Longitudinal cervicovaginal microbiome and virome alterations during ART and discordant shedding in women living with HIV.

Despite successful suppression of plasma HIV replication by antiretroviral therapy (ART), some women living with HIV (WLHIV) can still experience genital HIV shedding (discordant shedding). Female genital tract (FGT) microbiome and virome dynamics during long-term ART in WLHIV are poorly understood but might contribute to discordant HIV shedding, as the microbiome and virome are known to influence FGT health. To understand FGT microbial communities over time during ART usage and discordant shedding, we characterized the microbiome and virome in 125 cervicovaginal specimens collected over two years in 31 WLHIV in Lima, Peru. Intrapersonal bacterial microbiome variation was higher in HIV shedders compared to non-shedders. Cervicovaginal virome composition changed over time, particularly in non-shedders. Specifically, anellovirus relative abundance was inversely associated with ART duration and CD4 counts. Our results suggest that discordant HIV shedding is associated with FGT microbiome instability, and immune recovery during ART influences FGT virome composition.

A highly multiplexed droplet digital PCR assay to measure the intact HIV-1 proviral reservoir.

Quantifying the replication-competent HIV reservoir is essential for evaluating curative strategies. Viral outgrowth assays (VOAs) underestimate the reservoir because they fail to induce all replication-competent proviruses. Single- or double-region HIV DNA assays overestimate it because they fail to exclude many defective proviruses. We designed two triplex droplet digital PCR assays, each with 2 unique targets and 1 in common, and normalize the results to PCR-based T cell counts. Both HIV assays are specific, sensitive, and reproducible. Together, they estimate the number of proviruses containing all five primer-probe regions. Our 5-target results are on average 12.1-fold higher than and correlate with paired quantitative VOA (Spearman's ρ = 0.48) but estimate a markedly smaller reservoir than previous DNA assays. In patients on antiretroviral therapy, decay rates in blood CD4+ T cells are faster for intact than for defective proviruses, and intact provirus frequencies are similar in mucosal and circulating T cells.

Subclinical Genital Herpes Shedding in HIV/Herpes Simplex Virus 2-Coinfected Women during Antiretroviral Therapy Is Associated with an Increase in HIV Tissue Reservoirs and Potentially Promotes HIV Evolution.

Antigen (Ag)-specific immune responses to chronic infections, such as herpes simplex virus type 2 (HSV-2) in HIV/HSV-coinfected persons, may sustain HIV tissue reservoirs by promoting T-cell proliferation but are poorly studied in women on antiretroviral therapy (ART). Mixed anogenital swabs and cervical secretions were self-collected by nine HIV/HSV-2-coinfected women during ART for 28 days to establish subclinical HSV DNA shedding rates and detection of HIV RNA by real-time PCR. Typical herpes lesion site biopsy (TLSB) and cervical biopsy specimens were collected at the end of the daily sampling period. Nucleic acids (NA) isolated from biopsy specimens had HIV quantified and HIV envC2-V5 single-genome amplification (SGA) and T-cell receptor (TCR) repertoires assessed. Women had a median CD4 count of 537 cells/µl (IQR: 483 to 741) at enrollment and HIV plasma viral loads of <40 copies/ml. HSV DNA was detected on 12% of days (IQR: 2 to 25%) from anogenital specimens. Frequent subclinical HSV DNA shedding was associated with increased HIV DNA tissue concentrations and increased divergence from the most recent common ancestor (MRCA), an indicator of HIV replication. Distinct predominant TCR clones were detected in cervical and TLSB specimens in a woman with frequent HSV DNA shedding, with mixing of minor variants between her tissues. In contrast, more limited TCR repertoire mixing was observed in two women with less frequent subclinical HSV DNA shedding. Subclinical HSV shedding in HIV/HSV-coinfected women during ART may sustain HIV tissue reservoirs via Ag exposure or HIV replication. This study provides evidence supporting further study of interventions targeting suppression of Ag-specific immune responses as a component of HIV cure strategies.IMPORTANCE Persons with HIV infection are frequently coinfected with chronic herpesviruses, which periodically replicate and produce viable herpes virions, particularly in anogenital and cervical tissues. Persistent protein expression results in proliferation of CD8+ and CD4+ T cells, and the latter could potentially expand and sustain HIV tissue reservoirs. We found HSV genital shedding rates were positively correlated with HIV DNA concentrations and HIV divergence from ancestral sequences in tissues. Our work suggests that immune responses to common coinfections, such as herpesviruses, may sustain HIV tissue reservoirs during suppressive ART, suggesting future cure strategies should study interventions to suppress replication or reactivation of chronic herpes infections.

