Correction: A quality improvement initiative for COPD patients: A cost analysis.

[This corrects the article DOI: 10.1371/journal.pone.0235040.].

A quality improvement initiative for COPD patients: A cost analysis.

The objective of this analysis was to evaluate and report on the economic impact of implementing an integrated, quality, and operational improvement program on chronic obstructive pulmonary disease (COPD) care from acute through post-acute care settings. This initiative was established in a cohort of 12 hospitals in Alabama and sought to address COPD readmission through improved workflows pertaining to early diagnosis, efficient care transitions, and patient visibility across the entire care episode. Implementation of the initiative was influenced by lean principles, particularly cross-functional agreement of workflows to improve COPD care delivery and outcomes. A budget impact model was developed to calculate cost savings directly from objective data collected during this initiative. The model estimated payer annual savings over 5 years. Patients were classified for analysis based on whether or not they received noninvasive ventilation. Scenario analyses calculated savings for payers covering different COPD cohort sizes. The base case revealed annual per patient savings of $11,263 for patients treated through the quality improvement program versus traditional care. The model projected cumulative savings of $52 million over a 5-year period. Clinical incorporation of non-invasive ventilation (NIV) resulted in $20,535 annual savings per patient and projected $91 million over 5 years. We conclude that an integrated management program for COPD patients across the care continuum is associated with substantial cost savings and significantly reduced hospital readmissions.

Impact of Fluid Choice in Systemic Inflammatory Response Syndrome Patients on Hospital Cost Savings.

BACKGROUND: There is growing evidence of the benefits of intravenous fluid therapy with balanced crystalloids over 0.9% 'normal' saline. This analysis evaluated the economic impact of increasing usage of a calcium-free balanced crystalloid solution (BAL) in patients with systemic inflammatory response syndrome (SIRS) on an annual hospital budget. METHODS: An Excel®-based economic model was developed to estimate costs associated with increased BAL usage (i.e., use in a greater proportion of patients), from the US hospital perspective, over a 5-year time horizon. Clinical inputs were based on the results of a retrospective Electronic Health Record (EHR) database analysis identifying significantly fewer complications among SIRS patients receiving predominantly BAL versus saline. Complication-associated costs, adjusted to 2015, were obtained from published reports. Scenario analyses examined cost impacts for hospitals of various sizes, with different BAL adoption levels and rates. RESULTS: Base-case scenario analysis (300-bed hospital, 80% occupancy, current and year 5 BAL usage in 5 and 75% of SIRS patients, respectively, exponential year-over-year adoption) showed year 1 hospital savings of US$29,232 and cumulative 5-year savings of US$1.16M. Cumulative 5-year pharmacy savings were US$172,641. Scenario analyses demonstrated increasing cumulative 5-year savings with increasing hospital size, year 5 BAL usage in greater proportions of patients, and rapid/early BAL adoption. CONCLUSIONS: Increased BAL usage represents an opportunity for hospitals and pharmacy departments to reduce complication-related costs associated with managing SIRS patients. The model suggests that savings could be expected across a range of scenarios, likely benefiting hospitals of various sizes and with different adoption capabilities.

Cost Savings from Reduced Hospitalizations with Use of Home Noninvasive Ventilation for COPD.

