James F. Demarest

Analysis of the adult thymus in reconstitution of T lymphocytes in HIV-1 infection

Immunohistological analysis of the thymus in HIV infection. (a–d) Thymus from HIV-1+ patient no. 1 with no thymopoiesis. (e–h) Thymus from HIV-1+ patient no. 2 with areas of active thymopoiesis. (a) Hematoxylin and eosin stain of patient no. 1’s lymphoid thymus. ×13. (b) A similar area as in a, with thymic epithelium in immunohistological analysis reactive with antikeratin antibody (brown central areas). All keratin+ thymic epithelium (e) in the true thymus is collapsed (dark brown areas) and devoid of lymphocytes, with a surrounding infiltrate of blue mononuclear cells present in the thymic perivascular space (P). ×13. (c) Immunohistological stain of CD8+ T cells (brown cells; see arrows for examples) in the perivascular space (P) around a central empty thymic epithelial island (e). The dotted line surrounds thymic true epithelial thymus areas (e), and the short arrow points out a rare CD8+ T cell within the true epithelial thymus (e). ×66. (d) Many of the perivascular space (P) CD8+ cells are reactive with MAB TIA-1 (arrows) and therefore are mature effector cytotoxic T cells. ×66. e–h are from patient no. 2’s thymus. ×33. (e) Light microscopic view of patient no. 2’s thymus (hematoxylin and eosin stain with a Hassall’s body [h] in the thymus medulla). (f) Immunohistological analysis with antikeratin antibody, with areas of normal-appearing keratin+ thymic epithelium (brown areas) filled with lymphocytes (blue areas) intermingled with thymic epithelium (arrows). Most developing thymocytes are CD3+ T cells (arrows in g), many of which are normal CD1a+ cortical thymocytes (brown cells, arrows in h). A subset of these CD1a+, CD3+ immature thymocytes were actively dividing as determined by nuclear reactivity with MAB mib-1 (not shown). MAB, monoclonal antibody.

Role of lymphoid organs in the pathogenesis of human immunodeficiency virus (HIV) infection.

The pathogenic mechanisms of HIV disease are multifactorial and multi-phasic. The common denominator of the disease is the profound immunosuppression that occurs in the vast majority of infected patients. Studies in lymphoid tissues in HIV disease have provided considerable insight into the pathogenic processes involved from the earliest phases of infection through the advanced stages. Following primary infection, virus is disseminated throughout the body and seeds the lymphoid tissue where its replication is only incompletely suppressed and where a reservoir of virus is established. Extracellular virus is trapped within the FDC of the lymph node germinal centers and serves as a source of infection for cells which reside in or migrate through the lymph node throughout the course of infection even during the early and often prolonged asymptomatic period. Eventually, the architecture of the lymphoid tissue is destroyed, compounding the immune dysfunction that results from the depletion of CD4+ T cells. In this regard, the lymphoid tissue of LTNPs is relatively intact and viral burden and replication is considerably lower in the peripheral blood and lymph node mono-nuclear cells of LTNPs than in individuals whose disease progresses. Cytokines probably play a major role in the modulation of HIV expression in the milieu of the lymphoid tissue. Further understanding of the pathogenic mechanisms operative in the lymphoid tissues of HIV-infected individuals will have important implications in the design of therapeutic strategies involving both antiretroviral and immunomodulatory approaches.

Antiviral activity and safety of 873140, a novel CCR5 antagonist, during short-term monotherapy in HIV-infected adults.

Objective:873140 is a spirodiketopiperazine CCR5 antagonist with prolonged receptor binding and potent antiviral activity in vitro. This study evaluated plasma HIV RNA, safety, and pharmacokinetics following short-term monotherapy in HIV-infected adults. Design:Double-blind, randomized, placebo-controlled multi-center trial. Methods:Treatment-naive or experienced HIV-infected subjects with R5-tropic virus, CD4 cell count nadir > 200 × 106 cells/l, viral load > 5000 copies/ml and not receiving antiretroviral therapy for the preceding 12 weeks were enrolled. Forty subjects were randomized to one of four cohorts (200 mg QD, 200 mg BID, 400 mg QD, 600 mg BID) with 10 subjects (eight active, two placebo) in each cohort, and received treatment for 10 days. Serial HIV RNA, pharmacokinetics, and safety evaluations were performed through day 24. Results:Of the 40 subjects, 21 were treatment-experienced; 35 were male, 20 were non-white, and eight were coinfected with hepatitis C virus. Median baseline HIV RNA ranged from 4.26log10 to 4.46 log10. 873140 was generally well tolerated with no drug-related discontinuations. The most common adverse events were grade 1 gastrointestinal complaints that generally resolved within 1–3 days on therapy. No clinically significant abnormalities were observed on electrocardiogram or in laboratory parameters. Mean log changes in HIV RNA at nadir, and the percentage of subjects with > 1 log10 decrease were −0.12 (0%) for placebo, −0.46 (17%) for 200 mg once daily, −1.23 (75%) for 200 mg twice daily, −1.03 (63%) for 400 mg once daily, and −1.66 (100%) for 600 mg twice daily. An Emax relationship was observed between the area under the 873140 plasma concentration–time curve and change in HIV RNA. Conclusions:873140 demonstrated potent antiretroviral activity and was well tolerated. These results support further evaluation in Phase 2b/3 studies.