Genital Shedding of Human Immunodeficiency Virus Type-1 (HIV) When Antiretroviral Therapy Suppresses HIV Replication in the Plasma.

BACKGROUND: During antiretroviral treatment (ART) with plasma HIV RNA below the limit of quantification, HIV RNA can be detected in genital or rectal secretions, termed discordant shedding (DS). We hypothesized that proliferating cells produce virions without HIV replication. METHODS: ART-naive Peruvians initiating ART were observed for DS over 2 years. HIV env and pol genomes were amplified from DS. Antiretrovirals and cytokines/chemokines concentrations were compared at DS and control time points. RESULTS: Eighty-two participants had ART suppression. DS was detected in 24/82 (29%) participants: 13/253 (5%) cervicovaginal lavages, 20/322 (6%) seminal plasmas, and 6/85 (7%) rectal secretions. HIV RNA in DS specimens was near the limit of quantification and not reproducible. HIV DNA was detected in 6/13 (46%) DS cervicovaginal lavages at low levels. Following DNase treatment, 5/39 DS specimens yielded HIV sequences, all without increased genetic distances. Women with and without DS had similar plasma antiretroviral levels and DS in 1 woman was associated with inflammation. CONCLUSIONS: HIV RNA and DNA sequences and therapeutic antiretroviral plasma levels did not support HIV replication as the cause of DS from the genital tract. Rather, our findings infer that HIV RNA is shed due to proliferation of infected cells with virion production.

Phylogenetic Analyses Comparing HIV Sequences from Plasma at Virologic Failure to Cervix Versus Blood Sequences from Antecedent Antiretroviral Therapy Suppression.

Identifying tissue sources of HIV that rebound following "failure" of antiretroviral therapy (ART) is critical to evaluating cure strategies. To assess the role of the uterine cervix and peripheral blood mononuclear cells (PBMC) as viral reservoirs, nearest-neighbor phylogenetic analyses compared genetic relatedness of tissue sequences during ART suppression to those detected in plasma at viral rebound. Blood and genital tract specimens from a natural history cohort of HIV-infected women were collected over 5 years. HIV DNA sequences extracted from PBMC and cervical biopsies during ART suppression and plasma RNA from rebound (defined as HIV RNA >3 log10 copies/mL) were derived by single-genome amplification. Phylogenetic and nearest-neighbor analyses of HIV env sequences and drug resistance in pol sequences were compared between tissues. Nine instances of plasma viral rebound (median HIV RNA 3.6 log10 c/mL; IQR: 3.1-3.8) were detected in 7 of 57 women. Nearest-neighbor analyses found rebound plasma sequences were closer to uterine cervical sequences in 4/9 (44%), closer to PBMC in 3/9 (33%), and ambiguous in 2/9 (22%) cases. Rebound plasma clades (n = 27) shared identical sequences in seven instances with the cervix versus two with PBMC. Novel drug resistance mutations were detected in 4/9 (44%) rebounds. The observed tendency for greater sharing of identical HIV variants and greater nearest-neighbor association between rebounding plasma and uterine cervical versus PBMC sequences suggests that the uterine cervix may be a relevant HIV reservoir. The cervix, a readily accessible tissue in women that can be repeatedly sampled, could help assess the HIV reservoir when evaluating cure strategies.

Monotypic low-level HIV viremias during antiretroviral therapy are associated with disproportionate production of X4 virions and systemic immune activation.

