Jon H. Condra

Treatment with Indinavir, Zidovudine, and Lamivudine in Adults with Human Immunodeficiency Virus Infection and Prior Antiretroviral Therapy

BACKGROUND The new protease inhibitors are potent inhibitors of the human immunodeficiency virus (HIV), and in combination with other antiretroviral drugs they may be able to cause profound and sustained suppression of HIV replication. METHODS In this double-blind study, 97 HIV-infected patients who had received zidovudine treatment for at least 6 months and had 50 to 400 CD4 cells per cubic millimeter and at least 20,000 copies of HIV RNA per milliliter were randomly assigned to one of three treatments for up to 52 weeks: 800 mg of indinavir every eight hours; 200 mg of zidovudine every eight hours combined with 150 mg of lamivudine twice daily; or all three drugs. The patients were followed to monitor the occurrence of adverse events and changes in viral load and CD4 cell counts. RESULTS The decrease in HIV RNA over the first 24 weeks was greater in the three-drug group than in the other groups (P<0.001 for each comparison). RNA levels decreased to less than 500 copies per milliliter at week 24 in 28 of 31 patients in the three-drug group (90 percent), 12 of 28 patients in the indinavir group (43 percent), and none of 30 patients in the zidovudine-lamivudine group. The increase in CD4 cell counts over the first 24 weeks was greater in the two groups receiving indinavir than in the zidovudine-lamivudine group (P< or =0.01 for each comparison). The changes in the viral load and the CD4 cell count persisted for up to 52 weeks. All the regimens were generally well tolerated. CONCLUSIONS In most HIV-infected patients with prior antiretroviral therapy, the combination of indinavir, zidovudine, and lamivudine reduces levels of HIV RNA to less than 500 copies per milliliter for as long as one year.

