Chronic Viremia Research Articles

Modeling HIV-1 Mucosal Transmission and Prevention in Humanized Mice.

The new generation humanized mice (hu-mice) that permit continuous de novo generation of human hematopoietic cells have led to novel strategies in studying HIV-1 pathogenesis, prevention and therapies. HIV-1 infection of hu-mice results in chronic viremia and CD4+ T cell loss, thus mimicking key aspects of the disease progression. In addition, the new generation hu-mice are permissive for HIV-1 sexual transmission by vaginal and rectal routes thus allowing in vivo efficacy testing of new anti-HIV-1 drugs for prevention. Two leading models are currently being used, namely the hu-HSC mice and the BLT mice. Here we describe the methodology for generating both hu-HSC and BLT mice and their use in the study of HIV-1 transmission and prevention of infection by topical and oral administration of anti-retroviral drugs. Practical aspects of the methodologies are emphasized.

Risk Factor Analysis for the Development of Large Granular Lymphocytosis after Allogeneic Hematopoietic Cell Transplantation: Randomized Stratification and Propensity Score Analysis

IntroductionLarge granular lymphocytes (LGL) are a morphologically recognizable subpopulation of lymphocytes comprising an immunophenotypically heterogeneous population of activated CD3+ T cells and CD3- natural killer (NK) cells that mediate non-MHC-restricted cytotoxicity. Increased number of circulating LGL can be found as a response to viral infections, autoimmune disease or malignant neoplasms, as a result of chronic antigenic stimulation. Of interest, LGL lymphocytosis has been reported to occur following hematopoietic cell transplantation (HCT), with a variable incidence of up to 20%. This population display improved transplant outcomes with a lower incidence of non-relapse mortality and relapse (Kim, BMT, 2013; Nann-Rütti, BBMT, 2012). The aim of the present study is to determine the risk factors associated with the development of LGL lymphocytosis after allogeneic HCT.MethodsA total of 826 patients who underwent an allogeneic HCT at Princess Margaret Cancer Centre, Toronto, Canada from 2000 to 2012 were retrospectively analyzed. LGL lymphocytosis was defined as the presence of at least two of the followings: 1) Sustained peripheral blood lymphocyte count ≥3.0 x 109/L observed in at least three consecutive determinations over a period of 2-3 months; 2) Predominance (≥30%) of LGL lymphocytes in the peripheral blood, as assessed by morphologic or immunophenotypic criteria; 3) T-cell receptor monoclonality assessed by PCR. The patient population was divided into discovery and replication sets using 2 different methods: stratified randomization and propensity score matching, using relevant baseline variables such as donor type, CMV serostatus, conditioning, T-cell depletion.ResultsNo significant imbalances were found between the discovery and replication sets in terms of relevant baseline characteristics and clinical outcomes, for both the randomly divided patients and the propensity score matched groups. The overall incidence of LGL lymphocytosis was 14.5% at 3 years. The incidence of LGL lymphocytosis was similar across all subgroups of patients, both for the randomly divided groups and the propensity score matching (P-value not significant).A multivariable analysis of the risk factors for the development of LGL lymphocytosis was performed, including the following variables: grades 3-4 acute graft-versus-host-disease (GVHD), chronic GVHD, CMV viremia, CMV serostatus of the recipient, donor type, transplant year and T-cell depletion (TCD) for GVHD prophylaxis.In the stratified randomization analysis, the following risk factors were identified: 1) Discovery set: chronic GVHD (Hazard Ratio: 8.3, 95% CI: 3.1-22.6, P<0.001), CMV viremia (HR: 2.7, 95% CI: 1.5-4.7, P<0.001) and use of an unrelated donor (HR: 2.1, 95% CI: 1.2-3.7, P =0.01). 2) Replication set: chronic GVHD (HR: 38.9, 95% CI: 5.3-284.9, P<0.001) and CMV viremia (HR 3.8, 95% CI: 2.2-6.6, P<0.001).For the propensity score matching analysis the risk factors for the development of LGL lymphocytosis were the following: 1) Discovery set: chronic GVHD (HR: 22.9, 95% CI: 3.2-169.3, P =0.002) and CMV viremia (HR: 2.2, 95% CI: 1.1-4.2, P =0.02). 2) Replication set: chronic GVHD (HR: 28.9, 95% CI: 3.9-212.5, P<0.001), CMV viremia (HR: 3.6, 95% CI: 1.9-6.7, P<0.001).ConclusionsChronic GVHD and CMV viremia are strongly associated with the development of LGL lymphocytosis following allogeneic HCT. This may reflect a chronic antigenic stimulation in the setting of GVHD and CMV infection, leading to the expansion of LGL. However, the underlying mechanisms of LGL activation and expansion in the allogeneic HCT setting still remain unclear. Thus, further investigations are needed to elucidate these mechanisms in particular in the setting of GVHD. DisclosuresLipton:Pfizer: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Teva: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding. Kim:Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.

