Cytokines Elevated in HIV Elite Controllers Reduce HIV Replication In Vitro and Modulate HIV Restriction Factor Expression.

A subset of HIV-infected individuals termed elite controllers (ECs) maintain CD4+ T cell counts and control viral replication in the absence of antiretroviral therapy (ART). Systemic cytokine responses may differentiate ECs from subjects with uncontrolled viral replication or from those who require ART to suppress viral replication. We measured 87 cytokines in four groups of women: 73 ECs, 42 with pharmacologically suppressed viremia (ART), 42 with uncontrolled viral replication (noncontrollers [NCs]), and 48 HIV-uninfected (NEG) subjects. Four cytokines were elevated in ECs but not NCs or ART subjects: CCL14, CCL21, CCL27, and XCL1. In addition, median stromal cell-derived factor-1 (SDF-1) levels were 43% higher in ECs than in NCs. The combination of the five cytokines suppressed R5 and X4 virus replication in resting CD4+ T cells, and individually SDF-1ß, CCL14, and CCL27 suppressed R5 virus replication, while SDF-1ß, CCL21, and CCL14 suppressed X4 virus replication. Functional studies revealed that the combination of the five cytokines upregulated CD69 and CCR5 and downregulated CXCR4 and CCR7 on CD4+ T cells. The CD69 and CXCR4 effects were driven by SDF-1, while CCL21 downregulated CCR7. The combination of the EC-associated cytokines induced expression of the anti-HIV host restriction factors IFITM1 and IFITM2 and suppressed expression of RNase L and SAMHD1. These results identify a set of cytokines that are elevated in ECs and define their effects on cellular activation, HIV coreceptor expression, and innate restriction factor expression. This cytokine pattern may be a signature characteristic of HIV-1 elite control, potentially important for HIV therapeutic and curative strategies.IMPORTANCE Approximately 1% of people infected with HIV control virus replication without taking antiviral medications. These subjects, termed elite controllers (ECs), are known to have stronger immune responses targeting HIV than the typical HIV-infected subject, but the exact mechanisms of how their immune responses control infection are not known. In this study, we identified five soluble immune signaling molecules (cytokines) in the blood that were higher in ECs than in subjects with typical chronic HIV infection. We demonstrated that these cytokines can activate CD4+ T cells, the target cells for HIV infection. Furthermore, these five EC-associated cytokines could change expression levels of intrinsic resistance factors, or molecules inside the target cell that fight HIV infection. This study is significant in that it identified cytokines elevated in subjects with a good immune response against HIV and defined potential mechanisms as to how these cytokines could induce resistance to the virus in target cells.

Hepatic fibrosis and immune phenotype vary by HCV viremia in HCV/HIV co-infected subjects: A Women's interagency HIV study.

HCV and HIV independently lead to immune dysregulation. The mechanisms leading to advanced liver disease progression in HCV/HIV coinfected subjects remain unclear.In this cross-sectional study, we assessed the association of HCV viremia, liver fibrosis, and immune response patterns in well-characterized HIV phenotypes: Elite controllers (Elites), HIV controlled (ARTc), and HIV uncontrolled (ARTuc) matched by age and race. Groups were stratified by HCV RNA status. Regulatory T-cell frequencies, T-cell activation (HLADR+CD38+), apoptosis (Caspase-3+), and intracellular cytokines (interferon-γ, IL-2, IL-17) were assessed using multiparametric flow-cytometry. Liver fibrosis was scored by AST to platelet ratio index (APRI).We found liver fibrosis (APRI) was 50% lower in Elites and ARTc compared to ARTuc. Higher liver fibrosis was associated with significantly low CD4+ T cell counts (P < 0.001, coefficient r = -0.463). Immune activation varied by HIV phenotype but was not modified by HCV viremia. HCV viremia was associated with elevated CD8 T-cell Caspase-3 in Elites, ARTuc, and HIV- except ARTc. CD8 T-cell Caspase-3 levels were significantly higher in HCV RNA+ Elites (P = 0.04) and ARTuc (P = 0.001) and HIV- groups (P = 0.02) than ARTc. Importantly, ARTuc HCV RNA+ had significantly higher CD4 T-cell interleukin-17 levels than ARTuc HCV RNA- (P = 0.005).HIV control was associated with lower liver fibrosis in HCV/HIV co-infected women. HCV viremia is associated with an inflammatory CD4 TH-17 phenotype in absence of HIV control and higher frequency of pro-apoptosis CD8 T-cells critical to avert progression of HIV and HCV disease that is attenuated in ART controllers. Elite controllers with HCV viremia are more prone to CD8 T-cell apoptosis than ART controllers, which could have negative consequences over time, highlighting the importance of ART control in HCV/HIV coinfected individuals.

