat as typified by recent interest in avian subtypes of influenza. Based on the limitations associated with targeting virally-encoded molecules, we have taken an orthogonal approach of targeting host pathways in a manner that prevents viral propagation or spares the host from virus-mediated pathogenicity. To this end, we report herein the application of an improved technology for target discovery, Random Homozygous Gene Perturbation (RHGP), to identify host-oriented targets that are well-tolerated in normal cells but that prevent influenza-mediated killing of host cells. Improvements in RHGP facilitated a thorough screening of the entire genome, both for overexpression or loss of expression, to identify targets that render host cells resistant to influenza infection. We identify a set of host-oriented targets that prevent influenza killing of host cells and validate these targets using multiple approaches. These studies provide further support for a new paradigm to combat viral disease and demonstrate the power of RHGP to identify novel targets and mechanisms.

Rapamycin reduces CCR5 density levels on CD4 T cells, and this effect results in potentiation of enfuvirtide (T-20) against R5 strains of human immunodeficiency virus type 1 in vitro.

The CCR5 chemokine receptor plays a pivotal role in human immunodeficiency virus type 1 (HIV-1) infection. Several studies have suggested that CCR5 density levels in individuals are rate limiting for infection. In addition, CCR5 density levels influence the antiviral activity of the HIV-1 fusion inhibitor enfuvirtide (T-20) against R5 strains. In the present study we demonstrate that rapamycin (RAPA), a drug approved for the treatment of renal transplantation rejection, reduces CCR5 density levels on CD4 T cells and inhibits R5 HIV-1 replication. In addition, RAPA increased the antiviral activity of T-20 against R5 strains of the virus in a cell-cell fusion assay and as shown by quantification of early products of viral reverse transcription. Median-effect analysis of drug interaction between RAPA and T-20 in infectivity assays using donor peripheral blood mononuclear cells demonstrated that the RAPA-T-20 combination is synergistic against R5 strains of HIV-1 and this synergy translates into T-20 dose reductions of up to approximately 33-fold. Importantly, RAPA effects on replication levels and T-20 susceptibility of R5 strains of HIV-1 were observed at drug concentrations that did not inhibit cell proliferation. These results suggest that low concentrations of RAPA may potentiate the antiviral activity of T-20 against R5 strains of HIV-1, which are generally present throughout the course of infection and are less sensitive to T-20 inhibition than are X4 strains.