Generation and evaluation of simian-human immunodeficiency virus subtype AE challenge stocks (VIR2P.1166)

Abstract The majority of preclinical and clinical human immunodeficiency virus type 1 (HIV-1) vaccine development efforts have focused on HIV-1 subtype B strains from the western world. However, the overwhelming majority of HIV/AIDS-related deaths occur in the developing world and are from other HIV-1 subtype strains. Therefore, we sought to develop and generate the first mucosally transmissible simian-human immunodeficiency virus (SHIV) challenge stocks from HIV-1 subtype AE env sequences. The sequences were identified from acutely infected HIV-1 individuals from Southeast Asia. The challenge stocks efficiently infected rhesus monkeys, replicated to high levels during acute infection, and established chronic setpoint viremia. The SHIV challenge stocks should facilitate the evaluation of vaccines and other interventions aimed at controlling the spread of HIV-1 subtype AE infection.

Dominance of HLA-B restricted virus-specific CD8+ T cell epitopes in chronic HBV infection

Background and Aims: HBV-specific CD8+ T cells are essential for HBV clearance and are an attractive target for immunotherapeutic intervention. However, the HBV-specific CD8+ T cell repertoire is poorly characterized. Indeed, <10 HLA-B restricted HBV-specific CD8+ T cell epitopes are described to date, although recent findings in the field of HIV and HCV infection clearly demonstrate a dominant role of these epitopes. We therefore aimed to identify novel CD8+ T-cell epitopes in HBV-infection and also address the relative contribution of HLA-A versus HLA-B restricted epitopes. Methods: 30 patients with chronic HBV (genotype D) and low viremia were screened for HBV-specific CD8+ T-cell responses using overlapping peptides spanning all HBV proteins in a matrix setup in an elispot assay. Positive responses were confirmed by intracellular interferon gamma staining, optimal epitopes were fine-mapped and HLA restriction was determined. In addition, the autologous viral sequences corresponding to the identified epitopes were analyzed in the patients. Results: 10 out of 30 patients targeted at least one CD8+ T cell epitope (range: 1–10). In total, we identified 27 CD8+ T cell responses. Interestingly, responses targeted core, polymerase and X proteins, but completely spared the surface antigen. None of these responses targeted previously described HBV-specific CD8+ T cell epitopes. To date, we were able to fine-map 10 novel CD8+ T cell epitopes. Importantly, most of these epitopes were restricted by HLA-B alleles (4 x HLA-B*35; 2 x HLA-B*7; 1 x HLA-B*14; 1 x HLAB*15). In one epitope restricted by HLA-B*35:02 we found evidence for the selection of viral escape mutations. Conclusions: In our cohort, HBV-specific CD8+ T cell responses are dominantly restricted by HLA-B alleles. These findings indicate that previous studies on HBV-specific immunity mainly focussing e.g. on HLA-A*02 restricted CD8+ T cell epitopes may not necessarily be conferrable to the complete CD8+ T cell repertoire. They also suggest that HLA-B restricted CD8+ T cell epitopes may be important targets for immunotherapy. The absence of T-cell responses located in the surface-Protein implies a dominant role of the HBs antigen in immune regulation and persistence of HBV.

Generation and evaluation of clade C simian-human immunodeficiency virus challenge stocks.

