Biomedical subjects

Margherita Rosati

Publications and source records attributed to Margherita Rosati.

8 recordsLinked to original sources

PubMed2006

Long-lasting decrease in viremia in macaques chronically infected with simian immunodeficiency virus SIVmac251 after therapeutic DNA immunization.

Rhesus macaques chronically infected with highly pathogenic simian immunodeficiency virus (SIV) SIVmac251 were treated with antiretroviral drugs and vaccinated with combinations of DNA vectors expressing SIV antigens. Vaccination during therapy increased cellular immune responses. After the animals were released from therapy, the virus levels of 12 immunized animals were significantly lower (P = 0.001) compared to those of 11 animals treated with only antiretroviral drugs. Vaccinated animals showed a persistent increase in immune responses, thus indicating both a virological and an immunological benefit following DNA therapeutic vaccination. Several animals show a long-lasting decrease in viremia, suggesting that therapeutic vaccination may provide an additional benefit to antiretroviral therapy.

Animals↗

PubMed2006

Ability of herpes simplex virus vectors to boost immune responses to DNA vectors and to protect against challenge by simian immunodeficiency virus.

The immunogenicity and protective capacity of replication-defective herpes simplex virus (HSV) vector-based vaccines were examined in rhesus macaques. Three macaques were inoculated with recombinant HSV vectors expressing Gag, Env, and a Tat-Rev-Nef fusion protein of simian immunodeficiency virus (SIV). Three other macaques were primed with recombinant DNA vectors expressing Gag, Env, and a Pol-Tat-Nef-Vif fusion protein prior to boosting with the HSV vectors. Robust anti-Gag and anti-Env cellular responses were detected in all six macaques. Following intravenous challenge with wild-type, cloned SIV239, peak and 12-week plasma viremia levels were significantly lower in vaccinated compared to control macaques. Plasma SIV RNA in vaccinated macaques was inversely correlated with anti-Rev ELISPOT responses on the day of challenge (P value<0.05), anti-Tat ELISPOT responses at 2 weeks post challenge (P value <0.05) and peak neutralizing antibody titers pre-challenge (P value 0.06). These findings support continued study of recombinant herpesviruses as a vaccine approach for AIDS.

Animals↗

PubMed2006

RTE and CTE mRNA export elements synergistically increase expression of unstable, Rev-dependent HIV and SIV mRNAs.

Studies of retroviral mRNA export identified two distinct RNA export elements utilizing conserved eukaryotic mRNA export mechanism(s), namely the Constitutive Transport Element (CTE) and the RNA Transport Element (RTE). Although RTE and CTE are potent in nucleocytoplasmic mRNA transport and expression, neither element is as powerful as the Rev-RRE posttranscriptional control. Here, we found that whereas CTE and the up-regulatory mutant RTEm26 alone increase expression from a subgenomic gag and env clones, the combination of these elements led to a several hundred-fold, synergistic increase. The use of the RTEm26-CTE combination is a simple way to increase expression of poorly expressed retroviral genes to levels otherwise only achieved via more cumbersome RNA optimization. The potent RTEm26-CTE element could be useful in lentiviral gene therapy vectors, DNA-based vaccine vectors, and gene transfer studies of other poorly expressed genes.

Animals↗

PubMed2005

Rational design of a plasmid DNA vaccine capable of eliciting cell-mediated immune responses to multiple HIV antigens in mice.

Given the importance of the HIV-specific cell-mediated immune response in the early control and resolution of HIV infection and the observed correlation between pre-challenge vaccine elicited CTL responses and post challenge outcome in SHIV/rhesus macaque experiments, we sought to identify several candidate plasmid DNA (pDNA) vaccine designs capable of eliciting robust and balanced cell-mediated immune responses to multiple HIV-1 derived antigens in mice for further vaccine development. To rationally construct candidate vaccines for immunogenicity testing, we determined the relative immunogenicity of the individual HIV-derived vaccine antigens (env, gag, pol, nef, tat and vif) and the relative strength of various transcriptional control elements (HCMV, SCMV, HSV Lap1) in Balb/c mice. Next, a number of 1-, 2-, 3- and 4-vector pDNA vaccine designs were tested for their ability to elicit HIV-1 antigen-specific CMI responses. For these studies, Balb/c mice were immunized with a fixed total pDNA vaccine dose of 100 mcg in combination with 25 mcg plasmid-based murine IL-12 and tested for the induction of HIV-1 antigen-specific CMI responses by IFN-gamma ELISpot analysis. The results of this study indicate that all pDNA vaccine designs were capable of eliciting CMI responses to multiple HIV-1 antigens. As a result of this iterative comparative analysis, we have identified a number of pDNA vaccine candidates capable of eliciting potent, balanced CMI responses to multiple HIV-1 derived antigens. These results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection.

Animals↗

PubMed2005

Priming with plasmid DNAs expressing interleukin-12 and simian immunodeficiency virus gag enhances the immunogenicity and efficacy of an experimental AIDS vaccine based on recombinant vesicular stomatitis virus.