OBJECTIVE: During effective antiretroviral therapy (ART), low-level plasma viremias (LLV) (HIV RNA >30-1000 copies/ml) can be detected intermittently. We hypothesized that systemic inflammation is associated with LLV either as the cause or result of the production of virions from clonally expanded cells. METHODS: Prospective cohort study of HIV-infected ART-naive Peruvians enrolled prior to ART and followed for 2 years. Plasma HIV RNA and peripheral blood mononuclear cell (PBMC) HIV DNA concentrations were quantified pre-ART from individuals whose plasma HIV RNA was ART-suppressed. Inflammatory biomarker concentrations were measured pre and during ART. Single-genome amplification (SGA) derived HIV env and pol genotypes from pre-ART and LLV specimens. Antiretroviral levels during ART assessed adherence. Statistical associations and phylogenetic relationships were examined. RESULTS: Among 82 participants with median plasma HIV RNA less than 30 copies/ml, LLV were detected in 33 of 82 (40%), with a LLV median HIV RNA of 73 copies/ml. Participants with vs. without LLV had significantly higher pre-ART plasma HIV RNA (P < 0.001) and PBMC HIV DNA (P < 0.007); but, during ART, their antiretroviral drug levels were similar. LLV env sequences were monotypic in 17 of 28 (61%) and diverse in 11 of 28 (39%) participants. Those with the monotypic vs. diverse LLV pattern had elevated hsCRP and sCD163 (P = 0.004) and LLV with more X4 variants (P = 0.02). CONCLUSION: In individuals with monotypic LLV sequences, higher levels of pre-ART HIV DNA and RNA, systemic inflammation and X4 viruses suggest an interaction between inflammation and the production of virions from proliferating infected cells, and that naïve T cells may be a source of LLV.

Genetic analyses of HIV env associated with uveitis in antiretroviral-naive individuals.

OBJECTIVE: To analyze and compare HIV-1 env sequences from the eye to those from the blood of individuals with uveitis attributed to HIV with the goal of gaining insight into the pathogenesis of HIV-associated eye disease. DESIGN: A prospective case series of five HIV-infected antiretroviral-naive individuals with uveitis negative for other pathogens. METHODS: RNA from blood plasma and ocular aqueous humor was reverse transcribed using random hexamers. HIV env C2-V5 (HXB2: 6990-7668) sequences were generated by single-genome amplification using nested polymerase chain reaction followed by bidirectional Sanger sequencing. Sequence analyses by Geneious, Geno2Pheno, N-GLYCOSITE, DIVEIN, and HyPhy evaluated relationships between HIV in plasma and aqueous humor. RESULTS: A median of 20 (range: 13-22) plasma and 15 (range: 9-18) aqueous humor sequences were generated from each individual. The frequencies of sequences with predicted-N-linked-glycosylation sites and C-X-C chemokine receptor type 4 were comparable in aqueous humor and plasma of all five patients. Aqueous humor sequences had lower median genetic diversity compared with plasma across all patients, but similar divergence, in four of five patients. Aqueous humor HIV sequences were compartmentalized from plasma across subjects by Critchlow correlation coefficient, Slatkin and Maddison, nearest-neighbor statistic, and Fixation index. CONCLUSION: Among antiretroviral-naive individuals with uveitis attributed to HIV, the universal compartmentalization and decreased diversity of eye compared with blood sequences suggests time-limited passage of a small subset of variants from each patient's viral population into the eye tissues, followed by limited immune selection despite the inflammatory uveitis.

Biomarkers of inflammation in HIV-infected Peruvian men and women before and during suppressive antiretroviral therapy.

OBJECTIVE: Inflammatory biomarkers associated with cardiovascular disease are elevated in HIV-infected persons. These biomarkers improve with antiretroviral therapy (ART) but do not normalize to values observed in HIV-uninfected adults. Little is known regarding biomarkers of inflammation in HIV-infected Peruvians, in whom an increased burden of infectious diseases may exacerbate inflammation, and women, in whom sex difference may alter inflammation compared with men. METHODS: Peruvians initiating first-line ART were enrolled in a prospective observational study. Individuals with suppression of HIV RNA plasma loads to less than 30 copies/ml when determined quarterly over 24 months of ART, had biomarkers of inflammation and cellular activation measured pre-ART and at 24-months of ART, and evaluated for associations with sex and clinical parameters. RESULTS: Pre-ART high-sensitivity C-reactive protein (hsCRP) values of men were in the high-risk cardiovascular disease category (>3.0 mg/l) more frequently compared with women (P = 0.02); most women's values were in the low/average-risk categories. At 24 months of suppressive ART, hsCRP concentrations decreased in men (P = 0.03), but tended to increase in women, such that the proportion with high-risk hsCRP did not differ by sex. Pre-ART, soluble CD163 concentrations were higher in women compared with men (P = 0.02), and remained higher after 24 months of suppressive ART (P = 0.02). All other inflammatory biomarkers (P < 0.03) decreased across sexes. Biomarker concentrations were not associated with BMI or coinfections. CONCLUSION: Elevated inflammatory biomarkers persisted despite 24 months of suppressive ART in a subset of Peruvians, and to a greater extent in women compared with men. These findings suggest that lifestyle or pharmacologic interventions may be required to optimize the health of HIV-infected Peruvians, particularly women.