The development of a panel of mucosally transmissible simian-human immunodeficiency virus (SHIV) challenge stocks from multiple virus clades would facilitate preclinical evaluation of candidate HIV-1 vaccines and therapeutics. The majority of SHIV stocks that have been generated to date have been derived from clade B HIV-1 env sequences from viruses isolated during chronic infection and typically required serial animal-to-animal adaptation for establishing mucosal transmissibility and pathogenicity. To capture essential features of mucosal transmission of clade C viruses, we produced a series of SHIVs with early clade C HIV-1 env sequences from acutely HIV-1-infected individuals from South Africa. SHIV-327c and SHIV-327cRM expressed env sequences that were 99.7 to 100% identical to the original HIV-1 isolate and did not require in vivo passaging for mucosal infectivity. These challenge stocks infected rhesus monkeys efficiently by both intrarectal and intravaginal routes, replicated to high levels during acute infection, and established chronic setpoint viremia in 13 of 17 (76%) infected animals. The SHIV-327cRM challenge stock was also titrated for both single, high-dose intrarectal challenges and repetitive, low-dose intrarectal challenges in rhesus monkeys. These SHIV challenge stocks should facilitate the preclinical evaluation of vaccines and other interventions aimed at preventing clade C HIV-1 infection. We describe the development of two related clade C SHIV challenge stocks. These challenge stocks should prove useful for preclinical testing of vaccines and other interventions aimed at preventing clade C HIV-1 infection.

A-112 DNA and Protein Co-immunization Improves the Magnitude, Longevity, and mucosal dissemination of Immune Responses

Background: To increase effectiveness of DNA vaccines, we developed a method of simultaneous vaccination of DNA and protein. DNA & protein (inactivated viral particles) co-immunization induced both systemic and mucosal Ab responses, which correlated with slower virus acquisition; and potent T cell responses, which correlated with protection from chronic viremia. Different regimens of DNA & protein vaccines using purified SIV/HIV-1 Env were evaluated to further improve mucosal dissemination. Methods: Macaques were vaccinated with SIV/HIV DNAs via the IM route using in vivo electroporation followed by protein administered into the same muscle. Immune responses were monitored in blood and mucosal sites including vaginal and rectal secretions. Results: DNA & protein co-immunization induced rapid and high humoral responses while maintaining robust cellular responses typically obtained with DNA. We found high systemic and mucosal antibody binding titers against gp120 as well as scaffolded V1/V2 env region; high levels of Env-specific IgG in saliva; Env-specific IgG and IgA in rectal mucosa; robust humoral responses in vaginal and rectal secretions. Importantly, while IM administered DNA vaccine induced cellular responses disseminated to mucosal sites, we noted poor dissemination of the humoral responses. In contrast, co-immunization with Env led to the significant increases in mucosal Ab dissemination. Co-immunization also resulted in improved longevity of immune responses detectable for several years. Conclusions: Intramuscular DNA & protein co-delivery increases the magnitude and longevity of systemic and mucosal humoral immune responses in immunized macaques and is proposed as a practical and efficient method for human vaccination.

DNA and Protein Co-immunization Improves the Magnitude, Longevity, and Mucosal Dissemination of Immune Responses

OA24.04 Background: Determining the quality and longevity of vaccine-induced immune responses is essential for improving the prospects of AIDS vaccines. DNA and protein (inactivated viral particles) co-immunization regimen induced systemic and mucosal Ab responses, which correlated with slower virus acquisition upon challenge, and potent T cell responses providing protection against chronic viremia. We are evaluating different regimens of DNA&protein vaccines using purified SIV or HIV-1 Env to further dissect humoral and cellular responses including magnitude, breadth and mucosal dissemination. Methods: Macaques were vaccinated using DNA&protein co-immunization regimen in the presence of IL-12 DNA, coinjected into the same muscle. Humoral and cellular responses were monitored in blood and different tissues. Results: Co-immunization strategy of DNAp high binding titers against scaffolded V1/V2 env region; efficient dissemination to mucosal sites; high Env-specific IgG in saliva and Env-specific IgG and IgA in rectal mucosa. Analysis of cellular responses revealed the presence of cytotoxic memory T cells against several viral proteins. These cellular responses disseminated systemically as demonstrated by their presence not only in blood and lymphoid tissues, but also in bone marrow, liver, lung (effector site) and, importantly, rectal and vaginal mucosa. The longevity of the cellular responses induced by this co-immunization regimen was significantly improved, with SIV-specific T cells detected>5 yrs after the vaccination. Conclusions: Intramuscular DNA&protein co-delivery increases the magnitude and longevity of systemic and mucosal humoral immune responses in immunized macaques and is proposed as a practical and efficient method for human vaccination.

