DNA nanoparticles with core-shell morphology.

Mannobiose-modified polyethylenimines (PEI) are used in gene therapy to generate nanoparticles of DNA that can be targeted to the antigen-presenting cells of the immune system. We report that the sugar modification alters the DNA organization within the nanoparticles from homogenous to shell-like packing. The depth-dependent packing of DNA within the nanoparticles was probed using AFM nano-indentation. Unmodified PEI-DNA nanoparticles display linear elastic properties and depth-independent mechanics, characteristic of homogenous materials. Mannobiose-modified nanoparticles, however, showed distinct force regimes that were dependent on indentation depth, with 'buckling'-like response that is reproducible and not due to particle failure. By comparison with theoretical studies of spherical shell mechanics, the structure of mannobiosylated particles was deduced to be a thin shell with wall thickness in the order of few nanometers, and a fluid-filled core. The shell-core structure is also consistent with observations of nanoparticle denting in altered solution conditions, with measurements of nanoparticle water content from AFM images, and with images of DNA distribution in Transmission Electron Microscopy.

Nanomedicine applications towards the cure of HIV.

Combination antiretroviral therapy (cART) successfully suppresses HIV replication. However, daily and lifelong treatment is necessary to manage patient illness because cART neither eradicates infected cells from reservoirs nor reconstitutes HIV-specific immunity that could kill infected cells. Toward the cure of HIV, different nanomedicine classes have been developed with the following disease-modifying properties: to eradicate the virus by activation of latently infected CD4+ T-cells and reservoirs flushing; to kill the infected cells in the reservoirs by boosting of HIV-specific T cells; and to prevent infection by the use of microbicides with improved epithelial penetration and drug half-life. Preclinical and clinical trials consistently demonstrated that DermaVir, the most advanced nanomedicine, induces long-lasting memory T-cell responses and reduces viral load in comparison with placebo. DermaVir and the nanomedicine pipelines have the potential to improve the health of HIV-infected people at lower costs, to decrease antiretroviral drug exposure, and to contribute to the cure of HIV/AIDS. FROM THE CLINICAL EDITOR: Despite the leaps and bounds in the development of antiretroviral therapy, HIV remains a significant public health challenge. In this review, applications of nanomedicine- based technologies are discussed in the context of HIV treatment, including virus elimination by activation of latently infected CD4+ T-cells; infected cell elimination in the reservoirs by boosting HIV-specific T cells, and by preventing infection by the use of microbicides with improved epithelial penetration and drug half-life.

DermAll nanomedicine for allergen-specific immunotherapy.

Allergen-specific immunotherapy (ASIT) the only disease-modifying treatment for IgE-mediated allergies is characterized with long treatment duration and high risk of side effects. We investigated the safety, immunogenicity and efficacy of a novel ASIT, called DermAll, in an experimental allergic rhinitis model. We designed and characterized DermAll-OVA, a synthetic plasmid pDNA/PEIm nanomedicine expressing ovalbumin (OVA) as model allergen. DermAll-OVA was administered topically with DermaPrep device to target Langerhans cells. To detect the clinical efficacy of DermAll ASIT we quantified the nasal symptoms and characterized the immunomodulatory activity of DermAll ASIT by measuring cytokine secretion after OVA-stimulation of splenocytes and antibodies from the sera. In allergic mice DermAll ASIT was as safe as Placebo, balanced the allergen-induced pathogenic TH2-polarized immune responses, and decreased the clinical symptoms by 52% [32%, 70%] compared to Placebo. These studies suggest that DermAll ASIT is safe and should significantly improve the immunopathology and symptoms of allergic diseases. FROM THE CLINICAL EDITOR: A novel allergen-specific immunotherapy for IgE-mediated allergies is presented in this paper, using an experimental allergic rhinitis model and a synthetic plasmid pDNA/PEIm nanomedicine expressing ovalbumin as model allergen. Over 50% reduction of symptoms was found as the immune system's balance was favorably altered toward more TH2-polarized immune responses.