3-Year suppression of HIV viremia with indinavir, zidovudine, and lamivudine

The use of antiretroviral therapy that includes an HIV protease inhibitor has markedly decreased morbidity and mortality in HIV-infected persons (1-3). In addition, antiretroviral combination therapy that includes a protease inhibitor can suppress viral load levels for up to 2 years (4-8). However, the long-term durability and toxicity of these regimens are unknown. We present results after 3 years of follow-up in patients who received three-drug therapy with indinavir, zidovudine, and lamivudine in a previously reported study (4, 5). Methods Study Design The study was originally designed as a randomized, double-blind comparison of three antiretroviral regimens: indinavir (Crixivan, Merck & Co., Inc., West Point, Pennsylvania), 800 mg every 8 hours; zidovudine (Retrovir, Glaxo Wellcome, Research Triangle Park, North Carolina), 200 mg every 8 hours, with lamivudine (Epivir, Glaxo Wellcome), 150 mg every 12 hours; and all three drugs together at the same specified doses (4, 5). Patients were encouraged to drink at least 1.5 L of fluid per day. We report on the patients who were originally assigned to receive three-drug therapy. Institutional review boards at each site approved the study and amendments, and all patients gave informed consent. Study Participants Eligible patients were HIV-infected adults who had received at least 6 months of zidovudine therapy but had never taken lamivudine or an HIV protease inhibitor. Patients had serum viral load levels of at least 20 000 copies/mL (Amplicor HIV Monitor Test, Roche Diagnostic Systems, Branchburg, New Jersey) and CD4 counts between 50 to 400 cells/mm3 at screening. Assessments Patients had study visits at least every 4 weeks through week 52 and every 8 weeks through week 156. At baseline and at each visit, a history was taken, a physical examination was performed, and standardized laboratory tests were conducted without regard to food intake. Serum was processed, stored at 70 C, and subsequently assayed for HIV RNA by using the Amplicor and ultradirect assays (4, 5). T-lymphocyte subgroups were quantified by using flow cytometry. Genotypic analysis of serum HIV RNA was performed as described elsewhere (5). Individual investigators graded adverse events according to standardized guidelines. A drug-related adverse event was one that the investigator assessed as possibly, probably, or definitely related to the study therapy. Nephrolithiasis was defined as the passing of macroscopic stones or gravel or flank pain with or without associated hematuria. During follow-up, investigators assessed lipodystrophy at one time point from October to December 1998 (after approximately 2.5 to 3.5 years of treatment). Patients were considered to have lipodystrophy if they had one or more of the following features without evidence of hypercortisolemia: truncal or central obesity with or without thinning of the extremities; accumulation of body fat in the abdomen, the neck (buffalo hump), the retroperitoneum, the face, or the breasts; and accumulation or redistribution of body fat in some areas that was out of proportion to other body areas. Statistical Analysis Antiretroviral activity was assessed by calculating 1) the proportions (with 95% CIs) of patients whose HIV RNA levels were less than 500 copies/mL (Amplicor assay) and those whose HIV RNA levels were less than 50 copies/mL [ultradirect assay] and 2) the median changes (plus interquartile ranges) from baseline in log10HIV RNA levels (Amplicor assay) and CD4 cell counts over time. Analyses were performed on an intention-to-treat basis. Patients who withdrew early from the study were considered to have had virologic failure at subsequent time points, except for two patients who withdrew for reasons that were not related to therapy and had HIV RNA levels less than 500 copies/mL at the time of withdrawal, as described elsewhere (5). Because the analyses included patients with observed values and those with imputed values, the term contributing patients is used. Among patients with at least two measurements, those who never achieved an HIV RNA level less than 500 copies/mL were considered to have experienced virologic failure. Those who achieved an HIV RNA level less than 500 copies/mL were considered to have experienced virologic failure if they had two consecutive measurements of HIV RNA levels that were at least 500 copies/mL but did not have subsequent re-suppression while receiving the same three-drug regimen. Role of the Study Sponsor Employees of the industry sponsor participated in the study as co-investigators. After designing the study with the input of the other study investigators, these employees implemented the protocol and coordinated data collection and statistical analyses. All investigators interpreted the data, determined the content of the paper, and decided whether to submit the paper for publication. Results Study Participants Originally, 33 patients were randomly assigned to receive three-drug therapy with zidovudine, lamivudine, and indinavir. Median age was 40 years (range, 30 to 62 years). Thirty-one patients (94%) were men, and 2 (6%) were women; 26 (79%) were white, 2 (6%) were African American, 3 (9%) were Latin American, and 2 (6%) were members of other racial or ethnic groups. At study entry, patients had taken zidovudine for a median of 28 months (range, 6 to 92 months) and had a median baseline serum HIV RNA level of 41 900 copies/mL (range, 7550 to 219 040 copies/mL) and a median baseline CD4 count of 133 cells/mm3 (range, 35 to 433 cells/mm3). Of the 33 patients, 12 (36%) discontinued therapy within 3 years: 7 because of increased viral load levels; 2 because of need for contraindicated medications (rifampin and cytotoxic chemotherapy); and 1 each because of nausea, patient request, and investigator recommendation after resolution of urinary tract obstruction. Nine patients experienced virologic failure (6 in the first year, 0 in the second year, and 3 in the third year). Antiretroviral Activity The percentages of contributing patients whose HIV RNA level decreased from baseline to less than 500 copies/mL and less than 50 copies/mL, respectively, were 78% (95% CI, 60% to 90%) and 75% (CI, 56% to 88%) at 1 year, 78% (CI, 60% to 90%) and 66% (CI, 47% to 81%) at 2 years, and 68% (CI, 49% to 83%) (21 of 31 patients) and 65% (CI, 45% to 80%) (20 of 31 patients) at 3 years (Figure 1). Patients experienced a median change in HIV RNA level from baseline of 2.07 log10 copies/mL (interquartile range, 2.39 to 1.61 log10 copies/mL) at 1 year, 2.07 log10 copies/mL (interquartile range, 2.40 to 1.61 log10 copies/mL) at 2 years, and 1.99 log10 copies/mL (interquartile range, 2.32 to 1.31 log10 copies/mL) at 3 years (Figure 2). The median increase in CD4 counts from baseline was 155 cells/mm3 (interquartile range, 95 to 230 cells/mm3) at 1 year, 209 cells/mm3 (interquartile range, 117 to 339 cells/mm3) at 2 years, and 230 cells/mm3 (interquartile range, 150 to 316 cells/mm3) at 3 years (Figure 2). Figure 1. Proportion of patients with serum HIV RNA levels less than 500 copies/mL and less than 50 copies/mL during 3 years of treatment with indinavir, zidovudine, and lamivudine. Figure 2. Median changes in serum HIV RNA level and CD4 cell count from baseline during 3 years of treatment with indinavir, zidovudine, and lamivudine. Genotypic Analysis of Viral Resistance For the nine patients who experienced virologic failure by year 3, results of genotypic analyses performed at baseline and at the time of virologic failure were similar to the results of the 2-year analysis (5). Briefly, six patients had preexisting zidovudine resistance, as evidenced by the presence of the reverse transcriptase T215Y substitution combined with M41L (four patients), K70R (one patient), or D67N/K70R/K219Q (one patient). One patient developed zidovudine resistance (M41L), and all nine developed lamivudine resistance (M184V). Five patients acquired protease substitutions that were previously associated with indinavir resistance (9): M46L/V82A (four patients) and L90M (one patient). In two additional patients, evidence of new protease substitutions (L10V or L63P/S/A) appeared during treatment; however, the significance of substitutions at these two naturally occurring polymorphic sites is unclear. Adverse Events Four patients experienced a serious drug-related adverse event related to nephrolithiasis. Two of these patients experienced urinary tract obstruction, and one withdrew from the study 2 months after the adverse event resolved. Two of these patients also had other serious drug-related adverse events (pain and abdominal pain). In total, 12 of 33 patients (36%) had at least one episode of clinical nephrolithiasis during 3 years of treatment and 7 of 33 patients (21%) had more than one episode. Initial episodes of nephrolithiasis occurred from 24 weeks to 3 years of treatment. A total of 64.6 person-years of follow-up occurred before the first episode of nephrolithiasis or before censoring at 3 years. Therefore, the incidence of nephrolithiasis was 1.86 per 10 person-years of follow-up. Of the 21 patients in active follow-up, 4 (19%) fulfilled the clinical definition of lipodystrophy. When random, nonfasting specimens obtained throughout the study were used, serum triglyceride levels greater than 8.47 mmol/L (750 mg/dL) were documented at least once in 8 of 33 patients (24%) and levels greater than 13.55 mmol/L (1200 mg/dL) were documented in 2 of 33 patients (6%). Serum glucose levels greater than 13.88 mmol/L (250 mg/dL) occurred at least once in 1 of 33 patients (3%). Total serum cholesterol level was measured retrospectively in frozen samples obtained after 0.5, 1, 2, and 3 years of follow-up. Seven of 30 patients (23%) had total serum cholesterol levels of at least 6.21 mmol/L (240 mg/dL), and 1 of 30 patients (3%) had a level of at least 7.76 mmol/L (300 mg/dL) at least once. Discussion Evidence shows that it will be difficult