BACKGROUND: Although evidence suggests significant clinical benefits of home noninvasive ventilation (NIV) for management of severe chronic obstructive pulmonary disease (COPD), economic analyses supporting the use of this technology are lacking. OBJECTIVES: To evaluate the economic impact of adopting home NIV, as part of a multifaceted intervention program, for severe COPD. METHODS: An economic model was developed to calculate savings associated with the use of Advanced NIV (averaged volume assured pressure support with autoexpiratory positive airway pressure; Trilogy100, Philips Respironics, Inc., Murrysville, PA) versus either no NIV or a respiratory assist device with bilevel pressure capacity in patients with severe COPD from two distinct perspectives: the hospital and the payer. The model examined hospital savings over 90 days and payer savings over 3 years. The number of patients with severe COPD eligible for home Advanced NIV was user-defined. Clinical and cost data were obtained from a quality improvement program and published reports. Scenario analyses calculated savings for hospitals and payers covering different COPD patient cohort sizes. RESULTS: The hospital base case (250 patients) revealed cumulative savings of $402,981 and $449,101 over 30 and 90 days, respectively, for Advanced NIV versus both comparators. For the payer base case (100,000 patients), 3-year cumulative savings with Advanced NIV were $326 million versus no NIV and $1.04 billion versus respiratory assist device. CONCLUSIONS: This model concluded that adoption of home Advanced NIV with averaged volume assured pressure support with autoexpiratory positive airway pressure, as part of a multifaceted intervention program, presents an opportunity for hospitals to reduce COPD readmission-related costs and for payers to reduce costs associated with managing patients with severe COPD on the basis of reduced admissions.

The role of noninvasive ventilation in the management and mitigation of exacerbations and hospital admissions/readmissions for the patient with moderate to severe COPD (multimedia activity).

As seen in this CME online activity (available at http://journal.cme.chestnet.org/home-niv-copd), COPD is a common and debilitating disease and is currently the third leading cause of death in the United States. The role of noninvasive ventilation (NIV) in the management of severe, hypercapnic COPD has been controversial. However, it was concluded that current data would support the following recommendations. Patients with COPD with a waking Paco2 > 50 to 52 mm Hg, an overnight Paco2 > 55 mm Hg, or both who are symptomatic and compliant with other therapies should be eligible for NIV. In addition, multiple previous hospital admissions for COPD exacerbation, requiring noninvasive/invasive mechanical ventilation, strongly suggest a need for chronic NIV. Patients with COPD with a BMI > 30 kg/m2 respond particularly well to this therapy. When the decision is made to start NIV, this treatment is probably best initiated during a short hospitalization, although this can be accomplished in the clinic, home, or sleep laboratory if well-trained clinicians are available. Newer modes of NIV such as volume-assured pressure support, particularly with autotitrating expiratory positive airway pressure (EPAP), may create the opportunity for home NIV initiation easier for less experienced physicians. Regardless of the mode selected, inspiratory pressures must be in the 20 to 25 cm H2O range to meaningfully increase tidal volume, reduce work of breathing, and, importantly, reduce waking arterial Paco2. EPAP is currently set at 4 to 5 cm H2O, although future technologies may allow this to be individualized to maximally reduce auto-positive end expiratory pressure. The NIV device should have a backup rate although it is controversial as to whether this should be set at a high (18-20 breaths/min) vs a low (8-10 breaths/min) rate. The proper use of NIV in appropriately chosen patients with COPD can improve quality of life and increase survival. Ongoing studies are assessing if the frequency of future hospitalizations can be reduced with NIV. Thus, NIV should be strongly considered in any patients with COPD meeting the criteria described here.

Association between intravenous chloride load during resuscitation and in-hospital mortality among patients with SIRS.

PURPOSE: Recent data suggest that both elevated serum chloride levels and volume overload may be harmful during fluid resuscitation. The purpose of this study was to examine the relationship between the intravenous chloride load and in-hospital mortality among patients with systemic inflammatory response syndrome (SIRS), with and without adjustment for the crystalloid volume administered. METHODS: We conducted a retrospective analysis of 109,836 patients ≥ 18 years old that met criteria for SIRS and received fluid resuscitation with crystalloids. We examined the association between changes in serum chloride concentration, the administered chloride load and fluid volume, and the 'volume-adjusted chloride load' and in-hospital mortality. RESULTS: In general, increases in the serum chloride concentration were associated with increased mortality. Mortality was lowest (3.7%) among patients with minimal increases in serum chloride concentration (0-10 mmol/L) and when the total administered chloride load was low (3.5% among patients receiving 100-200 mmol; P < 0.05 versus patients receiving ≥ 500 mmol). After controlling for crystalloid fluid volume, mortality was lowest (2.6%) when the volume-adjusted chloride load was 105-115 mmol/L. With adjustment for severity of illness, the odds of mortality increased (1.094, 95% CI 1.062, 1.127) with increasing volume-adjusted chloride load (≥ 105 mmol/L). CONCLUSIONS: Among patients with SIRS, a fluid resuscitation strategy employing lower chloride loads was associated with lower in-hospital mortality. This association was independent of the total fluid volume administered and remained significant after adjustment for severity of illness, supporting the hypothesis that crystalloids with lower chloride content may be preferable for managing patients with SIRS.