Structure and biological activity of pathogen-like synthetic nanomedicines.

Here we characterize the structure, stability and intracellular mode of action of DermaVir nanomedicine that is under clinical development for the treatment of HIV/AIDS. This nanomedicine comprises pathogen-like pDNA/PEIm nanoparticles (NPs) having the structure and function resembling spherical viruses that naturally evolved to deliver nucleic acids to the cells. Atomic force microscopy demonstrated spherical 100 - 200 nm NPs with a smooth polymer surface protecting the pDNA in the core. Optical absorption determined both the NP structural stability and biological activity relevant to their ability to escape from the endosome and release the pDNA at the nucleus. Salt, pH and temperature influence nanomedicine shelf-life and intracellular stability. This approach facilitates the development of diverse polyplex nanomedicines where the delivered pDNA-expressed antigens induce immune responses to kill infected cells. FROM THE CLINICAL EDITOR: The authors investigated DermaVir nanomedicine comprised of pathogen-like pDNA/PEIm nanoparticles with structure and function resembling spherical viruses. DermaVir delivery of pDNA expresses antigens that induce immune responses to kill HIV infected cells.

Synergistic drug combinations designed to fully suppress SARS-CoV-2 in the lung of COVID-19 patients.

Despite new antivirals are being approved against SARS-CoV-2 they suffer from significant constraints and are not indicated for hospitalized patients, who are left with few antiviral options. Repurposed drugs have previously shown controversial clinical results and it remains difficult to understand why certain trials delivered positive results and other trials failed. Our manuscript contributes to explaining the puzzle: this might have been caused by a suboptimal drug exposure and, consequently, an incomplete virus suppression, also because the drugs have mostly been used as add-on monotherapies. As with other viruses (e.g., HIV and HCV) identifying synergistic combinations among such drugs could overcome monotherapy-related limitations. In a cell culture model for SARS-CoV-2 infection the following stringent criteria were adopted to assess drug combinations: 1) identify robust, synergistic antiviral activity with no increase in cytotoxicity, 2) identify the lowest drug concentration inhibiting the virus by 100% (LIC100) and 3) understand whether the LIC100 could be reached in the lung at clinically indicated drug doses. Among several combinations tested, remdesivir with either azithromycin or ivermectin synergistically increased the antiviral activity with no increase in cytotoxicity, improving the therapeutic index and lowering the LIC100 of every one of the drugs to levels that are expected to be achievable and maintained in the lung for a therapeutically relevant period of time. These results are consistent with recent clinical observations showing that intensive care unit admission was significantly delayed by the combination of AZI and RDV, but not by RDV alone, and could have immediate implications for the treatment of hospitalized patients with COVID-19 as the proposed "drug cocktails" should have antiviral activity against present and future SARS-CoV-2 variants without significant overlapping toxicity, while minimizing the onset of drug resistance. Our results also provide a validated methodology to help sort out which combination of drugs are most likely to be efficacious in vivo, based on their in vitro activity, potential synergy and PK profiles.

Topical DermaVir vaccine targeting dendritic cells.

DermaVir employs a topical, non-invasive method for vaccine delivery to dendritic cells. The vaccine product contains plasmid DNA as the active ingredient, encoding authentically expressed retroviral genes with appropriate safety modifications. The non-viral delivery system packages the DNA within pathogen-like nanoparticles and studies indicate that vaccine antigens are taken up by epidermal Langerhans cells, the precursors of dendritic cells. DermaVir loaded dendritic cells reach the draining lymph node target but not the bloodstream nor indiscriminately other organ systems. Safety data from DermaVir immunized infected macaques indicate improved survival, absence of apparent toxicities other than transient erythema and lack of recombination between the vaccine DNA and the infectious viral DNA integrated in the host genome. DermaVir represents a potential new approach for the treatment of HIV infection to be utilized either in conjunction with antiretroviral therapy or during structured treatment interruption.

CD4+ T cell-mediated presentation of non-infectious HIV-1 virion antigens to HIV-specific CD8+ T cells.