Spontaneous Clearance of Chronic HCV Infection: An Unusual Phenomenon

Introduction: In acute hepatitis C virus (HCV) infection, only about 25% of patients clear the infection through an immune response. Once chronic HCV infection is established in the remaining 75% of patients, spontaneous clearance of HCV infection is extremely uncommon. We present to you 5 such cases. We describe 5 consecutive patients at the Bronx VA Medical Center that spontaneously cleared chronic HCV infection within the last 10 years. The results are as follows: 3 out of 5 patients were chronically infected with HCV genotype 1. Spontaneous clearance occurred in 3 different scenarios: 1.) Two patients were HIV and HCV-co-infected (HIV infection was suppressed with antiretroviral therapy) and were never treated for hepatitis C. After many years of chronic HCV viremia, they spontaneously became HCV RNA undetectable and remained undetectable for up to 4 years. 2.) Two patients with chronic HCV mono-infection were each treated with antiviral therapy, which resulted in undetectable HCV RNA. After the end of therapy, they relapsed to high-level viremia and high ALT level, but spontaneously cleared HCV RNA to undetectable levels and a normal ALT. However, they relapsed again to high HCV RNA and high ALT. One patient subsequently cleared again and relapsed again. 3.) One HCV-mono-infected patient had received a liver transplant and was on immunosuppressive therapy. He never received HCV antiviral therapy. Two years after the liver transplant, his HCV RNA became undetectable, and it has remained undetectable since 3 years ago. Spontaneous clearance of chronic HCV infection is a very rare phenomenon, but we present 5 subjects in 1 medical center over a span of 10 years that exhibit this rare phenomenon, and in different scenarios: 1.) after relapse from antiviral therapy, 2.) post-liver transplantation, and 3.) in HIV/HCV co-infection.

Assessing the feasibility of hepatitis C virus vaccine trials: Results from the Hepatitis C Incidence and Transmission Study-community (HITS-c) vaccine preparedness study

Efficacy trials of preventive hepatitis C virus (HCV) vaccine candidates raise challenging scientific and ethical issues. Based on data from the first 3years of a community-based prospective observational study – the Hepatitis C Incidence and Transmission Study-community (HITS-c) – this paper examines the feasibility of conducting trials of candidate HCV vaccines with people who inject drugs (PWID) in Sydney, Australia. Of the 166 PWID confirmed HCV antibody negative and eligible for enrolment, 156 (94%) completed baseline procedures. Retention was high, with 89% of participants retained at 48weeks and 76% of participants completing at least 75% of study visits within 2weeks of schedule. The rate of primary HCV infection was 7.9/100py (95% CI 4.9, 12.7). Of the 17 incident cases, 16 completed at least one follow-up assessment and 12 (75%) had evidence of chronic viraemia with progression to chronic HCV infection estimated to be 6/100py. Power calculations suggest a chronic HCV infection rate of at least 12/100py (primary HCV infection rate 16/100py) will be required for stand-alone trials of highly efficacious candidates designed to prevent chronic infection. However, elevated primary HCV infection was observed among participants not receiving opioid substitution therapy who reported heroin as the main drug injected (26.9/100py, 95% CI 14.5, 50.0) and those who reported unstable housing (23.5/100py, 95% CI 7.6, 72.8), daily or more frequent injecting (22.7/100py, 95% CI 12.2, 42.2) and receptive syringe sharing (23.6/100py, 95% CI 9.8, 56.7) in the 6months prior to baseline. These data suggest that it is possible to recruit and retain at-risk PWID who adhere to study protocols and that modification of eligibility criteria may identify populations with sufficiently high HCV incidence. Results support the feasibility of large multi-centre HCV vaccine trials, including in the Australian setting.

Monitoring Human Immune Competence Using a Symbiont Virus: From Bench to Bedside.