HIV-1 shedding from the female genital tract is associated with increased Th1 cytokines/chemokines that maintain tissue homeostasis and proportions of CD8+FOXP3+ T cells.

BACKGROUND: HIV-1 shedding from the female genital tract is associated with increased sexual and perinatal transmission and has been broadly evaluated in cross-sectional studies. However, few longitudinal studies have evaluated how the immune microenvironment effects shedding. METHODS: Thirty-nine HIV-1-infected women had blood, cervicovaginal lavage, and biopsies of the uterine cervix taken quarterly for up to 5 years. Cytokines/chemokines were quantified by Luminex assay in cervicovaginal lavage, and cellular phenotypes were characterized using immunohistochemistry in cervical biopsies. Comparisons of cytokine/chemokine concentrations and the percent of tissue staining positive for T cells were compared using generalized estimating equations between non-shedding and shedding visits across all women and within a subgroup of women who intermittently shed HIV-1. RESULTS: Genital HIV-1 shedding was more common when plasma HIV-1 was detected. Cytokines associated with cell growth (interleukin-7), Th1 cells/inflammation (interleukin-12p70), and fractalkine were significantly increased at shedding visits compared with non-shedding visits within intermittent shedders and across all subjects. Within intermittent shedders and across all subjects, FOXP3 T cells were significantly decreased at shedding visits. However, there were significant increases in CD8 cells and proportions of CD8FOXP3 T cells associated with HIV-1 shedding. CONCLUSIONS: Within intermittent HIV-1 shedders, decreases in FOXP3 T cells at the shedding visit suggests that local HIV-1 replication leads to CD4 T-cell depletion, with increases in the proportion of CD8FOXP3 cells. HIV-1-infected cell loss may promote a cytokine milieu that maintains cellular homeostasis and increases immune suppressor cells in response to HIV-1 replication in the cervical tissues.

Human immunodeficiency viruses appear compartmentalized to the female genital tract in cross-sectional analyses but genital lineages do not persist over time.

BACKGROUND: Whether unique human immunodeficiency type 1 (HIV) genotypes occur in the genital tract is important for vaccine development and management of drug resistant viruses. Multiple cross-sectional studies suggest HIV is compartmentalized within the female genital tract. We hypothesize that bursts of HIV replication and/or proliferation of infected cells captured in cross-sectional analyses drive compartmentalization but over time genital-specific viral lineages do not form; rather viruses mix between genital tract and blood. METHODS: Eight women with ongoing HIV replication were studied during a period of 1.5 to 4.5 years. Multiple viral sequences were derived by single-genome amplification of the HIV C2-V5 region of env from genital secretions and blood plasma. Maximum likelihood phylogenies were evaluated for compartmentalization using 4 statistical tests. RESULTS: In cross-sectional analyses compartmentalization of genital from blood viruses was detected in three of eight women by all tests; this was associated with tissue specific clades containing multiple monotypic sequences. In longitudinal analysis, the tissues-specific clades did not persist to form viral lineages. Rather, across women, HIV lineages were comprised of both genital tract and blood sequences. CONCLUSIONS: The observation of genital-specific HIV clades only in cross-sectional analysis and an absence of genital-specific lineages in longitudinal analyses suggest a dynamic interchange of HIV variants between the female genital tract and blood.

Monitoring of HIV type 1 DNA load and drug resistance in peripheral blood mononuclear cells during suppressive antiretroviral therapy does not predict virologic failure.