HIV RNA levels in plasma and cervical-vaginal lavage fluid in elite controllers and HAART recipients.

OBJECTIVES: The introduction of HAART leads to control of HIV replication to less than 50 copies/ml, similar to levels in 'elite controllers'. Low-level viral replication may be one of the contributing factors to persistent immune activation/inflammation in HAART-treated individuals. There are still gaps in our knowledge of whether low-level replication persists in systemic versus mucosal sites. DESIGN AND METHODS: Participants for this study were recruited from the Women's Interagency HIV Study. We evaluated 33 'elite controllers' who naturally controlled HIV replication and 33 matched HAART-suppressed recipients. This study employed a sensitive target-capture transcription-mediated-amplification assay to compare low-level virus concentrations in plasma and cervical-vaginal lavage (CVL) samples from HIV-positive HAART recipients and 'elite controllers'. RESULTS: The median (interquartile range) plasma viral load signal/cut-off (S/Co) for 'elite controllers' was 10.5 (3.9-21.1), which was significantly (P < 0.001) higher than the S/Co for HAART recipients [2.0 (1-4.9)]. The majority of CVL samples from both groups had undetectable HIV RNA and the proportion of CVL samples with a cut-off more than 1.0 was not different between 'elite controllers' and HAART-suppressed recipients. CONCLUSION: This study demonstrated persistent low-level HIV replication in 'elite controllers', suggesting potential value of HAART treatment for these individuals. Absent or very low levels of HIV RNA in CVL indicate very low risk of secondary sexual transmission for both groups.

Asymptomatic HLA-A*02:01-restricted epitopes from herpes simplex virus glycoprotein B preferentially recall polyfunctional CD8+ T cells from seropositive asymptomatic individuals and protect HLA transgenic mice against ocular herpes.

Evidence from C57BL/6 mice suggests that CD8(+) T cells, specific to the immunodominant HSV-1 glycoprotein B (gB) H-2(b)-restricted epitope (gB498-505), protect against ocular herpes infection and disease. However, the possible role of CD8(+) T cells, specific to HLA-restricted gB epitopes, in protective immunity seen in HSV-1-seropositive asymptomatic (ASYMP) healthy individuals (who have never had clinical herpes) remains to be determined. In this study, we used multiple prediction algorithms to identify 10 potential HLA-A*02:01-restricted CD8(+) T cell epitopes from the HSV-1 gB amino acid sequence. Six of these epitopes exhibited high-affinity binding to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01-positive, HSV-1-seropositive ASYMP individuals, the most frequent, robust, and polyfunctional CD8(+) T cell responses, as assessed by a combination of tetramer, IFN-γ-ELISPOT, CFSE proliferation, CD107a/b cytotoxic degranulation, and multiplex cytokine assays, were directed mainly against epitopes gB342-350 and gB561-569. In contrast, in 10 HLA-A*02:01-positive, HSV-1-seropositive symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent clinical herpes disease) frequent, but less robust, CD8(+) T cell responses were directed mainly against nonoverlapping epitopes (gB183-191 and gB441-449). ASYMP individuals had a significantly higher proportion of HSV-gB-specific CD8(+) T cells expressing CD107a/b degranulation marker and producing effector cytokines IL-2, IFN-γ, and TNF-α than did SYMP individuals. Moreover, immunization of a novel herpes-susceptible HLA-A*02:01 transgenic mouse model with ASYMP epitopes, but not with SYMP epitopes, induced strong CD8(+) T cell-dependent protective immunity against ocular herpes infection and disease. These findings should guide the development of a safe and effective T cell-based herpes vaccine.

Vitamin D and insulin resistance in non-diabetic women's interagency HIV study participants.