Monitoring immune competence in transplant recipients would help tailor-made induction and maintenance immunosuppression: to date, no comprehensive test is available on the market. Torquetenovirus (TTV) is an orphan human virus with > 95% prevalence worldwide. Once acquired, it establishes chronic viremia (approximatively 3 log10 in healthy donors), which goes up to > 5 logs in every condition of immune suppression. Since 2008, our group first reported kinetics of TTV in myeloma and lymphoma patients receiving high-dose chemotherapy supported by autologous hematopoietic stem cell transplantation (Focosi et al, Transplantation, 2008). The clinical utility of TTV viremia determination was promptly shown by correlation with opportunistic CMV reactivations, and ability to predict time needed for return to normal immune competence. A very recent work (de Vlaminck et al, Cell, 2013) has shown that TTV is the only virus in the human plasma virome whose load changes significantly after heart or lung transplantation. The authors used shotgun sequencing, a very costly technique unapt for routine diagnostic use. We report here a single-centre, prospective trial enrolling 100 orthotopic liver transplant recipients and 100 kidney and/or pancreas transplant recipients followed-up for 2 years. Plasma samples were collected before transplantation, and at months 1, 3, 6, 12, and 24. A cheap, simple, universal real-time PCR was used to determine overall TTV viremia. Liver transplant recipients were stratified according to the intensity of maintenance immune suppression. Kidney and/or pancreas transplant recipients were stratified according to intensity of induction immune suppression. We report a significant association between TTV viremia and CMV reactivations, and biopsy-proven graft rejection.Figure: No Caption available.In conclusion, our data support the emerging concept that this marker can be of help for noninvasively confirm the clinical suspicion of graft rejection.

HCV adaptations to altered CD8+ T-cell immunity during pregnancy.

Robust, polyfunctional CD8+ T-cell responses targeted to multiple viral epitopes have been associated with successful resolution of acute HCV infection. In most individuals, however, immunity fails and chronic viremia ensues as CD8+ T cells become functionally exhausted or select viral variants with escape mutations in class I epitopes. During pregnancy, dysfunctional HCV-specific CD8+ T-cell responses may be further impaired by maternal–fetal tolerance processes such as the expansion of regulatory T-cell populations or hormonal changes. Recent evidence suggests that HCV adapts to relaxed maternal CD8+ T-cell pressure in pregnancy by shedding unfit escape mutations in certain class I epitopes, resulting in the selection of viruses with more efficient replication. Emergence of HCV variants with enhanced replicative capacity during pregnancy has ramifications for mother-to-child transmission, the primary route of HCV infection in children, and could potentially be relevant to other important, vertically transmissible, persistent viruses such as HBV and HIV. HCV has infected over 185 million people worldwide and persists in approximately 75% of these individuals, predisposing to liver inflammation, cirrhosis and hepatocellular carcinoma [1]. More than 1 million children are born to HCV-infected mothers each year, and 3–5% acquire the infection in utero or at delivery, making vertical transmission the leading route of pediatric HCV infection in the developed world [2]. Here, we review the critical role of CD8+ T-cell immunity in HCV infection, its modulation in pregnancy and recent evidence that HCV takes advantage of the unique immunologic niche of pregnancy to improve replicative fitness and potentially promote vertical transmission.

Characterization of CD4+ T-Cell Immune Activation and Interleukin 10 Levels Among HIV, Hepatitis C Virus, and HIV/HCV-Coinfected Patients

HIV/hepatitis C virus (HCV)-coinfected patients have accelerated liver disease compared with HCV monoinfection. In HIV-positive patients with viral suppression, data comparing inflammatory cytokines and immune activation between HIV/HCV coinfection with chronic hepatitis C (CHC) to HIV/HCV-seropositive patients with cleared HCV are limited. Fifty-nine age- and sex-matched patients were stratified: (1) HIV monoinfection (n = 15); (2) HCV monoinfection with CHC (n = 15); (3) HIV/HCV coinfection with CHC (n = 14); and (4) HIV/HCV seropositive with cleared HCV (n = 15). All HIV-positive patients had undetectable HIV viremia, and median CD4 was 420 cells per microliter. Liver fibrosis was assessed in each subject using transient elastography. Cells were collected for CD4 and CD8 immune activation (CD38/HLA-DR) markers via flow cytometry and plasma for luminex-multiplex cytokine assays. CD38⁺HLA-DR⁺ expression on CD4⁺ T cells was significantly increased in HIV/HCV coinfection with CHC (7%) versus HCV monoinfection (4%) (P = 0.012). CD4⁺ total HLA-DR⁺ expression was significantly increased in HIV/HCV coinfection with CHC (43%) versus HIV monoinfection (31%) (P = 0.010) and HIV/HCV seropositive with cleared HCV (38%) (P = 0.046). Total CD4⁺CD38⁺ and CD4⁺CD38⁺HLA-DR⁻ expression was significantly higher in HIV monoinfection (23% and 18%) than HCV moninfection (13%, P = 0.002% and 9%, P = 0.001, respectively). Interleukin 10 levels were significantly lower in HIV monoinfection versus HIV/HCV coinfection with CHC (P = 0.0002). In multivariate analysis, severe fibrosis was associated with lower expression of CD4⁺CD38⁺HLA-DR⁺ and CD4⁺ total CD38⁺ than mild-moderate fibrosis (P = 0.03 and 0.03, respectively). CD4 immune activation with HLA-DR⁺ expression in HIV/HCV coinfection with well-controlled HIV may arise from chronic HCV viremia. Conversely, CD4⁺CD38⁺ expression may be driven by underlying HIV infection. CD4 immune activation was unexpectedly found to be associated with decreased liver fibrosis.