Our objective was to determine whether monitoring HIV-1 DNA concentration or new resistance mutations in peripheral blood mononuclear cells (PBMCs) during effective antiretroviral therapy (ART) predicts virologic failure. A retrospective analysis used blood specimens and clinical data from three nevirapine containing arms of a four-arm, open-label, randomized trial comparing ART regimens in HIV-1-infected children who had failed mono- or dual-nucleoside therapy. Sensitive assays compared cell-associated HIV-1 DNA concentrations and nevirapine (NVP) and lamivudine (3TC) resistance mutations in children with plasma HIV-1 RNA <400 copies(c)/ml who did or did not experience subsequent virologic failure. Forty-six children were analyzed through the last available follow-up specimen, collected at 48 (n=16) or 96 (n=30) weeks of ART. Thirty-five (76%) had sustained viral suppression and 11 (24%) had plasma viral rebound to ≥ 400 c/ml (virologic failure detected at a median of 36 weeks). HIV-1 DNA levels at baseline, 24, 48, and 96 weeks of ART were similar in children who did vs. did not experience virologic failure (p=0.82). HIV-1 DNA levels did not increase prior to viral rebound. NVP resistance mutations were detected in 91% of subjects in the failure group vs. 3% in the suppressed group (p <0.0001). Among nine evaluable children, NVP mutations were first detected prior to virologic failure in two (22%), at viral rebound in five (56%), and after failure in two (22%) children. HIV-1 DNA concentrations did not predict virologic failure in this cohort. New drug resistance mutations were detected in the PBMCs of a minority of virologically suppressed children who subsequently failed ART.

Transmitted HIV resistance to first-line antiretroviral therapy in Lima, Peru.

Transmission of drug-resistant HIV (TDR) has been associated with virologic failure of "first-line," nonnucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral therapy (ART). A national ART program began in Peru in 2004. We evaluated the prevalence of TDR in individuals initiating ART and their virologic outcome during 2 years of ART. HIV-infected, ARV-naive subjects who met criteria to start ART in Lima, Peru were enrolled in a longitudinal observational study between July 2007 and February 2009. Blood plasma and cells obtained prior to ART initiation were assessed for antiretroviral (ARV) resistance by an oligonucleotide ligation assay (OLA) sensitive to 2% mutant at reverse transcriptase (RT) codons K103N, Y181C, G190A, and M184V and a subset by consensus sequencing. A total of 112 participants were enrolled; the mean CD4 was 134 ± 89 cells/µl and the median plasma HIV RNA was 93,556 copies/ml (IQR 62,776-291,364). Drug resistance mutations conferring high-level resistance to ARV were rare, detected in one of 96 (1%) evaluable participants. This subject had the Y181C mutation detected in both plasma and peripheral blood mononuclear cells (PBMCs) at a concentration of 100% by OLA and consensus sequencing; nevertheless nevirapine-ART suppressed her viral replication. Consensus sequencing of 37 (19%) participants revealed multiple polymorphisms that occasionally have been associated with low-level reductions in ARV susceptibility. A low prevalence of TDR was detected among Peruvians initiating ART. Given the increasing availability of ART, continuing surveillance is needed to determine if TDR increases and the mutant codons associated with virologic failure.

Genetic analyses of HIV-1 env sequences demonstrate limited compartmentalization in breast milk and suggest viral replication within the breast that increases with mastitis.

The concentration of human immunodeficiency virus type 1 (HIV-1) is generally lower in breast milk than in blood. Mastitis, or inflammation of the breast, is associated with increased levels of milk HIV-1 and risk of mother-to-child transmission through breastfeeding. We hypothesized that mastitis facilitates the passage of HIV-1 from blood into milk or stimulates virus production within the breast. HIV-1 env sequences were generated from single amplicons obtained from breast milk and blood samples in a cross-sectional study. Viral compartmentalization was evaluated using several statistical methods, including the Slatkin and Maddison (SM) test. Mastitis was defined as an elevated milk sodium (Na(+)) concentration. The association between milk Na(+) and the pairwise genetic distance between milk and blood viral sequences was modeled using linear regression. HIV-1 was compartmentalized within milk by SM testing in 6/17 (35%) specimens obtained from 9 women, but all phylogenetic clades included viral sequences from milk and blood samples. Monotypic sequences were more prevalent in milk samples than in blood samples (22% versus 13%; P = 0.012), which accounted for half of the compartmentalization observed. Mastitis was not associated with compartmentalization by SM testing (P = 0.621), but Na(+) was correlated with greater genetic distance between milk and blood HIV-1 populations (P = 0.041). In conclusion, local production of HIV-1 within the breast is suggested by compartmentalization of virus and a higher prevalence of monotypic viruses in milk specimens. However, phylogenetic trees demonstrate extensive mixing of viruses between milk and blood specimens. HIV-1 replication in breast milk appears to increase with inflammation, contributing to higher milk viral loads during mastitis.

Compartmentalization of HIV-1 within the female genital tract is due to monotypic and low-diversity variants not distinct viral populations.