A PCSK9-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces LDL cholesterol in vivo

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%–50% for over 2 weeks, despite its relatively short terminal half-life (t1/2 = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.

Drug Resistance and Predicted Virologic Responses to Human Immunodeficiency Virus Type 1 Protease Inhibitor Therapy

The extent to which human immunodeficiency virus (HIV) type 1 drug resistance compromises therapeutic efficacy is intimately tied to drug potency and exposure. Most HIV-1 protease inhibitors maintain in vivo trough levels above their human serum protein binding-corrected IC(95) values for wild-type HIV-1. However, these troughs are well below corrected IC(95) values for protease inhibitor-resistant viruses from patients experiencing virologic failure of indinavir and/or nelfinavir. This suggests that none of the single protease inhibitors would be effective after many cases of protease inhibitor failure. However, saquinavir, amprenavir, and indinavir blood levels are increased substantially when each is coadministered with ritonavir, with 12-h troughs exceeding corrected wild-type IC(95) by 2-, 7-, and 28-79-fold, respectively. These indinavir and amprenavir troughs exceed IC(95) for most protease inhibitor-resistant viruses tested. This suggests that twice-daily indinavir-ritonavir and, to a lesser extent, amprenavir-ritonavir may be effective for many patients with viruses resistant to protease inhibitors.

Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus.

The major envelope glycoprotein (gp350) of Epstein-Barr virus has been expressed and secreted in the yeast Saccharomyces cerevisiae as a 400-kDa glycoprotein. This is the first example of the secretion of such a large, heavily glycosylated heterologous protein in yeast. Since gp350 proved highly toxic to S. cerevisiae, initial cellular growth required repression of the expression of gp350. Using temperature- or galactose-inducible promoters, cells could be grown and the expression of gp350 then induced. After induction, the glycoprotein accumulated both intracellularly as well as in the culture medium. Only the most heavily glycosylated form was secreted, suggesting a role for N-linked glycans in directing secretion. The extent of O-linked glycosylation of the yeast-derived protein was similar to that of the mature viral gp350. N-linked glycosylation varied slightly depending upon culture conditions and host strain used and was more extensive than that associated with the mature viral gp350. Although there is no evidence that more than a single mRNA for the glycoprotein was expressed from the recombinant plasmid, variously sized glycoproteins accumulated in yeast at early stages after induction, probably reflecting intermediates in glycosylation. The yeast-derived glycoproteins reacted with animal and human polyclonal antibodies to gp350 as well as with a neutralizing murine monoclonal antibody to gp350, suggesting that this glycoprotein retains several epitopes of the native glycoprotein.