Vaccination with Vaxfectin® adjuvanted SIV DNA induces long-lasting humoral immune responses able to reduce SIVmac251 Viremia

We evaluated the immunogenicity and efficacy of Vaxfectin® adjuvanted SIV DNA vaccines in mice and macaques. Vaccination of mice with Vaxfectin® adjuvanted SIV gag DNA induced higher humoral immune responses than administration of unadjuvanted DNA, whereas similar levels of cellular immunity were elicited. Vaxfectin® adjuvanted SIVmac251 gag and env DNA immunization of rhesus macaques was used to examine magnitude, durability, and efficacy of humoral immunity. Vaccinated macaques elicited potent neutralizing antibodies able to cross-neutralize the heterologous SIVsmE660 Env. We found remarkable durability of Gag and Env humoral responses, sustained during ~2 y of follow-up. The Env-specific antibody responses induced by Vaxfectin® adjuvanted env DNA vaccination disseminated into mucosal tissues, as demonstrated by their presence in saliva, including responses to the V1-V2 region, and rectal fluids. The efficacy of the immune responses was evaluated upon intrarectal challenge with low repeated dose SIVmac251. Although 2 of the 3 vaccinees became infected, these animals showed significantly lower peak virus loads and lower chronic viremia than non-immunized infected controls. Thus, Vaxfectin® adjuvanted DNA is a promising vaccine approach for inducing potent immune responses able to control the highly pathogenic SIVmac251.

The γδ T-cell receptor repertoire is reconstituted in HIV patients after prolonged antiretroviral therapy

Determine whether reconstitution of Vγ2Vδ2 T cells in patients with HIV is due to new cell synthesis with recovery of the T-cell receptor repertoire or proliferative expansion of residual cells from the time of treatment initiation. Perform a cross-sectional analysis of the T-cell receptor complexity of Vγ2 chain in patients treated for HIV, natural virus suppressors who control viremia to undetectable levels, patients with chronic low-level viremia in the absence of therapy, and uninfected controls. Apply quantitative methods for repertoire analysis to assess the degree of Vδ2 repertoire loss or reconstitution. T-cell receptor Vγ2 chain DNA clones (up to 300 per patient sample) were sequenced and aligned to enumerate the antigen-reactive subset with Vγ2-Jγ1.2 rearrangements. Predominant shared (public) sequences in each patient were compared to a reference library of public sequences from uninfected controls to assess the extent of similarity. Repertoire comparisons were quantified through bioinformatics testing. Patients with prolonged virus suppression due to antiretroviral therapy reconstituted the Vγ2 T-cell repertoire to near-normal levels. Natural virus suppressors were similar to the treatment group. Severe defects in the Vγ2 T-cell receptor repertoire were observed in patients with chronic viremia despite the absence of overt disease. Prolonged HIV suppression with antiretroviral therapy leads to reconstitution of the Vγ2Vδ2 T-cell subset deleted in HIV disease. Direct evidence for repair of the T-cell receptor repertoire supports a view that treatment-associated immune reconstitution is due to new cell synthesis and not to expansion of residual cell populations.