We explored the relationship between vitamin D levels and insulin resistance (IR) among 1082 nondiabetic (754 HIV-infected) women enrolled in the Women's Interagency HIV study (WIHS), a large and well-established cohort of HIV infected and uninfected women in the US. Vitamin D levels 20-29 ng/mL were considered insufficient and <20 ng/mL deficient. IR was estimated using the homeostasis model assessment (HOMA) and a clinically significant cut-off ≥2.6 was used for HOMA-IR. In the unadjusted analysis, women who were vitamin D insufficient or deficient were 1.62 (95% CI: 1.01-2.61, p=0.05) and 1.70 (95% CI: 1.11-2.60, p=0.02) times more likely to have HOMA values≥2.6 compared to women with sufficient vitamin D. The association did not remain significant after adjustment for factors associated with IR. Among the 754 HIV-infected women, current PI use (OR 1.61, 95% CI: 1.13-2.28, p=0.008) remained independently associated with HOMA ≥2.6 while vitamin D insufficiency (OR 1.80, 95% CI: 0.99-3.27, p=0.05) was marginally associated with HOMA ≥2.6 after adjustment. Ethnicity, body mass index, smoking status, and hepatitis C status were independently associated with insulin resistance in HIV-infected and uninfected women. We found a marginally significant association between vitamin D insufficiency and insulin resistance among nondiabetic HIV-infected WIHS women.

Interleukin 10 responses are associated with sustained CD4 T-cell counts in treated HIV infection.

BACKGROUND: Inflammation persists in treated human immunodeficiency virus (HIV) infection and may contribute to an increased risk for non-AIDS-related pathologies. We investigated the correlation of cytokine responses with changes in CD4 T-cell levels and coinfection with hepatitis C virus (HCV) during highly active antiretroviral treatment (HAART). METHODS: A total of 383 participants in the Women's Interagency HIV Study (212 with HIV monoinfection, 56 with HCV monoinfection, and 115 with HIV/HCV coinfection) were studied. HIV-infected women had <1000 HIV RNA copies/mL, 99.7% had >200 CD4 T cells/µL; 98% were receiving HAART at baseline. Changes in CD4 T-cell count between baseline and 2-4 years later were calculated. Peripheral blood mononuclear cells (PBMCs) obtained at baseline were used to measure interleukin 1ß (IL-1ß), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 12 (IL-12), and tumor necrosis factor α (TNF-α) responses to Toll-like receptor (TLR) 3 and TLR4 stimulation. RESULTS: Undetectable HIV RNA (<80 copies/mL) at baseline and secretion of IL-10 by PBMCs were positively associated with gains in CD4 T-cell counts at follow-up. Inflammatory cytokines (IL-1ß, IL-6, IL-12, and TNF-α) were also produced in TLR-stimulated cultures, but only IL-10 was significantly associated with sustained increases in CD4 T-cell levels. This association was significant only in women with HIV monoinfection, indicating that HCV coinfection is an important factor limiting gains in CD4 T-cell counts, possibly by contributing to unbalanced persistent inflammation. CONCLUSIONS: Secreted IL-10 from PBMCs may balance the inflammatory environment of HIV, resulting in CD4 T-cell stability.

The profile of inflammatory cytokines in gingival crevicular fluid around healthy osseointegrated implants.

OBJECTIVE: Regardless of gingival health and subgingival microbiology, production of cytokines within peri-implant tissues may be different from that of teeth. The objective of this study was to describe the peri-implant levels of pro-inflammatory cytokines and subgingival microbiology in clinically healthy sites. MATERIALS AND METHODS: Subgingival plaque and gingival crevicular fluid (GCF) were obtained from 28 clinically healthy implants and 26 teeth selected from 24 individuals. Microbial composition was determined by selective anaerobic culture techniques. Pro-inflammatory cytokines were quantified by flow cytometry analysis of GCF. The concentration of cytokines between implants and teeth were compared with the independent t-test. RESULTS: The concentration of cytokines was higher in GCF from healthy implants than in teeth. The profile of cytokines was characteristic of an innate immune response. A more frequent detection of periodontopathic bacteria was observed in teeth than implants. Cultivable levels of periodontopathic bacteria were similar between implants and teeth. CONCLUSIONS: Despite gingival tissue health and scarce plaque accumulation, the profile of inflammatory cytokines in implant crevicular fluid was distinctive of an innate immune response and in higher concentration than in teeth. Other than bacterial stimulus, intrinsic factors related to implants may account for more cytokine production than teeth.

Vitamin D deficiency in HIV-infected and HIV-uninfected women in the United States.