Outcomes of secondary hyperparathyroidism in chronic kidney disease and the direct costs of treatment.

BACKGROUND: There has been an emphasis over the last several years to identify and treat chronic kidney disease (CKD) and its complications as they evolve rather than waiting until the patient reaches end-stage renal disease (ESRD), also known as CKD stage 5. The number of patients who will be identified and prescribed therapies for complications such as secondary hyperparathyroidism (SHPT) is greater than initially proposed. OBJECTIVE: To review the pathways, complications, management, and estimated treatment costs of CKD-related SHPT. METHODS: An electronic literature search of MEDLINE (January 1980 through January 2007) was conducted for English-language publications using the base search term secondary hyperparathyroidism. To refine subsequent searches, the authors added Boolean operators to the following secondary and tertiary search terms: parathyroid hormone, chronic kidney disease, renal osteodystrophy, adynamic bone disease, vascular calcification, cardiovascular disease, vitamin D, vitamin D analogs, hypercalcemia, hyperphosphatemia, calcimimetics, costs, prevalence, and economics. RESULTS: The initial MEDLINE search produced 278 relevant articles. After refining the search terms, the authors triaged the results for English-language publications relevant to the discussion of SHPT and its complications in CKD, eliminating 149 publications. The remaining 129 publications were accepted for review. These articles represent a growing body of primarily observational evidence that demonstrates that elevated intact parathyroid hormone (PTH) levels cause deleterious physiological results across a variety of organ systems, including the cardiovascular and skeletal systems. Specific complications associated with SHPT are left ventricular hypertrophy (LVH), renal osteodystrophy (ROD), and extraskeletal calcification. Medical management of the PTH/vitamin D/calcium and phosphorus imbalances in SHPT focus on regulating PTH levels via vitamin D therapy. The class of calcimimetics is a newer treatment modality that has favorable effects on biochemical laboratory values, such as serum calcium and phosphorus levels, but current data do not show differences on hard endpoint patient-oriented outcomes compared with standard generic agents. The direct drug costs in April 2007 U.S. dollars of treating CKD-associated elevations in PTH in predialysis patients range from $8.40 per patient per week ($437 per year) for oral generic calcitriol to $88.90 per patient per week ($4,623 per year) for oral paricalcitol (expressed as 85% of average wholesale price [AWP] for brand drugs or 70% of AWP for generic drugs). The direct drug costs of treating SHPT in hemodialysis patients range from $80.20 per patient per week ($4,170 per year) for generic calcitriol (IV) to $278.46 per patient per week ($14,480 per year) for oral cinacalcet. CONCLUSIONS: SHPT causes skeletal and cardiovascular complications in CKD patients. Calcitriol therapy is effective in managing PTH levels, but efforts to reduce the associated hypercalcemia and hyperphosphatemia have led to the development of newer, yet more expensive, vitamin D analogs. With the lack of evidence to support comparative superior outcomes in end-organ disease among SHPT therapy alternatives, future research is still needed to clearly identify which newer agents are most competitive with the historical gold standard of calcitriol therapy.

Contribution of T-cell receptor repertoire breadth to the dominance of epitope-specific CD8+ T-lymphocyte responses.