Efficient Mucosal Transmissibility but Limited Pathogenicity of R5 SHIVSF162P3N in Chinese-Origin Rhesus Macaques

Infection of rhesus macaques (RMs) of Indian origin with simian immunodeficiency virus or simian-HIV (SHIV) provided powerful tools to study HIV-1 transmission and disease and for testing the efficacy of novel drugs, vaccines, and prevention strategies. In developing alternative nonhuman primate AIDS models for the CCR5 (R5)-tropic SHIVSF162P3N, we characterized virus transmission and infection in Chinese-origin RMs. Virologic, immunologic, and pathogenic evaluations of R5 SHIVSF162P3N infection in Chinese RMs challenged intrarectally (ir) or intravaginally were performed and compared with those previously observed in Indian-origin rhesus exposed to the same inoculum dose and via similar route. R5 SHIVSF162P3N transmits efficiently across mucosal surfaces in Chinese RMs. The magnitude and kinetics of early virus dissemination after ir inoculation in the Chinese macaques were similar to those observed in Indian rhesus, but a trend toward increased SHIVSF162P3N vaginal infectivity and rapid virus spread was seen in the Chinese macaques compared with the Indian-origin animals. Once infected, however, set point viremia in the ir- and intravaginal-infected Chinese rhesus was significantly lower and the animals survived longer compared with infected Indian rhesus. The R5 SHIVSF162P3N/Chinese RM infection model is suitable for studies of mucosal HIV-1 transmission and protection, but the high frequency of spontaneous control of chronic viremia and reduced virulence with SHIVSF162P3N in this macaque subspecies may limit its utility in studying HIV-1 pathogenesis and in evaluating vaccines and antiretrovirals that rely on reduction in chronic viral load or AIDS development as an experimental end point.

Impact of antibody quality and anamnestic response on viremia control post-challenge in a combined Tat/Env vaccine regimen in rhesus macaques

Previously, priming rhesus macaques with Adenovirus type 5 host range mutant-recombinants encoding Tat and Env and boosting with Tat and Env protein in MPL-SE controlled chronic viremia by 4 logs following homologous intravenous SHIV89.6P challenge. Here we evaluated Tat, Env, and Tat/Env regimens for immunogenicity and protective efficacy using clade C Env, alum adjuvant, and a heterologous intrarectal SHIV1157ipd3N4 challenge. Despite induction of strong cellular and humoral immunity, Tat/Env group T and B-cell memory responses were not significantly enhanced over Tat- or Env-only groups. Lack of viremia control post-challenge was attributed to lower avidity Env antibodies and no anamnestic ADCC response or SHIV1157ipd3N4 neutralizing antibody development post-challenge. Poor biologic activity of the Tat immunogen may have impaired Tat immunity. In the absence of sterilizing immunity, strong anamnestic responses to heterologous virus can help control viremia. Both antibody breadth and optimal adjuvanticity are needed to elicit high-quality antibody for protective efficacy.

Characterization of plasmablasts in the blood of HIV-infected viremic individuals: evidence for nonspecific immune activation.

Terminal differentiation of B cells and hypergammaglobulinemia are hallmarks of B-cell hyperactivity in HIV disease. Plasmablasts are terminally differentiating B cells that circulate transiently in the blood following infection or vaccination; however, in HIV infection, they arise early and are maintained at abnormally high levels in viremic individuals. Here we show that only a small fraction of plasmablasts in the blood of viremic individuals is HIV specific. Assessment of plasmablast immunoglobulin isotype distribution revealed increased IgG(+) plasmablasts in early and most prominently during chronic HIV viremia, contrasting with a predominantly IgA(+) plasmablast profile in HIV-negative individuals or in aviremic HIV-infected individuals on treatment. Of note, IgG is the predominant immunoglobulin isotype of plasmablasts that arise transiently in the blood following parenteral immunization. Serum immunoglobulin levels were also elevated in HIV-infected viremic individuals, especially IgG, and correlated with levels of IgG(+) plasmablasts. Several soluble factors associated with immune activation were also increased in the sera of HIV-infected individuals, especially in viremic individuals, and correlated with serum immunoglobulin levels, particularly IgG. Thus, our data suggest that while plasmablasts in the blood may contribute to the HIV-specific immune response, the majority of these cells are not HIV specific and arise early, likely from indirect immune-activating effects of HIV replication, and reflect over time the effects of chronic antigenic stimulation. Such B-cell dysregulation may help explain why the antibody response is inadequate in HIV-infected individuals, even during early infection.