HIV Type 1 from a Patient with Baseline Resistance to CCR5 Antagonists Uses Drug-Bound Receptor for Entry

CCR5 antagonists are a new class of antiretroviral drugs that block viral entry by disrupting interactions between the viral envelope (Env) glycoprotein and coreceptor. During the CCR100136 (EPIC) Phase IIb study of the CCR5 antagonist aplaviroc (APL) in treatment-naive individuals, a patient was identified who harbored virus strains that exhibited partial resistance to APL at the time of virologic failure. Retrospectively, it was found that APL resistance was present at baseline as well. To investigate the mechanism of APL resistance in this patient, we cloned HIV-1 env genes from plasma obtained at baseline and after virologic failure. Approximately 85% of cloned Envs were functional, and all exhibited partial resistance to APL. All Envs were R5-tropic, were partially resistant to other CCR5 antagonists including maraviroc on cells with high CCR5 expression, but remained sensitive to the fusion inhibitor enfuvirtide. Competition studies with natural CCR5 ligands revealed that the mechanism of drug resistance entailed the use of the drug-bound conformation of CCR5 by the Env proteins obtained from this individual. The degree of drug resistance varied between Env clones, and also varied depending on the cell line used or the donor from whom the primary T cells were obtained. Thus, both virus and host factors contribute to CCR5 antagonist resistance. This study shows that R5 HIV-1 strains resistant to CCR5 inhibitors can arise in patients, confirming a mechanism of resistance previously characterized in vitro. In addition, some patients can harbor CCR5 antagonist-resistant viruses prior to treatment, which may have implications for the clinical use of this new class of antiretrovirals.

HIV-1 Resistance to CCR5 Antagonists Associated with Highly Efficient Use of CCR5 and Altered Tropism on Primary CD4+ T Cells

ABSTRACT We previously reported on a panel of HIV-1 clade B envelope (Env) proteins isolated from a patient treated with the CCR5 antagonist aplaviroc (APL) that were drug resistant. These Envs used the APL-bound conformation of CCR5, were cross resistant to other small-molecule CCR5 antagonists, and were isolated from the patients pretreatment viral quasispecies as well as after therapy. We analyzed viral and host determinants of resistance and their effects on viral tropism on primary CD4+ T cells. The V3 loop contained residues essential for viral resistance to APL, while additional mutations in gp120 and gp41 modulated the magnitude of drug resistance. However, these mutations were context dependent, being unable to confer resistance when introduced into a heterologous virus. The resistant virus displayed altered binding between gp120 and CCR5 such that the virus became critically dependent on the N′ terminus of CCR5 in the presence of APL. In addition, the drug-resistant Envs studied here utilized CCR5 very efficiently: robust virus infection occurred even when very low levels of CCR5 were expressed. However, recognition of drug-bound CCR5 was less efficient, resulting in a tropism shift toward effector memory cells upon infection of primary CD4+ T cells in the presence of APL, with relative sparing of the central memory CD4+ T cell subset. If such a tropism shift proves to be a common feature of CCR5-antagonist-resistant viruses, then continued use of CCR5 antagonists even in the face of virologic failure could provide a relative degree of protection to the TCM subset of CD4+ T cells and result in improved T cell homeostasis and immune function.

Dual Pressure from Antiretroviral Therapy and Cell-Mediated Immune Response on the Human Immunodeficiency Virus Type 1 Protease Gene

ABSTRACT Human immunodeficiency virus (HIV)-specific CD8+ T-lymphocyte pressure can lead to the development of viral escape mutants, with consequent loss of immune control. Antiretroviral drugs also exert selection pressures on HIV, leading to the emergence of drug resistance mutations and increased levels of viral replication. We have determined a minimal epitope of HIV protease, amino acids 76 to 84, towards which a CD8+ T-lymphocyte response is directed. This epitope, which is HLA-A2 restricted, includes two amino acids that commonly mutate (V82A and I84V) in the face of protease inhibitor therapy. Among 29 HIV-infected patients who were treated with protease inhibitors and who had developed resistance to these drugs, we show that the wild-type PR82V76-84 epitope is commonly recognized by cytotoxic T lymphocytes (CTL) in HLA-A2-positive patients and that the CTL directed to this epitope are of high avidity. In contrast, the mutant PR82A76-84 epitope is generally not recognized by wild-type-specific CTL, or when recognized it is of low to moderate avidity, suggesting that the protease inhibitor-selected V82A mutation acts both as a CTL and protease inhibitor escape mutant. Paradoxically, the absence of a mutation at position 82 was associated with the presence of a high-avidity CD8+ T-cell response to the wild-type virus sequence. Our results indicate that both HIV type 1-specific CD8+ T cells and antiretroviral drugs provide complex pressures on the same amino acid sequence of the HIV protease gene and, thus, can influence viral sequence evolution.