Dominant epitope-specific CD8(+) T-lymphocyte responses play a central role in controlling viral spread. We explored the basis for the development of this focused immune response in simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys through the use of two dominant (p11C and p199RY) and two subdominant (p68A and p56A) epitopes. Using real-time PCR to quantitate T-cell receptor (TCR) variable region beta (Vbeta) family usage, we show that CD8(+) T-lymphocyte populations specific for dominant epitopes are characterized by a diverse Vbeta repertoire, whereas those specific for subdominant epitopes employ a dramatically more focused Vbeta repertoire. We also demonstrate that dominant epitope-specific CD8(+) T lymphocytes employ TCRs with multiple CDR3 lengths, whereas subdominant epitope-specific cells employ TCRs with a more restricted CDR3 length. Thus, the relative dominance of an epitope-specific CD8(+) T-lymphocyte response reflects the clonal diversity of that response. These findings suggest that the limited clonal repertoire of subdominant epitope-specific CD8(+) T-lymphocyte populations may limit the ability of these epitope-specific T-lymphocyte populations to expand and therefore limit the ability of these cell populations to contribute to the control of viral replication.

Compensatory substitutions restore normal core assembly in simian immunodeficiency virus isolates with Gag epitope cytotoxic T-lymphocyte escape mutations.

The evolution of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) as they replicate in infected individuals reflects a balance between the pressure on the virus to mutate away from recognition by dominant epitope-specific cytotoxic T lymphocytes (CTL) and the structural constraints on the virus' ability to mutate. To gain a further understanding of the strategies employed by these viruses to maintain replication competency in the face of the intense selection pressure exerted by CTL, we have examined the replication fitness and morphological ramifications of a dominant epitope mutation and associated flanking amino acid substitutions on the capsid protein (CA) of SIV/simian-human immunodeficiency virus (SHIV). We show that a residue 2 mutation in the immunodominant p11C, C-M epitope (T47I) of SIV/SHIV not only decreased CA protein expression and viral replication, but it also blocked CA assembly in vitro and virion core condensation in vivo. However, these defects were restored by the introduction of upstream I26V and/or downstream I71V substitutions in CA. These findings demonstrate how flanking compensatory amino acid substitutions can facilitate viral escape from a dominant epitope-specific CTL response through the effects of these associated mutations on the structural integrity of SIV/SHIV.

Bax does not have to adopt its final form to drive T cell death.

The introduction of antigen into animals causes antigen-specific T cells to divide and then die. Activated T cell death requires either of the death effector molecules, Bak or Bax. When T cells die, Bak and Bax change their conformations, a phenomenon that is thought to be required for Bak or Bax to drive cell death. Here we show that Bak changes conformation before activated T cells die, as detected by an antibody specific for a peptide near the NH2 terminus of Bak, but Bax does not change its shape markedly until after the cells are dead, as detected by an antibody specific for a peptide near the NH2 terminus of Bax. This latter finding is also true in activated T cells that lack Bak and are therefore dependent on Bax to die. This result suggests that Bax does not have to adopt its final, completely unfolded form until after the cells are dead.

Use of molecular beacons for rapid, real-time, quantitative monitoring of cytotoxic T-lymphocyte epitope mutations in simian immunodeficiency virus.

Immune pressure on lentiviruses exerted by cytotoxic T lymphocytes (CTL) selects for virus CTL epitope mutations. Currently employed methods for monitoring emerging CTL epitope mutations rely on the labor-intensive and time-consuming techniques of virus population or clonal sequencing. Here we describe the development of a high-throughput quantitative reverse transcription-PCR assay that facilitates large-scale CTL epitope monitoring. This approach utilizes both sequence-specific molecular beacons and the sequence-independent double-stranded DNA binding dye Sybr Green. We show that this assay detects single-nucleotide mutations in an immunodominant CTL epitope in viral RNA isolated from both viral culture supernatants and plasma samples from simian immunodeficiency virus (SIV)-infected rhesus monkeys. Furthermore, mutant viruses can be detected even when they represent as few as 500 mutant copies in a sample containing 10,000 total copies. This real-time PCR technique for evaluating CTL epitope mutations may prove to be a useful tool for monitoring the genetic drift of human immunodeficiency virus and SIV in infected individuals.