BACKGROUND: Vitamin D deficiency is of increasing concern in HIV-infected persons because of its reported association with a number of negative health outcomes that are common in HIV. We undertook this study to determine the prevalence and predictors of vitamin D deficiency among a nationally representative cohort of middle-aged, ethnically diverse, HIV-infected and HIV-uninfected women enrolled in the Women's Interagency HIV Study (WIHS). METHODS: Vitamin D testing was performed by Quest Diagnostics on frozen sera using the liquid chromatography/mass spectroscopy method. Vitamin D deficiency was defined as 25(OH)D ≤20 ng/mL. Comparisons of continuous and categorical characteristics among HIV-infected and HIV-uninfected women were made by Wilcoxon tests and Pearson χ tests, respectively. RESULTS: One thousand seven hundred seventy-eight women (1268 HIV positive) were studied. Sixty-three percent had vitamin D deficiency (60% HIV positive vs. 72% HIV negative; P < 0.001). Multivariable predictors of vitamin D deficiency were being African American (adjusted odds ratio 3.02), Hispanic (adjusted odds ratio 1.40), body mass index (adjusted odds ratio 1.43), age (adjusted odds ratio 0.84), HIV positive (adjusted odds ratio 0.76), glomerular filtration rate <90·mL·min (adjusted odds ratio 0.94), and WIHS sites Los Angeles (adjusted odds ratio 0.66) and Chicago (adjusted odds ratio 0.63). In the HIV-positive women, multivariate predictors were undetectable HIV RNA (adjusted odds ratio 0.69), CD4 50-200 cells per cubic millimeter (adjusted odds ratio 1.60), CD4 <50 cells per cubic millimeter (adjusted odds ratio 1.94), and recent protease inhibitor use (adjusted odds ratio 0.67). CONCLUSIONS: In this study of more than 1700 women in the United States, most women with or without HIV infection had low vitamin D levels and African American women had the highest rates of vitamin D deficiency. An understanding of the role that vitamin D deficiency plays in non-AIDS-related morbidities is planned for investigation in WIHS.

Factors associated with hepatitis C viremia in a large cohort of HIV-infected and -uninfected women.

BACKGROUND: Co-infection with hepatitis C virus (HCV) is common among HIV-infected women. OBJECTIVE: To further our understanding of the risk factors for HCV viremia and the predictors of HCV viral load among women. STUDY DESIGN: We investigated sociodemographic, immunologic, and virologic factors associated with presence and level of HCV viremia among 1049 HCV-seropositive women, 882 of whom were HIV-infected and 167 HIV-uninfected at their entry into the Women's Interagency HIV Study. RESULTS: Plasma HCV RNA was detected in 852 (81%) of these 1049 women (range: 1.2-7.8 log(10)copies/ml). HCV-viremic women were more likely to have an HIV RNA level >100,000 copies/ml (P=0.0004), to have reported smoking (P=0.01), or to be Black (P=0.005). They were less likely to have current or resolved hepatitis B infection. HCV RNA levels were higher in women who were >35 years old, or HIV-infected. Current smoking and history of drug use (crack/freebase cocaine, marijuana, amphetamines, or heroin) were each associated with both presence and level of viremia. CONCLUSIONS: Substance abuse counseling aimed at eliminating ongoing use of illicit drugs and tobacco may reduce clinical progression, improve response to treatment, and decrease HCV transmission by lowering levels of HCV viremia in women.

Microbiology and cytokine levels around healthy dental implants and teeth.

BACKGROUND: Elicitation of the relationship of periodontopathogens and pro-inflammatory cytokines to bone resorption and formation is significant to a growing body of research known as osteoimmunology. It is essential that clinically healthy peri-implant and periodontal sites are studied to contribute comparison data for investigations that are addressing diseased sites. PURPOSE: The purpose of this study was to describe levels of selected pro-inflammatory cytokines in clinically healthy peri-implant and periodontal sites, and to examine whether cytokine levels may be related to specific bacterial/viral pathogens. MATERIALS AND METHODS: Eleven subjects (mean age 56.2 +/- 10) participated in the study. Subgingival microbial samples were cultured for periodontopathic bacteria. Gingival crevicular fluid samples were analyzed by nested polymerase chain reaction for Cytomegalovirus (HCMV) and were tested for the quantification of Interleukin (IL)-8, IL-1beta, IL-6, IL-10, Tumor Necrosis Factor (TNF)-alpha, and IL-12p70 using flow cytometry (FACS). Findings for microbiota composition and cytokine levels were compared between implants and teeth (chi square, Kruskall-Wallis, Mann-Whitney; p < or = .05). RESULTS: Both the frequency (%) and levels (%) of periodontopathic bacteria were higher around teeth than implants. The concentration (picogram per milliliter) of cytokines was more prominent around implants than teeth, reaching nearly twofold differences in some instances. Cytokine levels were higher when the sites analyzed were positive for any bacteria tested. HCMV was not detected. CONCLUSIONS: Pro-inflammatory cytokine production was unrelated to heavy bacterial challenge. Nevertheless, when periodontopathic bacteria were detected by culture, cytokine levels were increased around both implants and teeth. Studies are needed to investigate the pro-inflammatory cytokines (especially IL-1beta and TNF-alpha) produced in spite of minimal bacterial accumulation.