Of the various approaches being developed as prophylactic HIV vaccines, those based on a heterologous plasmid DNA prime, live vector boost vaccination regimen appear especially promising in the nonhuman primate/simian-human immunodeficiency virus (SHIV) challenge model. In this study, we sought to determine whether a series of intramuscular priming immunizations with a plasmid DNA vaccine expressing SIVgag p39, in combination with plasmid expressed rhesus IL-12, could effectively enhance the immunogenicity and postchallenge efficacy of two intranasal doses of recombinant vesicular stomatitis virus (rVSV)-based vectors expressing HIV-1 env 89.6P gp160 and SIVmac239 gag p55 in rhesus macaques. In macaques receiving the combination plasmid DNA prime, rVSV boost vaccination regimen we observed significantly increased SIVgag- specific cell-mediated and humoral immune responses and significantly lower viral loads postintravenous SHIV89.6P challenge relative to macaques receiving only the rVSV vectored immunizations. In addition, the plasmid DNA prime, rVSV boost vaccination regimen also tended to increase the preservation of peripheral blood CD4+ cells and reduce the morbidity and mortality associated with SHIV89.6P infection. An analysis of immune correlates of protection after SHIV89.6P challenge revealed that the prechallenge SHIV-specific IFN-gamma ELISpot response elicited by vaccination and the ability of the host to mount a virus-specific neutralizing antibody response postchallenge correlated with postchallenge clinical outcome. The correlation between vaccine-elicited cell-mediated immune responses and an improved clinical outcome after SHIV challenge provides strong justification for the continued development of a cytokine-enhanced plasmid DNA prime, rVSV vector boost immunization regimen for the prevention of HIV infection.

AIDS Vaccines↗

PubMed2005

DNA vaccines expressing different forms of simian immunodeficiency virus antigens decrease viremia upon SIVmac251 challenge.

We have tested the efficacy of DNA immunization as a single vaccination modality for rhesus macaques followed by highly pathogenic SIVmac251 challenge. To further improve immunogenicity of the native proteins, we generated expression vectors producing fusion of the proteins Gag and Env to the secreted chemokine MCP3, targeting the viral proteins to the secretory pathway and to a beta-catenin (CATE) peptide, targeting the viral proteins to the intracellular degradation pathway. Macaques immunized with vectors expressing the MCP3-tagged fusion proteins developed stronger antibody responses. Following mucosal challenge with pathogenic SIVmac251, the vaccinated animals showed a statistically significant decrease in viral load (P = 0.010). Interestingly, macaques immunized with a combination of vectors expressing three forms of antigens (native protein and MCP3 and CATE fusion proteins) showed the strongest decrease in viral load (P = 0.0059). Postchallenge enzyme-linked immunospot values for Gag and Env as well as gag-specific T-helper responses correlated with control of viremia. Our data show that the combinations of DNA vaccines producing native and modified forms of antigens elicit more balanced immune responses able to significantly reduce viremia for a long period (8 months) following pathogenic challenge with SIVmac251.

AIDS Vaccines↗

PubMed2003

Use of interleukin 15 to enhance interferon-gamma production by antigen-specific stimulated lymphocytes from rhesus macaques.

The enzyme-linked immune spot (ELISPOT) assay is receiving increased attention as a means for quantifying antigen-specific CD8 T-cell responses in rhesus macaques. Further improving the sensitivity of this assay could aid in the evaluation of vaccine candidates and/or immune therapeutic candidates. Interleukin (IL)-15 has been demonstrated to stimulate expansion of human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) and to regulate homeostatic proliferation of CD8+ memory cells. We evaluated the in vitro effect of IL-15 to increase the detection of interferon-gamma (IFN-gamma) production by antigen-specific stimulated lymphocytes from a group of rhesus macaques exposed to simian-human immunodeficiency virus (SHIV) and a second group infected with SIVmac251, before and after antiretroviral treatment (ART). Results from these studies demonstrate that the presence of IL-15 during stimulation in a peptide-based ELISPOT assay greatly enhanced IFN-gamma production in both SHIV and simian immunodeficiency virus (SIV)-infected macaques. IFN-gamma production was mainly mediated by CD8 lymphocytes. The optimal concentrations of IL-15 that give enhancement of IFN-gamma production to specific antigen, without a significant increase in the spontaneous IFN-gamma release, ranged from 0.5 to 2.5 ng/ml. The mean number of IFN-gamma spots was increased 3.1- to 3.6-fold in response to SIV gag or HIV env peptide pools, respectively, in peripheral blood mononuclear cells (PBMC) from SHIV-infected macaques. Similarly, in SIV-infected macaques, IL-15 increased the mean number of IFN-gamma spots 2.7-fold in response to both SIV gag and env peptide pools. In samples obtained after ART in the same macaques, the increase factor was 2.5 for SIV gag and 1.8 for the env peptide pools. Thus, the sensitivity of the ELISPOT assay can be enhanced by addition of IL-15. This modified assay will be useful for detection of low frequencies of IFN-gamma producing cells in rhesus macaques.

Animals↗

PubMed2002

Persistent HIV-1 infection of natural killer cells in patients receiving highly active antiretroviral therapy.

We have identified a subset of CD56(+)CD3(-) human natural killer (NK) cells that express CD4 and the HIV coreceptors CCR5 and CXCR4. These cells can be productively infected in vitro by both CCR5- and CXCR4-using molecular clones of HIV-1 in a CD4-dependent manner. Analysis of HIV-infected persons showed that viral DNA is present in purified NK cells, and virus could be rescued from these cells after in vitro cultivation. Longitudinal analysis of the HIV-1 DNA levels in NK cells from patients after 1-2 years of highly active antiretroviral therapy indicated that NK cells remain persistently infected and account for a substantial amount of the viral DNA in peripheral blood mononuclear cells. These results demonstrate that a subset of non-T cells with NK markers are persistently infected and suggest that HIV infection of NK cells is important for virus persistence. The properties of the virus reservoir in these cells should be considered in attempts to further optimize antiretroviral therapies.

Anti-HIV Agents↗