Use of Cellular HIV DNA to Predict Virologic Response to Maraviroc: Performance of Population-Based and Deep Sequencing

BACKGROUND A tropism test is required before administration of the antiretroviral drug maraviroc. However, plasma RNA testing is not possible in patients with undetectable plasma viral loads. Here we assess genotypic testing of cellular human immunodeficiency virus (HIV) DNA from peripheral blood mononuclear cells (PBMCs) to predict virologic responses in treatment-experienced patients beginning maraviroc-containing regimens. METHODS PBMC samples from 181 maraviroc recipients at study entry in MOTIVATE or A4001029 (51% R5 by original Trofile). The V3 loop was amplified in triplicate from cellular HIV DNA, and matching plasma RNA (n = 156). Sequencing was performed using standard population-based methods and next-generation deep sequencing, with tropism assessment as previously defined. RESULTS Genotypic DNA-based tropism testing from the cellular compartment had 78%-81% sensitivity relative to RNA-based Trofile at the same time point. Cell-based genotypic tropism methods and plasma-based phenotypic and genotypic methods were predictive of virologic response. However, when classifications were discordant, the outcomes favored the plasma predictions over the DNA ones. CONCLUSIONS Genotypic determination of HIV tropism can be performed using cell-derived viral DNA, and is a predictor of virologic success on maraviroc in therapy-experienced patients. However, the PBMC compartment appears to be a suboptimal predictor compared to plasma.

HIV-1 infection in the lymphoid organs

AimTo develop a model of HIV disease progression. MethodComparative analysis of viral burden and replication between peripheral blood and lymphoid organs and of the changes in viral distribution in the lymphoid tissue. ResultsIn early-stage disease HIV-1-infected cells were sequestered in the lymphoid tissue, and the viral particles were concentrated and trapped in the germinal centers. The dichotomy in viral burden and viral replication between peripheral blood and lymphoid tissue was related to the histopathologic abnormalities associated with different stages of disease. ConclusionsThese histopathologic abnormalities may not only explain the changes in viral distribution observed in the lymphoid tissue in different stages of the disease, but may also reflect different functional states of the immune system during the progression of HIV-1 infection from early- to late-stage disease.

Virologic Failure in Therapy-Naïve Subjects on Aplaviroc plus Lopinavir-Ritonavir: Detection of Aplaviroc Resistance Requires Clonal Analysis of Envelope

ABSTRACT The CCR100136 (EPIC) study evaluated the antiviral activity of the novel CCR5 entry inhibitor aplaviroc in combination with lopinavir-ritonavir in drug-naïve human immunodeficiency virus type 1-infected subjects. Although the trial was stopped prematurely due to idiosyncratic hepatotoxicity, 11 subjects met the protocol-defined virologic failure criteria. Clonal analyses of the viral envelope tropism, aplaviroc susceptibility, and env sequencing were performed on plasma at day 1 and at the time of virologic failure. Molecular evolutionary analyses were also performed. Treatment-emergent resistance to aplaviroc or lopinavir-ritonavir was not observed at the population level. However, aplaviroc resistance was detected prior to therapy at both the clonal and population levels in one subject with virologic failure and in six subjects in a minority (<50%) of clones at day 1 or at the time of virologic failure. Reduced aplaviroc susceptibility manifested as a 50% inhibitory concentration curve shift and/or a plateau. Sequence changes in the clones with aplaviroc resistance were unique to each subject and scattered across the envelope coding region. Clones at day 1 and at the time of virologic failure were not phylogenetically distinct. Two subjects with virologic failure had a population tropism change from CCR5- to dual/mixed-tropic during treatment. Virologic failure during a regimen of aplaviroc and lopinavir-ritonavir may be associated with aplaviroc resistance, only at the clonal level, and/or, infrequently, tropism changes.

Immunologic and virologic analyses of an acutely HIV type 1-infected patient with extremely rapid disease progression

The immunologic and virologic factors that impact on the rate of disease progression after acute infection with human immunodeficiency virus (HIV) type 1 are poorly understood. A patient with an extraordinarily rapid disease course leading to AIDS-associated death within 6 months of infection was studied intensively for the presence of anti-HIV immune reactivities as well as changes in the genetic and biologic properties of virus isolates. Although altered humoral responses were evident, the most distinctive immunologic feature was a nearly complete absence of detectable HIV-specific CTL responses. In addition to a rapid decline in CD3+CD4+ cells, elevated percentages of CD8+CD45RA+ and CD8+CD57+ cells and diminished CD8+CD45R0+ and CD8+CD28+ cells were evident. Primary viral isolates recovered throughout the course of infection exhibited limited sequence diversity. Cloned viral envelopes were found to have unusually broad patterns of coreceptor usage for cell-cell fusion, although infectivity studies yielded no evidence of infection via these alternative receptors. The infectivity studies demonstrated that these isolates and their envelopes maintained an R5 phenotype throughout the course of disease. The absence of demonstrable anti-HIV CTL reactivities, coupled with a protracted course of seroconversion, highlights the importance of robust HIV-specific immune responses in the control of disease progression.