Neutralizing antibodies to adenovirus serotype 5 vaccine vectors are directed primarily against the adenovirus hexon protein.

The utility of recombinant adenovirus serotype 5 (rAd5) vector-based vaccines for HIV-1 and other pathogens will likely be limited by the high prevalence of pre-existing Ad5-specific neutralizing Abs (NAbs) in human populations. However, the immunodominant targets of Ad5-specific NAbs in humans remain poorly characterized. In this study, we assess the titers and primary determinants of Ad5-specific NAbs in individuals from both the United States and the developing world. Importantly, median Ad5-specific NAb titers were >10-fold higher in sub-Saharan Africa compared with the United States. Moreover, hexon-specific NAb titers were 4- to 10-fold higher than fiber-specific NAb titers in these cohorts by virus neutralization assays using capsid chimeric viruses. We next performed adoptive transfer studies in mice to evaluate the functional capacity of hexon- and fiber-specific NAbs to suppress the immunogenicity of a prototype rAd5-Env vaccine. Hexon-specific NAbs were remarkably efficient at suppressing Env-specific immune responses elicited by the rAd5 vaccine. In contrast, fiber-specific NAbs exerted only minimal suppressive effects on rAd5 vaccine immunogenicity. These data demonstrate that functionally significant Ad5-specific NAbs are directed primarily against the Ad5 hexon protein in both humans and mice. These studies suggest a potential strategy for engineering novel Ad5 vectors to evade dominant Ad5-specific NAbs.

Dynamic immune responses maintain cytotoxic T lymphocyte epitope mutations in transmitted simian immunodeficiency virus variants.

Viral escape from cytotoxic T lymphocytes (CTLs) can undermine immune control of human immunodeficiency virus 1. It is therefore important to assess the stability of viral mutations in CTL epitopes after transmission to naive hosts. Here we demonstrate the persistence of mutations in a dominant CTL epitope after transmission of simian immunodeficiency virus variants to major histocompatibility complex-matched rhesus monkeys. Transient reversions to wild-type sequences occurred and elicited CTLs specific for the wild-type epitope, resulting in immunological pressure that rapidly reselected the mutant viruses. These data suggest that mutations in dominant human immunodeficiency virus 1 CTL epitopes may accumulate in human populations with limited major histocompatibility complex heterogeneity by a mechanism involving dynamic CTL control of transiently reverted wild-type virus.

Fitness costs limit viral escape from cytotoxic T lymphocytes at a structurally constrained epitope.

The intense selection pressure exerted by virus-specific cytotoxic T lymphocytes (CTL) on replicating human immunodeficiency virus and simian immunodeficiency virus results in the accumulation of CTL epitope mutations. It has been assumed that fitness costs can limit the evolution of CTL epitope mutations. However, only a limited number of studies have carefully examined this possibility. To explore the fitness costs associated with viral escape from p11C, C-M-specific CTL, we constructed a panel of viruses encoding point mutations at each position of the entire p11C, C-M epitope. Amino acid substitutions at positions 3, 4, 5, 6, 7, and 9 of the epitope significantly impaired virus replication by altering virus production and Gag protein expression as well as by destabilizing mature cores. Amino acid substitutions at position 2 of the epitope were tolerated but required reversion or additional compensatory mutations to generate replication-competent viruses. Finally, while amino acid substitutions at positions 1 and 8 of the p11C, C-M epitope were functionally tolerated, these substitutions were recognized by p11C, C-M-specific CTL and therefore provided no selection advantage for the virus. Together, these data suggest that limited sequence variation is tolerated by the region of the capsid encoding the p11C, C-M epitope and therefore that only a very limited number of mutations can allow successful viral escape from the p11C, C-M-specific CTL response.

Engineered T-cell receptor tetramers bind MHC-peptide complexes with high affinity.