BACKGROUND: The mechanism of chronic immune activation and impairment of HIV-specific immune responses during chronic infection is not fully understood. However, it is known that high immune activation leads to more rapid progression to AIDS. We hypothesize that CD4(+) T cell-mediated viral antigen presentation contributes to this pathologic immune activation in HIV-infected individuals. METHODS: HIV-specific T cells, responding to noninfectious HIV-1 virions as antigen, were measured by flow cytometric assays. These experimental conditions reflect the in vivo condition where noninfectious HIV-1 represents more than 99% of the antigens. RESULTS: CD4(+) T cells purified from HIV-infected individuals were capable of cross presenting exogenous noninfectious HIV-1 virions to HIV-1-specific CD8(+) T cells. Cross presentation required the entry of HIV-1 to CD4(+) T cells and antigen translocation from endoplasmic reticulum to the Golgi complex. Blocking CD4(+) mediated activation of HIV-specific CD8(+) T cells and redirecting the viral antigens to antigen presenting cells improved HIV-specific T cell responses. CONCLUSIONS: One possible cause of chronic immune activation and impairment of HIV-1 specific T cell responses is represented by HIV-1 harboring CD4(+) T cells cross presenting HIV-1 antigen to activate CD8(+) T cells. This new mechanism provides the first evidence that cross presentation of noninfectious HIV-1 virions play a role in the immunopathogenesis of HIV-1 infection.

Optimal suppression of HIV replication by low-dose hydroxyurea through the combination of antiviral and cytostatic ('virostatic') mechanisms.

BACKGROUND: The hydroxyurea-didanosine combination has been shown to limit immune activation (a major pathogenic component of HIV/AIDS) and suppress viral load by both antiviral and cytostatic ('virostatic') activities. Virostatics action represent a novel approach to attack HIV/AIDS from multiple directions; however, the use of these drugs is limited by the lack of understanding of their dose-dependent mechanism of action and by fear of pancreatic toxicity, even though a large review of ACTG studies has shown that hydroxyurea does not increase the incidence of pancreatitis. METHODS: In vitro cytostatic and cytotoxic activity, inhibition of viral replication and immune activation by pharmacologically attainable plasma concentrations of hydroxyurea (10-100 micromol/l) and didanosine (1-5 micromol/l) were analyzed by cell proliferation, viability, apoptosis and infection assays using peripheral blood mononuclear cells. In vivo, 600, 900 and 1200 mg daily doses of hydroxyurea in combination with standard doses of didanosine and stavudine were studied in 115 randomized chronically infected patients. RESULTS: A cytostatic low (10 micromol/l) concentration of hydroxyurea inhibited cell proliferation and HIV replication in vitro. A gradual switch from cytostatic to cytotoxic effects was observed by increasing hydroxyurea concentration to 50-100 micromol/l, predicting that lower doses of hydroxyurea would be less toxic and more potent in vivo. The clinical results confirmed that 600 mg hydroxyurea was better tolerated, had fewer side effects and was more potent in suppressing HIV replication than the higher doses. CONCLUSIONS: A bimodal, dose-dependent, cytostatic-cytotoxic switch is an immune-based mechanism explaining the apparent paradox that lowering the dose of hydroxyurea to 600 mg daily induces maximal antiviral suppression in HIV-infected patients.

Hydroxyurea exerts a cytostatic but not immunosuppressive effect on T lymphocytes.