DNA and virus particle vaccination protects against acquisition and confers control of viremia upon heterologous simian immunodeficiency virus challenge.

We have previously shown that macaques vaccinated with DNA vectors expressing SIVmac239 antigens developed potent immune responses able to reduce viremia upon high-dose SIVmac251 challenge. To further improve vaccine-induced immunity and protection, we combined the SIVmac239 DNA vaccine with protein immunization using inactivated SIVmac239 viral particles as protein source. Twenty-six weeks after the last vaccination, the animals were challenged intrarectally at weekly intervals with a titrated dose of the heterologous SIVsmE660. Two of DNA-protein coimmunized macaques did not become infected after 14 challenges, but all controls were infected by 11 challenges. Vaccinated macaques showed modest protection from SIVsmE660 acquisition compared with naïve controls (P = 0.050; stratified for TRIM5α genotype). Vaccinees had significantly lower peak (1.6 log, P = 0.0048) and chronic phase viremia (P = 0.044), with 73% of the vaccinees suppressing viral replication to levels below assay detection during the 40-wk follow-up. Vaccine-induced immune responses associated significantly with virus control: binding antibody titers and the presence of rectal IgG to SIVsmE660 Env correlated with delayed SIVsmE660 acquisition; SIV-specific cytotoxic T cells, prechallenge CD4(+) effector memory, and postchallenge CD8(+) transitional memory cells correlated with control of viremia. Thus, SIVmac239 DNA and protein-based vaccine protocols were able to achieve high, persistent, broad, and effective cellular and humoral immune responses able to delay heterologous SIVsmE660 infection and to provide long-term control of viremia. These studies support a role of DNA and protein-based vaccines for development of an efficacious HIV/AIDS vaccine.

Rituximab Provides Steroid-Sparing Therapy in New-Onset Chronic Graft-Versus-Host Disease

s / Biol Blood Marrow Transplant 19 (2013) S129eS149 S140 3.15 vs.9.34%; p1⁄40.045). As reported in chronic HIV viremia, expanded CD21B cells in cGVHD patients also expressed higher inhibitory receptors namely FCRL4, CD22 and CD85J, chemokine receptors CD11c, CXCR3 and lower CD62L and CCR7 compared with naive (CD27-CD21+CD19+) and classical memory CD27+CD21+CD19+) B cells. Moreover, B cells in patients with cGVHD had a lower proliferative potential following B-cell receptor crosslinking compared with B cells from patients with no GvHD or HC, a feature of exhaustion associated with high expression of inhibitory receptors. Finally, the frequencies of circulating CD21B cells correlated with cGvHD grade (r1⁄40.67; P 1⁄4 .002) but not with time from SCT (r1⁄40.28; P 1⁄4 .15). These data suggest that CD21B cells may play a role in the pathogenesis of cGVHD and could be used as as a marker of disease severity.

Decoupling activation and exhaustion of B cells in spontaneous controllers of HIV infection

To define the impact of chronic viremia and associated immune activation on B-cell exhaustion in HIV infection. Progressive HIV infection is marked by B-cell anergy and exhaustion coupled with dramatic hypergammaglobulinemia. Although both upregulation of CD95 and loss of CD21 have been used as markers of infection-associated B-cell dysfunction, little is known regarding the specific profiles of dysfunctional B cells and whether persistent viral replication and its associated immune activation play a central role in driving B-cell dysfunction. Multiparameter flow cytometry was used to define the profile of dysfunctional B cells. The changes in the expression of CD21 and CD95 were tracked on B-cell subpopulations in patients with differential control of viral replication. : Although the emergence of exhausted, CD21 tissue-like memory B cells followed similar patterns in both progressors and controllers, the frequency of CD21 activated memory B cells was lower in spontaneous controllers. Our results suggest that the loss of CD21 and the upregulation of CD95 occur as separate events during the development of B-cell dysfunction. The loss of CD21 is a marker of B-cell exhaustion induced in the absence of appreciable viral replication, whereas the upregulation of CD95 is tightly linked to persistent viral replication and its associated immune activation. Thus, these dysfunctional profiles potentially represent two functionally distinct states within the B-cell compartment.