Ex vivo selection and expansion of cells based on expression of a mutated inosine monophosphate dehydrogenase 2 after HIV vector transduction: effects on lymphocytes, monocytes, and CD34+ stem cells.

Hematopoietic progenitor cells (HPCs) represent an ideal target for gene therapy treatment of human immunodeficiency virus (HIV) infection. However, gene delivery into quiescent HPCs by retroviral or lentiviral vectors remains relatively poor. We evaluated a selection scheme based on the expression of a variant of inosine monophosphate dehydrogenase 2 (IMPDH2), the rate-limiting enzyme in the de novo purine biosynthesis pathway. As lymphocytes depend more than other cell types on de novo synthesis of purines, IMPDH inhibitors such as mycophenolic acid (MPA) can selectively expand lymphocytes overexpressing the enzymes. We used HIV vectors to deliver an IMPDH variant into T cells and HPCs. We showed that the transduced T cells became resistant to MPA selection. By expressing a short hairpin RNA gene targeted to the HIV gag transcript, the MPA-selected T cells became resistant to HIV-1 infection. Monocyte/macrophages derived from the transduced HPCs differentiated normally and exhibited normal function as measured by B7 up-regulation and phagocytosis when stimulated. Our results suggest that this system may be applicable as a selection strategy to enrich transduced T lymphocytes and mononuclear cells in vivo for HIV gene therapy.

Reduced type 1 and type 2 cytokines in antiviral memory T helper function among women coinfected with HIV and HCV.

Bias in cytokine responses has been proposed as a contributing mechanism to pathogenesis in persistent HIV or hepatitis C virus (HCV) infections. We investigated whether coinfection with HCV modifies the profile of antigen-specific cytokine secretion in women persistently infected with HIV compared to women with single HIV or HCV infection. The T helper response to HIV, HCV and cytomegalovirus (CMV) as a positive viral control was dominated by type 1 cytokines (interleukin- [IL] 2, interferon- [IFN] gamma and tumor necrosis factor- [TNF] alpha), with IFN-gamma as the most abundantly secreted. IL-4, IL-5 and IL-10 were low in healthy controls and patients. Robust CMV-specific responses contrasted with curtailed HCV-specific responses in HCV-infected women. The overall anti-viral profile was dominated by Th1 cytokines even in coinfected women but both type 1 and type 2 responses were reduced in HIV-infected women and more extensively in women with HCV/HIV coinfection.

Making dendritic cells from the inside out: lentiviral vector-mediated gene delivery of granulocyte-macrophage colony-stimulating factor and interleukin 4 into CD14+ monocytes generates dendritic cells in vitro.

We have evaluated a one-hit lentiviral transduction approach to genetically modifying monocytes in order to promote autocrine and paracrine production of factors required for their differentiation into immature dendritic cells (DCs). High-titer third-generation self-inactivating lentiviral vectors expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) efficiently achieved simultaneous and persistent codelivery of the transgenes into purified human CD14+ monocytes. Coexpression of GM-CSF and IL-4 in CD14+ cells was sufficient to induce their differentiation into a DC-like phenotype, as evidenced by their morphology, immature immunophenotypic profile (CD14-, CD1a+, CD80+, CD86+, MHC-I+, MHC-II+), and their ability to further develop into a mature phenotype (CD83+) on further treatment with soluble CD40 ligand. Mixed lymphocyte reactions showed that the T cell-stimulating activity of lentivirus-modified DCs was superior to that of DCs grown by conventional methods. Lentivirus-modified DCs displayed efficient antigen-specific, MHC class I-restricted stimulation of autologous CD8+ T cells, as shown by IFN-gamma production and CTL assays. DCs coexpressing GM-CSF and IL-4 could be kept metabolically active and viable in culture for 14 days in the absence of exogenously added growth factors, unlike conventionally produced DCs. Coexpression of FLT3 ligand did not improve the viability, expansion, or immunologic performance of lentivirus-modified DCs. This article demonstrates the proof-of-concept to genetically convert monocytes to DC-type antigen-presenting cells with lentiviral vectors.