OBJECTIVE: To demonstrate that, despite a dose-dependent cytostatic effect, hydroxyurea (HU) does not have immunosuppressive effects. METHODS: The effects of HU on T lymphocyte proliferation parameters, activation phenotype and cytokine production were examined in vitro after exposure to clinically relevant concentrations of HU (10, 50, and 100 micromol/l). The effects of HU in vivo on CD4 T cell counts, viral load, activation phenotype and virus-specific response were examined in 17 Rhesus macaques infected with SIV(mac251) and randomized into three groups: untreated controls; treated with (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA) and didanosine (ddI) only; and treated with PMPA, didanosine, and HU. RESULTS: The in vitro inhibition of T lymphocyte proliferation confirmed the cytostatic effect of HU, with a linear dose-dependent effect; however, no relevant differences were found in the expression of activation markers between treated and untreated controls. Both T helper type 1 and type 2 cytokine production were enhanced by HU. Consistent with the in vitro results, a blunted increase of peripheral CD4 T cells was observed in vivo in the HU group, without relevant effects on the expression of activation markers, and SIV-specific T cell responses were not affected by HU. CONCLUSIONS: Hyper-proliferation of T-lymphocytes is a major factor contributing to HIV pathogenesis. HU exerts a cytostatic effect on T lymphocytes, without altering their activation and apparently without having an immunosuppressive effect. The increase in cytokine production at the single cell level might compensate for the decrease in the percentage of activated CD4 T lymphocytes, without overall impairment of HIV-specific immune responses.

Control of viral rebound through therapeutic immunization with DermaVir.

OBJECTIVE: To reconstitute immune responses capable of eliminating infected cells and suppressing viral load during chronic retroviral infection. DESIGN: : A topical, DNA-based therapeutic immunization (DermaVir) was designed to express most of the regulatory and structural viral genes in dendritic cells. METHODS: DermaVir alone and in combination with antiretroviral drugs was tested in chronically SIV-infected macaques. RESULTS: DermaVir provided virological, immunological and clinical benefit for SIV-infected macaques during chronic infection and AIDS. In combination with antiretroviral drugs, DermaVir augmented SIV-specific T-cell responses and enhanced control of viral load rebound during treatment interruptions. CONCLUSIONS: The results indicate the feasibility of therapeutic immunization even in immune compromised hosts, and suggest that DermaVir can complement antiretroviral drugs to sustain suppression of HIV-1 replication.

DermaVir: a novel topical vaccine for HIV/AIDS.

Human immunodeficiency virus (HIV) vaccines have the potential to improve antiretroviral drug treatment by inducing cytotoxic killing of HIV-infected cells. Prophylactic vaccines utilize new antigens to initiate immunity; however, in HIV-infected individuals the load of viral antigen is not the limiting factor for the restoration of immune responses. Here we describe a novel immunization strategy with DermaVir that improves viral antigen presentation using dendritic cells (DC). DermaVir contains a distinctive plasmid DNA expressing all HIV proteins except integrase to induce immune responses with broad specificity. The DNA is formulated to a mannosilated particle to target antigen-presenting cells and to protect the DNA from intracellular degradation. After topical application, DermaVir-transduced cells migrate from the skin to the draining lymph node and interdigitate as DermaVir-expressing, antigen-presenting DC. We compared the immunogenicity of topical and ex vivo DC-based DermaVir vaccinations in naive rhesus macaques. Both vaccinations induced simian immunodeficiency virus-specific CD4 helper and CD8 memory T cells detected by an in vivo skin test and an in vitro intracellular cytokine-based assay. Topical DermaVir vaccination represents an improvement upon existing ex vivo DC-based immunization technologies and may provide a new therapeutic option for HIV-infected patients.

Quantification of HIV-specific CD8 T cells by in vitro stimulation with inactivated viral particles.

OBJECTIVES: HIV-specific CD8 T cells play a central role in the immune control of virus replication. To further understand the role of CD8 T cells in clinical settings, there is a need for a diagnostic assay that quantifies HIV-specific CD8 T cells in all HIV-infected individuals. DESIGN: and methods: The CD8VIR (CD8 T cell-mediated virus-specific immune response) assay was designed to mimic viral load rebound by adding replication defective HIV particles to peripheral blood mononuclear cells. Antigen presenting cells processed the virus and presented most of the viral epitopes to T cells. Activated HIV-specific CD8 T cells were quantified by flow cytometry analysis as CD3CD8 IFNgamma producing T cells. RESULTS: The CD8VIR assay reproducibly detected a large proportion of functional HIV-specific CD8 T cells responding to viral load rebound. The whole HIV particle stimulation used in the CD8VIR assay was comparable to the sum of Gag, Pol, Env and Nef stimulations. The percentage of HIV-specific CD8 T cells also significantly correlated with the percentage of Gag-specific cytotoxicity measured by the traditional Cr release assay. HIV-specific CD8 T cells correlated with immune control of HIV in chronically infected patients. CONCLUSIONS: The CD8VIR assay quantifies the majority of HIV-specific CD8 T cells capable of killing HIV-infected cells during viral load rebound. This simple, versatile and reproducible assay can be performed from the specimen submitted for CD4 analysis. Upon clinical validation, the CD8VIR assay can be a new diagnostic tool to predict the control of viral load rebound after treatment interruption.