In this study we extend tetramerization technology to T-cell receptors (TCRs). We identified TCR alpha beta pairs in the absence of accessory molecules, ensuring isolation of high-affinity TCRs that maintain stable binding characteristics after tetramerization. Subtle changes in cognate peptide levels bound to the class I molecule were accurately reflected by parallel changes in the mean fluorescence intensity of cells that bound TCR tetramers, allowing us to accurately assess the binding affinity of a panel of peptides to major histocompatibility complex (MHC) class I. Using a TCR tetramer specific for the Mamu-A(*)01 allele, we identified animals expressing this restricting class I allele from a large cohort of outbred rhesus macaques. TCR tetramers should facilitate analysis of the MHC-peptide interface and, more generally, the design of immunotherapeutics and vaccines.

Structural constraints on viral escape from HIV- and SIV-specific cytotoxic T-lymphocytes.

Cytotoxic T lymphocytes (CTL) play a central role in controlling lentiviral infections in both humans and monkeys. While they contain the spread of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), CTL are not capable of fully eradicating virus following infection. Ongoing viral replication can therefore lead to the accumulation of viral mutations within CTL epitopes that can undermine cellular immune control of virus. Here we review the importance of CTL in controlling HIV/SIV infection and how immunologic pressure exerted by effector T cells selects for viral variants that escape CTL recognition. We review two examples of viral escape from CTL at highly conserved epitopes that illustrate the extraordinary capacity of lentiviruses to adapt to their immunologic environment despite structural constraints on the ability of the virus to accommodate mutations.

Subsets of memory cytotoxic T lymphocytes elicited by vaccination influence the efficiency of secondary expansion in vivo.

Vaccine-elicited cytotoxic T lymphocytes (CTL) should be long-lived memory cells that can rapidly expand in number following re-exposure to antigen. The present studies were initiated to analyze the ability of plasmid interleukin-12 (IL-12) to augment CTL responses in mice when delivered during the peak phase of an immune response elicited by a plasmid human immunodeficiency virus type 1 gp120 DNA vaccine. Delivery of plasmid IL-12 on day 10 postimmunization resulted in a robust expansion of gp120-specific CD8+ T cells, as measured by tetramer, gamma interferon ELISPOT, and functional-killing assays. Interestingly, this delayed administration of plasmid IL-12 had no significant effect on antigen-specific CD4(+)-T-cell and antibody responses. Phenotypic analyses suggested that administration of plasmid IL-12 near the time of the peak CTL response activated and expanded antigen-specific effector cells, preventing their loss through apoptosis. However, this IL-12-augmented population of gp120-specific CD8+ T cells did not efficiently expand following gp120 boost immunization, suggesting that these effector cells would be of little utility in expanding to contain a viral infection. Analyses of the phenotypic profile and anatomic distribution of the plasmid IL-12-augmented CTL population indicated that these lymphocytes were primarily effector memory rather than central memory T cells. These observations suggest that CTL-based vaccines should elicit central memory rather than effector memory T cells and illustrate the importance of monitoring the phenotype and functionality of vaccine-induced, antigen-specific CTL.

Simian-human immunodeficiency virus escape from cytotoxic T-lymphocyte recognition at a structurally constrained epitope.

Virus-specific cytotoxic T lymphocytes (CTL) exert intense selection pressure on replicating simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) in infected individuals. The immunodominant Mamu-A(*)01-restricted Gag p11C, C-M epitope is highly conserved among all sequenced isolates of SIV and therefore likely is structurally constrained. The strategies used by virus isolates to mutate away from an immunodominant epitope-specific CTL response are not well defined. Here we demonstrate that the emergence of a position 2 p11C, C-M epitope substitution (T47I) in a simian-human immunodeficiency virus (SHIV) strain 89.6P-infected Mamu-A(*)01(+) monkey is temporally correlated with the emergence of a flanking isoleucine-to-valine substitution at position 71 (I71V) of the capsid protein. An analysis of the SIV and HIV-2 sequences from the Los Alamos HIV Sequence Database revealed a significant association between any position 2 p11C, C-M epitope mutation and the I71V mutation. The T47I mutation alone is associated with significant decreases in viral protein expression, infectivity, and replication, and these deficiencies are restored to wild-type levels with the introduction of the flanking I71V mutation. Together, these data suggest that a compensatory mutation is selected for in SHIV strain 89.6P to facilitate the escape of that virus from CTL recognition of the dominant p11C, C-M epitope.