Predominant type 1 CMV-specific memory T-helper response in humans: evidence for gender differences in cytokine secretion.

Cell-mediated memory immune responses to viral antigens are important for protection against viruses causing persistent or acute infections. This study compared the cytokine profile of memory T-helper cells specific for cytomegalovirus (CMV) in healthy CMV-seropositive men and women. The cytokine response reflected T(H)1 bias, with dominant secretion of interferon (IFN)-gamma along with moderate levels of tumor necrosis factor-alpha, interleukin (IL)-10, and IL-2. Analyzed by gender, women had higher and significant spontaneous release of IFN-gamma and CMV-specific IL-2 secretion. Similar analysis with herpes simplex virus-1 showed a trend toward higher cytokine responsiveness in women, but the differences were not statistically significant. In contrast, men had statistically significant higher influenza virus-specific tumor necrosis factor-alpha secretion. IL-4 and IL-5, both T(H)2 cytokines, were low for all three viruses. The results show a predominant T(H)1 antiviral cytokine T-help memory response with significant differences linked to gender. Such differences may have an impact in the design of immunization strategies against CMV.

Attenuated poxviruses generate clinically relevant frequencies of CMV-specific T cells.

Immunotherapeutic approaches to limit cytomegalovirus (CMV) morbidity and mortality after hematopoietic stem cell transplants (HSCTs) are currently under investigation as alternatives to antiviral drugs. In this context, we have inserted full-length and ubiquitin-modified CMV phosphoprotein 65 (pp65), phosphoprotein 150 (pp150), and immediate early protein 1 (IE1) immunodominant antigens into the virulent Western Reserve strain of vaccinia virus (VV) and the highly attenuated strain, modified vaccinia Ankara (MVA). Recombinant (r) VV or rMVA stimulated vigorous expansion of CMV-specific CD8+ T cells in CMV-positive donor peripheral blood mononuclear cells (PBMCs), which showed minimal alloreactivity and high levels of HLA tetramer binding, cytokine production, and cytotoxicity. Ubiquitinated antigens had a profound effect when expressed in VV. Single antigen rMVA expressing pp65 or IE1, either ubiquitin-modified or native, stimulated both cytotoxic T lymphocyte (CTL) populations to be expanded up to 500-fold in a 60-mL blood draw from the same donor. This result demonstrates the clinical feasibility of simultaneously amplifying multiple CMV-CTL populations. Transgenic HLA A2.1 (HHD II) mice, immunized with the same rMVA as used with human PBMCs, produced a robust cytotoxic response to both CMV pp65 and IE1. The specificity of the vigorous immunologic response to rMVA, both in vitro and in vivo, makes them candidates for clinical evaluation in the context of adoptive immunotherapy for hematopoietic stem cell transplant (HSCT) recipients or donor vaccination.

Relevance of peptide avidity to the T cell receptor for cytomegalovirus-specific ex vivo CD8 T cell cytotoxicity.

CD8(+) T cells contribute to the control of viral infection by several effector mechanisms, including lysis of virally infected cells and interferon (IFN)-gamma secretion. Ex vivo cytotoxicity and potent secretion of IFN-gamma in response to cytomegalovirus (CMV) epitope peptides was seen in freshly prepared unstimulated peripheral blood mononuclear cells from human immunodeficiency virus-infected patients with high T cell receptor (TCR)/peptide avidity. Lymphocytes with low TCR/peptide avidity had no ex vivo cytotoxicity, secreted minimal IFN-gamma, and could not recognize autologous infected targets. Despite this, ex vivo responding and nonresponding patients had substantial frequencies of tetramer-positive and IFN-gamma-secreting lymphocytes. Levels of activation and memory markers were also similar in tetramer-positive populations of both groups. However, cytolytic capacity remained in nonresponders; their lymphocytes regained cytotoxicity after in vitro stimulation with peptide without coactivators or interleukin-2. High-avidity CD8(+) T cells are likely important in viral control, and their generation should be a goal of therapeutic vaccination.