Structured treatment interruptions in HIV/AIDS therapy.

Structured treatment interruption (STI) has been investigated for three distinct clinical scenarios: during acute infection with the goal of immune reconstitution and auto immunization; during chronic infection, to decrease the amount and toxicity of antiretroviral drugs; and during virologic failure to restore response to subsequent antiretroviral therapy. The potential costs and benefits of STI should be determined.

Strategies to decrease viral load rebound, and prevent loss of CD4 and onset of resistance during structured treatment interruptions.

BACKGROUND: Toxicity and other drug adherence-related factors have contributed to decreased compliance to antiretroviral regimens amongst HIV-infected patients. Irregular therapy disruption causes loss of CD4 T cells, onset of drug resistance and rapid rebound of plasma viral load (VL). However, an appropriate choice of drugs and properly scheduled structured treatment interruptions (STIs) may limit VL rebound, maintain CD4 counts and minimize resistance. METHODS: We conducted a clinical study of STIs, RIGHT 901, involving 60 drug-naive patients with chronic HIV infection (CD4 >300, VL >10,000) randomized to receive didanosine-stavudine-indinavir (IDV group) or didanosine-stavudine-hydroxyurea (HU group), for 12 weeks. Subsequently, all patients were randomized again to start STI (short induction) or to continue the therapy for an additional 24 weeks before starting STI (long induction). Both groups underwent four STI cycles and then stopped therapy as long as viraemia remained below 10,000 copies/ml before reinitiating another four cycles of STI. RESULTS: During continuous therapy VLs were suppressed at similar rates in both the HU and IDV groups, while a blunted CD4 count was documented in the HU group. Following the first stop median VL rebounded close to baseline values in both groups, however, during the following STI median VL rebound decreased in the HU group, while in the IDV group VL continued to rebound to values close to baseline, and the difference between the two groups was statistically significant. Moreover, patients treated with HU had a constant and stable CD4 increase during STI, whereas CD4 counts fluctuated in the IDV group, with sharp falls during treatment interruptions and partial CD4 recovery following treatment restart. Even in the presence of IDV resistance predisposing mutations at baseline, no genotypic change in the protease sequence was observed during STI. A relevant mutation in the reverse transcriptase sequence (K70R) emerged in one patient interrupting treatment after 36 weeks of continuous therapy and in one patient after four STI cycles. Side effects (no major events) were similar among groups. CONCLUSIONS: An appropriate choice of STI schedule and regimens containing drugs less prone to resistance and/or able to prevent CD4 fluctuation may contribute to optimizing STI for chronically infected patients with respect to limiting viral rebound, improving CD4 counts and maintaining a resistance profile comparable to continuous highly active antiretroviral therapy.

Vaccinia assay for the rapid detection of functional HIV-specific CD8+ cytotoxic T lymphocytes.