Low frequency of cytotoxic T lymphocytes against the novel HLA-A*0201-restricted JC virus epitope VP1(p36) in patients with proven or possible progressive multifocal leukoencephalopathy.

JC virus (JCV)-specific cytotoxic T lymphocytes (CTL) in peripheral blood are associated with a favorable outcome in patients with progressive multifocal leukoencephalopathy (PML). However, the frequency of these cells in the peripheral blood mononuclear cells (PBMC) of PML patients is unknown. To develop a highly sensitive assay for detecting the cellular immune response against this virus, we performed a CTL epitope mapping study of JCV VP1 major capsid protein by using overlapping peptides. A novel HLA-A*0201-restricted epitope, the VP1(p36) peptide SITEVECFL, was characterized. The cellular immune response against JCV was assessed in 32 study subjects. By combining the results of the (51)Cr release assay on pooled peptides and staining with the HLA-A*0201/JCV VP1(p36) tetramer, VP1-specific CTL were detected in 10 of 11 PML survivors (91%) versus only 1 of 11 PML progressors (9%, P = 0.0003). VP1-specific CTL were also detected in two of two patients recently diagnosed with PML and in four of four human immunodeficiency virus-positive patients with possible PML. The frequency of CTL specific for the novel VP1(p36) and the previously described VP1(p100) epitopes was determined. In two patients, the frequency of CTL specific for the VP1(p36) or VP1(p100) epitopes, as determined by fresh blood tetramer staining (FBTS), ranged from 1/6,000 to 1/24,000 PBMC. A CTL sorting technique combining tetramer staining and selection with immunomagnetic beads allowed the detection of epitope-specific CTL in two cases that were determined to be negative by FBTS. The phenotype of these CTL in vivo was consistent with activated memory cells. These data suggest that, although present in low numbers, JCV-specific CTL may be of central importance in the containment of JCV spread in immunosuppressed individuals.

Codon usage optimization of HIV type 1 subtype C gag, pol, env, and nef genes: in vitro expression and immune responses in DNA-vaccinated mice.

Codon usage optimization of human immunodeficiency virus type 1 (HIV-1) structural genes has been shown to increase protein expression in vitro as well as in the context of DNA vaccines in vivo; however, all optimized genes reported thus far are derived from HIV-1 (group M) subtype B viruses. Here, we report the generation and biological characterization of codon usage-optimized gag, pol, env (gp160, gp140, gp120), and nef genes from a primary (nonrecombinant) HIV-1 subtype C isolate. After transfection into 293T cells, optimized subtype C genes expressed one to two orders of magnitude more protein (as determined by immunoblot densitometry) than the corresponding wild-type constructs. This effect was most pronounced for gp160, gp140, Gag, and Pol (>250-fold), but was also observed for gp120 and Nef (45- and 20-fold, respectively). Optimized gp160- and gp140-derived glycoproteins were processed, incorporated into virus particles, and mediated virus entry when expressed in trans to complement an env-minus HIV-1 provirus. Mice immunized with optimized gp140 DNA developed antibody as well as CD4+ and CD8+ T cell immune responses that were orders of magnitude greater than those of mice immunized with wild-type gp140 DNA. These data confirm and extend previous studies of codon usage optimization of HIV-1 genes to the most prevalent group M subtype. Our panel of matched optimized and wild-type subtype C genes should prove valuable for studies of protein expression and function, the generation of subtype-specific immunological reagents, and the production of DNA-based sub-unit vaccines directed against a broader spectrum of viruses.