Matthew T. Trivett

Persistent Human Papillomavirus Infection Is Associated with a Generalized Decrease in Immune Responsiveness in Older Women

The development of cervical cancer and its precursors are linked to persistent infection with oncogenic types of human papillomavirus (HPV). Host immune responses seem to be determinants of risk for this disease. However, little is known about the immunologic determinants of HPV persistence. Here, we examined the association between lymphoproliferative responses to antigens/mitogens and persistent HPV infection in women older than 45 years. Women included in this study were participants in a 10,000-woman population-based cohort study of cervical neoplasia in Costa Rica. Women older than 45 years and HPV DNA positive at a screening visit were selected as cases (n = 283). We selected a comparably sized control group of HPV DNA-negative women, matched to cases on age and time since enrollment (n = 261). At an additional clinical visit, women were cytologically and virologically rescreened, and cervical and blood specimens were collected. Proliferative responses to phytohemagglutinin (PHA), influenza virus (Flu), and HPV16 virus-like particle (VLP) were lower among women with persistent HPV infection [median counts per minute (cpm): 72,849 for PHA, 1,241 for Flu, and 727 for VLP] than for the control group (median cpm: 107,049 for PHA, 2,111 for Flu, and 2,068 for VLP). The decreases were most profound in women with long-term persistence and were only observed for the oldest age group (>/=65 years). Our results indicate that an impairment in host immunologic responses is associated to persistent HPV infection. The fact that effects were evident for all studied stimuli is suggestive of a generalized effect.

Long-lasting humoral and cellular immune responses and mucosal dissemination after intramuscular DNA immunization.

Naïve Indian rhesus macaques were immunized with a mixture of optimized plasmid DNAs expressing several SIV antigens using in vivo electroporation via the intramuscular route. The animals were monitored for the development of SIV-specific systemic (blood) and mucosal (bronchoalveolar lavage) cellular and humoral immune responses. The immune responses were of great magnitude, broad (Gag, Pol, Nef, Tat and Vif), long-lasting (up to 90 weeks post third vaccination) and were boosted with each subsequent immunization, even after an extended 90-week rest period. The SIV-specific cellular immune responses were consistently more abundant in bronchoalveolar lavage (BAL) than in blood, and were characterized as predominantly effector memory CD4(+) and CD8(+) T cells in BAL and as both central and effector memory T cells in blood. SIV-specific T cells containing Granzyme B were readily detected in both blood and BAL, suggesting the presence of effector cells with cytolytic potential. DNA vaccination also elicited long-lasting systemic and mucosal humoral immune responses, including the induction of Gag-specific IgA. The combination of optimized DNA vectors and improved intramuscular delivery by in vivo electroporation has the potential to elicit both cellular and humoral responses and dissemination to the periphery, and thus to improve DNA immunization efficacy.

Fixation and cryopreservation of whole blood and isolated mononuclear cells: Influence of different procedures on lymphocyte subset analysis by flow cytometry†

Immunophenotyping of whole blood (WB) and isolated peripheral blood mononuclear cells (PBMCs) is a common tool used to evaluate immune system changes in clinical studies. The development of methods that would allow preservation of samples for flow cytometric analysis is important for the extension of this technology to field testing in settings where the equipment might be not readily accessible.

Distribution, Persistence, and Efficacy of Adoptively Transferred Central and Effector Memory-Derived Autologous Simian Immunodeficiency Virus-Specific CD8+ T Cell Clones in Rhesus Macaques during Acute Infection

Plasma viremia decreases coincident with the appearance of virus-specific CD8+ T cells during acute HIV or SIV infection. This finding, along with demonstrations of viral mutational escape from CD8+ T cell responses and transient increase in plasma viremia after depletion of CD8+ T cells in SIV-infected monkeys strongly suggest a role for CD8+ T cells in controlling HIV/SIV. However, direct quantitative or qualitative correlates between CD8+ T cell activity and virus control have not been established. To directly assess the impact of large numbers of virus-specific CD8+ T cells present at time of SIV infection, we transferred in vitro expanded autologous central and effector memory-derived Gag CM9-, Nef YY9-, and Vif WY8-specific CD8+ T cell clones to acutely infected rhesus macaques. The cells persisted in PBMCs between 4 and 9 d, but were not detected in gut-associated lymphoid tissue or lymph nodes. Interestingly, a high frequency of the infused cells localized to the lungs, where they persisted at high frequency for >6 wk. Although persisting cells in the lungs were Ag reactive, there was no measurable effect on virus load. Sequencing of virus from the animal receiving Nef YY9-specific CD8+ T cells demonstrated an escape mutation in this epitope <3 wk postinfection, consistent with immune selection pressure by the infused cells. These studies establish methods for adoptive transfer of autologous SIV-specific CD8+ T cells for evaluating immune control during acute infection and demonstrate that infused cells retain function and persist for at least 2 mo in specific tissues.