BACKGROUND: Compartmentalization of HIV-1 between the genital tract and blood was noted in half of 57 women included in 12 studies primarily using cell-free virus. To further understand differences between genital tract and blood viruses of women with chronic HIV-1 infection cell-free and cell-associated virus populations were sequenced from these tissues, reasoning that integrated viral DNA includes variants archived from earlier in infection, and provides a greater array of genotypes for comparisons. METHODOLOGY/PRINCIPAL FINDINGS: Multiple sequences from single-genome-amplification of HIV-1 RNA and DNA from the genital tract and blood of each woman were compared in a cross-sectional study. Maximum likelihood phylogenies were evaluated for evidence of compartmentalization using four statistical tests. Genital tract and blood HIV-1 appears compartmentalized in 7/13 women by >/=2 statistical analyses. These subjects' phylograms were characterized by low diversity genital-specific viral clades interspersed between clades containing both genital and blood sequences. Many of the genital-specific clades contained monotypic HIV-1 sequences. In 2/7 women, HIV-1 populations were significantly compartmentalized across all four statistical tests; both had low diversity genital tract-only clades. Collapsing monotypic variants into a single sequence diminished the prevalence and extent of compartmentalization. Viral sequences did not demonstrate tissue-specific signature amino acid residues, differential immune selection, or co-receptor usage. CONCLUSIONS/SIGNIFICANCE: In women with chronic HIV-1 infection multiple identical sequences suggest proliferation of HIV-1-infected cells, and low diversity tissue-specific phylogenetic clades are consistent with bursts of viral replication. These monotypic and tissue-specific viruses provide statistical support for compartmentalization of HIV-1 between the female genital tract and blood. However, the intermingling of these clades with clades comprised of both genital and blood sequences and the absence of tissue-specific genetic features suggests compartmentalization between blood and genital tract may be due to viral replication and proliferation of infected cells, and questions whether HIV-1 in the female genital tract is distinct from blood.

Monotypic human immunodeficiency virus type 1 genotypes across the uterine cervix and in blood suggest proliferation of cells with provirus.

Understanding the dynamics and spread of human immunodeficiency virus type 1 (HIV-1) within the body, including within the female genital tract with its central role in heterosexual and peripartum transmission, has important implications for treatment and vaccine development. To study HIV-1 populations within tissues, we compared viruses from across the cervix to those in peripheral blood mononuclear cells (PBMC) during effective and failing antiretroviral therapy (ART) and in patients not receiving ART. Single-genome sequences of the C2-V5 region of HIV-1 env were derived from PBMC and three cervical biopsies per subject. Maximum-likelihood phylogenies were evaluated for differences in genetic diversity and compartmentalization within and between cervical biopsies and PBMC. All subjects had one or more clades with genetically identical HIV-1 env sequences derived from single-genome sequencing. These sequences were from noncontiguous cervical biopsies or from the cervix and circulating PBMC in seven of eight subjects. Compartmentalization of virus between genital tract and blood was observed by statistical methods and tree topologies in six of eight subjects, and potential genital lineages were observed in two of eight subjects. The detection of monotypic sequences across the cervix and blood, especially during effective ART, suggests that cells with provirus undergo clonal expansion. Compartmentalization of viruses within the cervix appears in part due to viruses homing to and/or expanding within the cervix and is rarely due to unique viruses evolving within the genital tract. Further studies are warranted to investigate mechanisms producing monotypic viruses across tissues and, importantly, to determine whether the proliferation of cells with provirus sustain HIV-1 persistence in spite of effective ART.

Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials.

Interferon-gamma (IFN-gamma) ELISpot and intracellular cytokine staining (ICS) assays are routinely employed in clinical HIV vaccine trials to identify antigen-specific T cells in cryopreserved peripheral blood mononuclear cells (PBMC). Several parameters involved in blood collection, processing and shipping may influence immunological function of the resulting cells, including anticoagulant type, time from venipuncture to PBMC isolation/cryopreservation, method of PBMC isolation and procedure for sample shipping. We examined these parameters in single and multiple site studies, and found the length of time from venipuncture to cryopreservation is the most important parameter affecting performance of T cells in immunological assays. Comparing blood processed at 24 h after venipuncture with that processed within 8 h, we observed on average a modest reduction in PBMC viability ( approximately 8% decrease), a greater loss in cell recovery ( approximately 32%), and between 36-56% loss in IFN-gamma T cell frequencies by ELISpot assay. We also describe three cold shipping methods that maintain immunological function in appropriately cryopreserved PBMC. These data indicate that cryopreservation of PBMC should occur within 8 h of venipuncture for optimal performance. This narrow window for specimen processing has important implications in selecting and monitoring clinical sites with laboratory capacity to perform these procedures in future clinical trials.