Clinically effective HIV-1 protease inhibitors

For 10 years now, the HIV-1 protease has been recognized as an attractive target for antiviral therapy, and the discovery of potent inhibitors has been the focus of many research groups. There are currently four HIV protease inhibitors approved in the USA for use in treating AIDS, and several compounds are in various phases of clinical development. All of these compounds represent important advances and result from intensive research efforts. This review outlines the approaches taken by the research groups in discovering their clinical compounds, the problems encountered during this process and their subsequent solutions. Limited clinical data are also presented for each compound.

A proprotein convertase subtilisin-like/kexin type 9 (PCSK9) C-terminal domain antibody antigen-binding fragment inhibits PCSK9 internalization and restores low density lipoprotein uptake.

PCSK9 binds to the low density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with subnanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of β-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9ΔC, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.

Comprehensive mutant enzyme and viral variant assessment of human immunodeficiency virus type 1 reverse transcriptase resistance to nonnucleoside inhibitors.

The nonnucleoside reverse transcriptase (RT) inhibitors comprise a class of structurally diverse compounds that are functionally related and specific for the human immunodeficiency virus type 1 RT. Viral variants resistant to these compounds arise readily in cell culture and in treated, infected human. Therefore, the eventual clinical usefulness of the nonnucleoside inhibitors will rely on a thorough understanding of the genetic and biochemical bases for resistance. A study was performed to assess the effects of substitutions at each RT amino acid residue that influences the enzymes susceptibility to the various nonnucleoside compounds. Single substitutions were introduced into both purified enzyme and virus. The resulting patterns of resistance were markedly distinct for each of the tested inhibitors. For instance, a > 50-fold loss of enzyme susceptibility to BI-RG-587 was engendered by any of four individual substitutions, while the same level of relative resistance to the pyridinone derivatives was mediated only by substitution at residue 181. Similarly, substitution at residue 181. Similarly, substitution at residue 106 had a noted effect on virus resistance to BI-RG-587 but not to the pyridinones. The opposite effect was mediated by a substitution at residue 179. Such knowledge of nonucleoside inhibitor resistance profiles may help in understanding the basis for resistant virus selection during clinical studies of these compounds.

Cross-Reactivity of Anti–HIV-1 T Cell Immune Responses among the Major HIV-1 Clades in HIV-1–Positive Individuals from 4 Continents

BACKGROUND The genetic diversity of human immunodeficiency virus type 1 (HIV-1) raises the question of whether vaccines that include a component to elicit antiviral T cell immunity based on a single viral genetic clade could provide cellular immune protection against divergent HIV-1 clades. Therefore, we quantified the cross-clade reactivity, among unvaccinated individuals, of anti-HIV-1 T cell responses to the infecting HIV-1 clade relative to other major circulating clades. METHODS Cellular immune responses to HIV-1 clades A, B, and C were compared by standardized interferon- gamma enzyme-linked immunospot assays among 250 unvaccinated individuals, infected with diverse HIV-1 clades, from Brazil, Malawi, South Africa, Thailand, and the United States. Cross-clade reactivity was evaluated by use of the ratio of responses to heterologous versus homologous (infecting) clades of HIV-1. RESULTS Cellular immune responses were predominantly focused on viral Gag and Nef proteins. Cross-clade reactivity of cellular immune responses to HIV-1 clade A, B, and C proteins was substantial for Nef proteins (ratio, 0.97 [95% confidence interval, 0.89-1.05]) and lower for Gag proteins (ratio, 0.67 [95% confidence interval, 0.62-0.73]). The difference in cross-clade reactivity to Nef and Gag proteins was significant (P<.0001). CONCLUSIONS Cross-clade reactivity of cellular immune responses can be substantial but varies by viral protein.

Factors Influencing the Emergence of Resistance to Indinavir: Role of Virologic, Immunologic, and Pharmacologic Variables

A major problem with the use of human immunodeficiency virus type 1 (HIV-1) protease inhibitors as monotherapy has been an unacceptably high rate of emergence of resistance. To examine possible influences on the time to emergence of resistance, 24-week data were examined from five studies in which indinavir had been administered as monotherapy or as a component of combination therapy. Monotherapy data indicated a correlation between the level of HIV-1 RNA achieved and the risk of emergence of resistance: the lower the level, the lower the risk. When combination and monotherapy regimens were compared, the group receiving indinavir + lamivudine + zidovudine had a significantly lower risk of resistance, even after adjusting for the minimum HIV-1 RNA level achieved. The findings indicate that if at all possible, HIV-1-infected patients should receive combination chemotherapy to minimize the emergence of resistance to the protease inhibitor portion of the regimen. The goal of therapy should be to decrease the HIV-1 RNA load to a less-than-detectable level.