Human immunodeficiency virus (HIV)-specific T cells play a critical role in anti-virus immunity. Therefore, during the clinical development of immune-based therapies, it is important to perform a diagnostic test that rapidly quantifies and characterizes cellular immune responses. For detection of functional HIV-specific CD8(+) cytotoxic T cells (CTL), we used a rapid vaccinia assay that employs recombinant vaccinia virus-infected peripheral blood mononuclear cells (PBMC). This assay was compared with the traditional 51Cr-release CTL assay and with the peptide pool-based enzyme-linked immunospot assay (ELISPOT). We demonstrated a close correlation between these assays by using identical antigens in parallel assays. However, the vaccinia assay was the least expensive and time- and labor-consuming test. Regarding sensitivity, the vaccinia assay was similar to both the peptide pool-based ELISPOT and CTL assays. We showed that human histocompatibility leukocyte antigen (HLA)-Dr(+) cells are responsible for presenting recombinant vaccinia antigens to T cells. The recombinant vaccinia-based assay is a rapid, sensitive and quantitative test and is thus suitable for the detection of functional antigen-specific CD8(+) CTL in large-scale clinical studies.

Gene therapy approaches to HIV infection.

The HIV pandemic represents a new challenge to biomedical research. What began as a handful of recognized cases among homosexual men in the US has become a global pandemic of such proportions that it clearly ranks as one of the most destructive viral scourges in history. In the past few years new treatments and drugs have been developed and tested, but the development of a new generation of therapies remains a major priority, because of the lack of chemotherapeutic drugs or vaccines that show long-term efficacy in vivo. Recently, gene therapeutic strategies for the treatment of patients with HIV infection have received increased attention because they are able to offer the possibility of simultaneously targeting multiple sites in the HIV genome, thereby minimizing the production of resistant virus. Recombinant genes for gene therapy can be classified as expressing interfering proteins (intracellular antibodies, dominant negative proteins) or interfering RNAs (antisense RNAs, ribozymes, RNA decoys). The latter group offers the advantage of avoiding the stimulation of host immune response which might progressively decrease the efficacy of proteins. The stumbling block to achieving lasting antiviral effects is still represented by the lack of efficient gene transfer techniques capable of generating persistent transgene expression and a high number of transduced cells relative to untransduced cells. Novel delivery vectors, such as lentiviruses, might overcome some of these shortcomings. The use of recombinant genes to generate immunity is a very promising concept that is rapidly expanding. Since the immune system can significantly amplify the response to tiny amounts of antigen, DNA vaccines can indeed be delivered by exploiting traditional gene therapy approaches without the need of high transduction efficiency.

Methods of using interleukin 2 to enhance HIV-specific immune responses.

Interleukin 2 (IL-2) in combination with highly active antiretroviral therapy (HAART) can significantly increase CD4+ T cell counts but does not improve HIV-specific T cell-mediated immune responses that are associated with the control of viral replication. To characterize the immunomodulatory activity of IL-2 in HIV-infected individuals we studied the virus-specific immune response (VIR) by intracellular cytokine expression (interferon gamma, IFN-gamma) after mimicking HIV rebound in peripheral blood mononuclear cells (PBMCs). We found that the whole virus used as HIV antigen was able to activate HIV-specific T cells in the presence of a low concentration (50 IU/ml) of IL-2. Interestingly, increasing concentrations of IL-2 (400 or 1000 IU/ml) in combination with the same amount of HIV doubled the number of HIV-specific T cells. These cells were functionally intact because all of the IFN-gamma-producing CD8+ T cells contained perforin, a marker for cytotoxic T lymphocytes (CTLs). Induction of HIV-specific immune responses by IL-2 was not detected in the absence of HIV antigen both in vitro and in patients treated with HAART, indicating that IL-2 can amplify HIV-specific T cells in the presence of HIV antigen. Therefore, a combination of IL-2 with either structured treatment interruption, which results in a controlled viral load rebound, or with therapeutic vaccination is expected to improve HIV-specific T cell-mediated immune responses.

2nd International Symposium on Molecular Diagnostics and Skin Gene Therapy.