Trafficking, Persistence, and Activation State of Adoptively Transferred Allogeneic and Autologous Simian Immunodeficiency Virus-Specific CD8+ T Cell Clones during Acute and Chronic Infection of Rhesus Macaques

Despite multiple lines of evidence suggesting their involvement, the precise role of CD8+ T cells in controlling HIV replication remains unclear. To determine whether CD8+ T cells can limit retroviral replication in the absence of other immune responses, we transferred 1–13 × 109 allogeneic in vitro expanded SIV-specific CD8+ T cell clones matched for the relevant restricting MHC-I allele into rhesus macaques near the time of i.v. SIV challenge. Additionally, in vitro expanded autologous SIV-specific CD8+ T cell clones were infused 4–9 mo postinfection. Infused cells did not appreciably impact acute or chronic viral replication. The partially MHC-matched allogeneic cells were not detected in the blood or most tissues after 3 d but persisted longer in the lungs as assessed by bronchoalveolar lavage (BAL). Autologous cells transferred i.v. or i.p. were found in BAL and blood samples for up to 8 wk postinfusion. Interestingly, despite having a nominally activated phenotype (CD69+HLA-DR+), many of these cells persisted in the BAL without dividing. This suggests that expression of such markers by T cells at mucosal sites may not reflect recent activation, but may instead identify stable resident memory T cells. The lack of impact following transfer of such a large number of functional Ag-specific CD8+ T cells on SIV replication may reflect the magnitude of the immune response required to contain the virus.

Role of DC-SIGN in the activation of dendritic cells by HPV-16 L1 virus-like particle vaccine.

Dendritic cell‐specific intercellular adhesion molecule‐grabbing non‐integrin (DC‐SIGN), a specific C‐type lectin expressed on DC, binds and transmits different pathogens to susceptible cells. In the present study, we examined the role of DC‐SIGN in the capture of human papillomavirus (HPV) pseudovirions and activation of DC. We demonstrate that HPV virus‐like particles (VLP) bind to DC‐SIGN expressed on transfected Raji cells and that antibodies against DC‐SIGN block this interaction. DC take up VLP, which activate expression of costimulatory markers and cytokines/chemokines. Although our results indicate that DC‐SIGN is not the major receptor for VLP in DC, this interaction contributes to the activation of DC surface antigens (HLA class I) and of various cytokines/chemokines, particularly TNF‐α, IL‐6, and RANTES. Induction of these markers in DC by VLP was significantly abrogated when binding to DC‐SIGN was blocked by anti‐DC‐SIGN antibodies. These results suggest that DC‐SIGN has a functional role in DC activation induced by HPV‐16 L1‐VLP, and thus highlight new aspects of DC interactions with HPV VLP.

Cytotoxic Capacity of SIV-Specific CD8+ T Cells against Primary Autologous Targets Correlates with Immune Control in SIV-Infected Rhesus Macaques

Although the study of non-human primates has resulted in important advances for understanding HIV-specific immunity, a clear correlate of immune control over simian immunodeficiency virus (SIV) replication has not been found to date. In this study, CD8+ T-cell cytotoxic capacity was examined to determine whether this function is a correlate of immune control in the rhesus macaque (RM) SIV infection model as has been suggested in chronic HIV infection. SIVmac251-infected human reverse transcriptase (hTERT)-transduced CD4+ T-cell clone targets were co-incubated with autologous macaque effector cells to measure infected CD4+ T-cell elimination (ICE). Twenty-three SIV-infected rhesus macaques with widely varying plasma viral RNA levels were evaluated in a blinded fashion. Nineteen of 23 subjects (83%) were correctly classified as long-term nonprogressor/elite controller (LTNP/EC), slow progressor, progressor or SIV-negative rhesus macaques based on measurements of ICE (weighted Kappa 0.75). LTNP/EC had higher median ICE than progressors (67.3% [22.0–91.7%] vs. 23.7% [0.0–58.0%], p = 0.002). In addition, significant correlations between ICE and viral load (r = −0.57, p = 0.01), and between granzyme B delivery and ICE (r = 0.89, p<0.001) were observed. Furthermore, the CD8+ T cells of LTNP/EC exhibited higher per-cell cytotoxic capacity than those of progressors (p = 0.004). These findings support that greater lytic granule loading of virus-specific CD8+ T cells and efficient delivery of active granzyme B to SIV-infected targets are associated with superior control of SIV infection in rhesus macaques, consistent with observations of HIV infection in humans. Therefore, such measurements appear to represent a correlate of control of viral replication in chronic SIV infection and their role as predictors of immunologic control in the vaccine setting should be evaluated.