Spontaneous T cell apoptosis in feline immunodeficiency virus (FIV)-infected cats is inhibited by IL2 and anti-B7.1 antibodies.

Lymph node (LN) T cells from feline immunodeficiency virus (FIV)-infected cats have an increased expression of B7 co-stimulatory molecules as well as their ligand CTLA4, resembling an activation phenotype shown to induce anergy and apoptosis in activated T cells. In addition, LN T cells from FIV-infected cats also show increased spontaneous apoptosis compared to uninfected animals. The apoptosis observed in these animals occurs primarily in T cells expressing B7 and CTLA4, suggesting a role for B7 and CTLA4 interactions in the induction of anergy/apoptosis. In order to investigate the role of B7 and CTLA4 interactions on T cell apoptosis in LN T cells from FIV-infected cats, we performed blocking experiments by measuring T cell apoptosis in LN T cell cultures treated with anti-feline B7.1, B7.2, and CTLA4 specific antibodies, as well as interleukin (IL)-2. The addition of IL2, the primary cytokine produced by B7/CD28 interactions, resulted in a significant decrease of T cell apoptosis in cultured LN cells as assessed by two-color flow cytometry and TUNEL assay. The addition of anti-B7.1 antibodies significantly inhibited T cell apoptosis in FIV-infected cats with low-level plasma viremia, while addition of anti-B7.2 and anti-CTLA4 antibodies had no affect. These results suggest a role of B7 signaling in the increased spontaneous apoptosis observed in LN T cells from FIV-infected animals.

B7+CTLA4+ T cells engage in T-T cell interactions that mediate apoptosis: a model for lentivirus-induced T cell depletion.

Apoptosis in lymph node (LN) T cells of feline immunodeficiency virus (FIV)-infected cats is associated with cells co-expressing B7.1 and B7.2 costimulatory molecules, and their ligand CTLA4. To study the possibility of B7.1/B7.2-CTLA4 mediated T-T interactions and the predicted induction of T cell apoptosis in vitro, costimulatory molecules were up-regulated on CD4+ and CD8+ T cells by mitogen stimulation. B7.1 expression on in vitro stimulated CD4+ and CD8+ cells increased within 24h; B7.2 and CTLA4 expression increased after 48-72 h. Apoptosis, as analyzed by terminal deoxynucleotidyl transferase (transferase nick end labeling, TUNEL)-based staining followed by three color flow cytometric analysis, correlated to the cells expressing B7 and/or CTLA4. Blocking experiments revealed that CD4+ and CD8+ T cell apoptosis could be significantly inhibited with anti-B7 antibodies. As FIV infection results in immune activation with a T cell phenotype similar to that of the in vitro activated T cells, the data support the hypothesis that the chronic expansion of B7+CTLA4+ LN T cells in infected cats allows for T-T cell interactions resulting in T cell depletion and eventually the development of AIDS.

Evaluation of T lymphocytes in captive african lions (Panthera leo) infected with feline immunodeficiency virus.

OBJECTIVE: To determine whether FIV infection in captive African lions is associated with changes in immune cell variables similar to those detected in domestic cats infected with FIV. ANIMALS: 5 captive African lions naturally infected with FIV (FIV+) and 5 lions not infected with FIV (FIV-). PROCEDURE: Peripheral blood samples were collected from FIV+ lions during annual examinations conducted during a 7-year period and at a single time point from the FIV- lions. From results of CBC and flow cytometry, lymphocyte subsets were characterized and compared. RESULTS: Flow cytometric analysis revealed that the percentage and absolute number of CD4+ and CD8+ T cells were significantly lower in FIV+ lions, compared with these values in FIV- lions. In FIV+ lions, severe depletion in the absolute number of CD4+ and CD8+ T cells was detected, although this did not correlate with clinical signs. Muscle wasting was the most consistent clinical sign of infection. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that FIV+ African lions develop lymphocyte deficiencies, including significant decreases in the absolute number of CD4+ and CD8+ T cells; these findings of immune dysfunction are similar to those defined for FIV+ domestic cats. It is important to monitor the number of CD4+ T cells in infected animals as a measure of disease progression.