The 2nd International Symposium on Molecular Diagnostics and Skin Gene Therapy was held on a beautiful spring weekend at one of the youngest universities in the world, the Heinrich-Heine University, founded 1965. The conference attracted more than 340 participants from 24 different countries, including ethicists, basic researchers, virologists, immunologists, molecular biologists and clinicians, as well as numerous students. The event was organised under the auspices of the European Gene Therapy Society by U Hengge of the Department of Dermatology at Heinrich-Heine University. As Dr J Glorioso, President of the American Society for Gene Therapy, pointed out in his welcome address, the skin is a new target for gene therapy, with the potential for in vivo gene transfer to the central nervous system or to the lymphoid organs, for example. As such, Düsseldorf was a perfect location for such a meeting, because the most important biomedical research investments in Germany have been in neurology and dermatology. Many scientific challenges facing this young field of medicine were presented, but there was also much discussion of ethical, regulatory and safety issues with respect to gene therapy.

APC-targeted immunization for the treatment of HIV-1.

Therapeutic immunization may be thought of as an adjunct to highly active antiretroviral therapy to prime the immune system and possibly correct for immunological defects. Most therapeutic vaccine strategies currently under investigation aim to increase HIV-specific cellular responses. This may be most successfully accomplished by utilizing professional antigen-presenting cells. Autologous dendritic cells may be isolated, cultured, loaded with antigen and re-injected into the subject (ex vivo) or antigen may be directly delivered in situ to Langerhans cells or dermal dendritic cells, which are located respectively at the epidermal and dermal layer of the skin. Once Langerhans cells or dermal dendritic cells have incorporated the antigen, they are expected to mature and migrate to the lymph node to present antigen and stimulate naive T-cells. Exciting results have been obtained in nonhuman primates with both ex vivo and topical antigen-presenting cell-based therapeutic immunization.

T-cell receptor excision circles (TREC) in SHIV 89.6p and SIVmac251 models of HIV-1 infection.

T-cell receptor excision circles (TREC) may be a useful surrogate marker in HIV-1 infection for evaluating the likelihood of continued clinical stability and/or the response to therapeutics, including vaccines. Analysis of TREC in SHIV and SIV models of HIV-1 infection may provide additional information concerning the utility of TREC as a marker. We measured TREC in peripheral blood mononuclear cells (PBMC) from rhesus macaques in SHIV89.6p (n = 20) and SIVmac251 (n = 11) models of HIV-1 infection. TREC were also evaluated in tissues in the SIVmac251 model at end-point. In the SHIV89.6p model, TREC in PBMC were significantly lower at 12 weeks postinfection compared to preinfection levels. The decrease in TREC correlated with the decline in CD4+ T cells (r(s) = 0.496; P = 0.026), which in turn correlated inversely with serum viral loads at end-point (r(s) = -0.517; P = 0.019). Macaques that controlled SHIV89.6p infection to some degree (n = 6) had higher TREC at study end-point (P = 0.017). In the SIVmac251 model, TREC in PBMC were significantly reduced after 17 months of infection (P = 0.012) despite receiving highly active antiretroviral therapy (HAART) consisting of didanosine (ddI) and (R)-9-(2-phosphonylmethoxypropyl)-adenine (PMPA) when not cycling off therapy during scheduled treatment interruptions (STI). However, macaques that received continuous hydroxyurea (HU) in addition to the HAART regimen had higher end-point TREC compared to the non-HU group (P = 0.041), and the reduction in TREC observed at end-point within the HU group was not significant. In the SIVmac251 model, TREC correlated with the percentage of CD4+ T cells (r(s) = 0.426; P = 0.048) and CD4+CD28+ T cells (r(s) = 0.624; P = 0.002), and inversely with CD8+ T cells (r(s) = -0.622; P = 0.002), CD8+CD28- T cells (r(s) = -0.516; P = 0.014), and serum viral loads (r(s) = -0.627; P = 0.039). High levels of TREC were observed in the thymus, levels comparable to PBMC were seen in the lymph node, and low but detectable levels of TREC were present in bone marrow. The use of correlates of TREC as covariates in ANCOVA revealed that the decline in TREC in the SHIV 89.6p model reflected the decline in the percentage of CD4+ T-cells due to viral cytopathogenicity. In the SIVmac251 model, the decline in TREC was related to increased immune activation and proliferation due to viral replication, as reflected by decreases in percentages of CD4+CD28+ T cells and increases in CD8+ and CD8+CD28- T cells.