Transduction of SIV-specific TCR genes into rhesus macaque CD8+ T cells conveys the ability to suppress SIV replication.

Background The SIV/rhesus macaque model for HIV/AIDS is a powerful system for examining the contribution of T cells in the control of AIDS viruses. To better our understanding of CD8+ T-cell control of SIV replication in CD4+ T cells, we asked whether TCRs isolated from rhesus macaque CD8+ T-cell clones that exhibited varying abilities to suppress SIV replication could convey their suppressive properties to CD8+ T cells obtained from an uninfected/unvaccinated animal. Principal Findings We transferred SIV-specific TCR genes isolated from rhesus macaque CD8+ T-cell clones with varying abilities to suppress SIV replication in vitro into CD8+ T cells obtained from an uninfected animal by retroviral transduction. After sorting and expansion, transduced CD8+ T-cell lines were obtained that specifically bound their cognate SIV tetramer. These cell lines displayed appropriate effector function and specificity, expressing intracellular IFNγ upon peptide stimulation. Importantly, the SIV suppression properties of the transduced cell lines mirrored those of the original TCR donor clones: cell lines expressing TCRs transferred from highly suppressive clones effectively reduced wild-type SIV replication, while expression of a non-suppressing TCR failed to reduce the spread of virus. However, all TCRs were able to suppress the replication of an SIV mutant that did not downregulate MHC-I, recapitulating the properties of their donor clones. Conclusions Our results show that antigen-specific SIV suppression can be transferred between allogenic T cells simply by TCR gene transfer. This advance provides a platform for examining the contributions of TCRs versus the intrinsic effector characteristics of T-cell clones in virus suppression. Additionally, this approach can be applied to develop non-human primate models to evaluate adoptive T-cell transfer therapy for AIDS and other diseases.

Generation of Alloantigen-Stimulated Anti—Human Immunodeficiency Virus Activity Is Associated with HLA-A*02 Expression

Stimulation of peripheral blood mononuclear cells (PBMC) with allogeneic PBMC (ALLO) can result in activity that inhibits the replication of human immunodeficiency virus (HIV). The present study demonstrates that strong anti-HIV activity is dependent on expression of HLA-A*02 by the responding PBMC. Anti-HIV activity was equally effective against 2 primary isolates that use different coreceptors. Neither ALLO-stimulated cell proliferation nor cytokine and beta-chemokine production was associated with the expression of HLA-A*02. ALLO-stimulated production of strong anti-HIV activity required intact PBMC and was not inhibited by monoclonal antibodies directed against nonpolymorphic regions of human leukocyte antigens (HLAs). Anti-HIV activity was generated by ALLO-stimulated CD4(+) cells, CD8(+) T lymphocytes, and monocytes from HLA-A*02-positive patients. These findings provide the first evidence that the production of an HIV inhibitory factor or factors is associated with certain HLA genes and raise new possibilities concerning the role of the major histocompatibility complex in controlling viral infections via alloantigen stimulation.

Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Single-Nucleotide Polymorphisms Identified in Clinical Samples Can Affect MicroRNA Processing, Level of Expression, and Silencing Activity

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) encodes 12 pre-microRNAs that can produce 25 KSHV mature microRNAs. We previously reported single-nucleotide polymorphisms (SNPs) in KSHV-encoded pre-microRNA and mature microRNA sequences from clinical samples (V. Marshall et al., J. Infect. Dis., 195:645–659, 2007). To determine whether microRNA SNPs affect pre-microRNA processing and, ultimately, mature microRNA expression levels, we performed a detailed comparative analysis of (i) mature microRNA expression levels, (ii) in vitro Drosha/Dicer processing, and (iii) RNA-induced silencing complex-dependent targeting of wild-type (wt) and variant microRNA genes. Expression of pairs of wt and variant pre-microRNAs from retroviral vectors and measurement of KSHV mature microRNA expression by real-time reverse transcription-PCR (RT-PCR) revealed differential expression levels that correlated with the presence of specific sequence polymorphisms. Measurement of KSHV mature microRNA expression in a panel of primary effusion lymphoma cell lines by real-time RT-PCR recapitulated some observed expression differences but suggested a more complex relationship between sequence differences and expression of mature microRNA. Furthermore, in vitro maturation assays demonstrated significant SNP-associated changes in Drosha/DGCR8 and/or Dicer processing. These data demonstrate that SNPs within KSHV-encoded pre-microRNAs are associated with differential microRNA expression levels. Given the multiple reports on the involvement of microRNAs in cancer, the biological significance of these phenotypic and genotypic variants merits further studies in patients with KSHV-associated malignancies.