Use of transgenic HLA A*0201/Kb and HHD II mice to evaluate frequency of cytomegalovirus IE1-derived peptide usage in eliciting human CD8 cytokine response.

Unlike the pp65 protein of human cytomegalovirus (CMV), which has an immunodominant peptide, pp65(495-503), recognized by human CD8(+) cells in the context of HLA A*0201, the fine peptide specificity for CMV IE1 has shown no such immunodominance. With the use of transgenic HLA A*0201/Kb and HHD II mice, a selected pool of IE1 peptides, including IE1(p256-264), IE1(p297-304), and IE1(p316-324), were shown to stimulate cytolytic T-lymphocyte lysis in the context of HLA A*0201. Based on an intracellular gamma interferon response, IE1(p297-304), a previously unrecognized CD8 epitope, triggered a prominent response to CMV IE1 in HLA A*0201 subjects.

Relative dominance of HLA-B*07 restricted CD8+ T-lymphocyte immune responses to human cytomegalovirus pp65 in persons sharing HLA-A*02 and HLA-B*07 alleles.

CD8(+) T-cell responses to three human cytomegalovirus (CMV) pp65 epitopes were studied in panels of healthy seropositive HLA-A*02/HLA-B*07 individuals, and HLA-A*02 donors mismatched for HLA-B*07. The majority of the latter had significant responses to a HLA-A*02-restricted epitope within the CMV pp65 antigen. By contrast, the strongest responses to CMV in the first group were to HLA-B*07-restricted epitopes. Similar immunodominance of HLA-B*07 over HLA-A*02 was found in two immunocompromised HIV-infected HLA-A*02/HLA-B*07 patients, and in the reconstituting immune system of three stem cell transplant recipients. In vitro stimulation of peripheral blood mononuclear cells (PBMC) from two immunocompetent HLA-A*02/HLA-B*07 individuals indicated that cytotoxic T lymphocyte (CTL) precursors specific for both HLA-A*02 and HLA-B*07 restricted epitopes were present and could be expanded by stimulation with the cognate peptides. However, if stimulation was performed by antigen presenting cells infected with recombinant vaccinia expressing full-length native pp65, only HLA-B*07 epitope-specific cells were seen. In one patient the HLA-B*07 dominance was partially broken by using recombinant vaccinia expressing ubiquitinated pp65, suggesting that enhanced protein processing can reveal weaker immune responses. Our results indicate that CMV-specific cellular immune responses restricted by HLA-B*07 dominate those restricted by HLA-A*02 in both immunocompetent and immunocompromised individuals. This may have significant consequences for the design of epitope-specific vaccines.

Preclinical development of an adjuvant-free peptide vaccine with activity against CMV pp65 in HLA transgenic mice.

Epitope vaccines have shown promise for inducing cellular immune responses in animal models of infectious disease. In cases where cellular immunity was augmented, peptide vaccines composed of covalently linked minimal cytotoxic T-lymphocyte (CTL) and T-helper (T(H)) epitopes generally showed the most efficacy. To address a clinical vaccine strategy for cytomegalovirus (CMV) in the context of HCT (hematopoietic cell transplantation), we observed that linking the synthetically derived pan-DR epitope peptide (PADRE) or one of several tetanus T(H) epitopes to the immunodominant human leukocyte antigen (HLA) A*0201-restricted CTL epitope from CMV-pp65 to create a fusion peptide caused robust cytotoxic cellular immune responses in HLA A*0201/K(b) transgenic mice. Significantly, the fusion peptides are immunogenic when administered in saline solution by either subcutaneous or intranasal routes. CpG-containing single-stranded DNA (ss-oligodeoxynucleotide [ODN]) added to the fusion peptides dramatically up-regulated immune recognition by either route. Notably, target cells that either expressed full-length pp65 protein from vaccinia viruses or were sensitized with the CTL epitope encoded in the vaccine were recognized by splenic effectors from immunized animals. Visualization of murine peptide-specific CTL by flow cytometry was accomplished using an HLA A*0201 tetramer complexed with the pp65(495-503) CTL epitope. T(H)-CTL epitope fusion peptides in combination with CpG ss-ODN represent a new strategy for parenteral or mucosal delivery of vaccines in a safe and effective manner that has applicability for control or prophylaxis of infectious disease, especially in situations such as vaccination of donors or recipients of HCT, where highly inflammatory adjuvants are not desired.