J. Michael Kilby

Antibody neutralization and escape by HIV-1

Neutralizing antibodies (Nab) are a principal component of an effective human immune response to many pathogens, yet their role in HIV-1 infection is unclear. To gain a better understanding of this role, we examined plasma from patients with acute HIV infection. Here we report the detection of autologous Nab as early as 52 days after detection of HIV-specific antibodies. The viral inhibitory activity of Nab resulted in complete replacement of neutralization-sensitive virus by successive populations of resistant virus. Escape virus contained mutations in the env gene that were unexpectedly sparse, did not map generally to known neutralization epitopes, and involved primarily changes in N-linked glycosylation. This pattern of escape, and the exceptional density of HIV-1 envelope glycosylation generally, led us to postulate an evolving ‘glycan shield’ mechanism of neutralization escape whereby selected changes in glycan packing prevent Nab binding but not receptor binding. Direct support for this model was obtained by mutational substitution showing that Nab-selected alterations in glycosylation conferred escape from both autologous antibody and epitope-specific monoclonal antibodies. The evolving glycan shield thus represents a new mechanism contributing to HIV-1 persistence in the face of an evolving antibody repertoire.

Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection

The precise identification of the HIV-1 envelope glycoprotein (Env) responsible for productive clinical infection could be instrumental in elucidating the molecular basis of HIV-1 transmission and in designing effective vaccines. Here, we developed a mathematical model of random viral evolution and, together with phylogenetic tree construction, used it to analyze 3,449 complete env sequences derived by single genome amplification from 102 subjects with acute HIV-1 (clade B) infection. Viral env genes evolving from individual transmitted or founder viruses generally exhibited a Poisson distribution of mutations and star-like phylogeny, which coalesced to an inferred consensus sequence at or near the estimated time of virus transmission. Overall, 78 of 102 subjects had evidence of productive clinical infection by a single virus, and 24 others had evidence of productive clinical infection by a minimum of two to five viruses. Phenotypic analysis of transmitted or early founder Envs revealed a consistent pattern of CCR5 dependence, masking of coreceptor binding regions, and equivalent or modestly enhanced resistance to the fusion inhibitor T1249 and broadly neutralizing antibodies compared with Envs from chronically infected subjects. Low multiplicity infection and limited viral evolution preceding peak viremia suggest a finite window of potential vulnerability of HIV-1 to vaccine-elicited immune responses, although phenotypic properties of transmitted Envs pose a formidable defense.

Emergence of Resistant Human Immunodeficiency Virus Type 1 in Patients Receiving Fusion Inhibitor (T-20) Monotherapy

ABSTRACT The synthetic peptide T-20 (enfuvirtide) represents the first of a new class of antiretroviral compounds to demonstrate in vivo potency by targeting a step in viral entry. T-20 inhibits a conformational change in the human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein (gp41) that is required for fusion between HIV-1 and target cell membranes. The initial phase I clinical trial of T-20 treatment for HIV-infected patients thus provided a unique opportunity to evaluate the emergence of resistant virus in vivo to this novel class of antiretroviral agents. All four patients who received an intermediate dose of T-20 (30 mg twice daily) had an initial decline in plasma viral load over the first 10 days but a rising trend by day 14, suggestive of selection for resistant virus. Plasma virus derived from patients enrolled in all dosage groups of the phase I T-20 trial was analyzed by population sequencing before and after treatment. While no mutations were found within a highly conserved 3-amino-acid sequence (GIV) known to be critical for fusion at baseline, after 14 days of therapy, virus from one patient in the 30-mg dose group (30-1) developed a mutation in this motif, specifically an aspartic acid (D) substitution for glycine (G) at position 36. Multiple env clones were derived from the plasma virus of all four patients in the 30-mg dosage group. Sequence analysis of 49 clones derived from the plasma of patient 30-1 on day 14 revealed that 25 clones contained the G36D mutation, while 8 contained the V38A mutation. Dual mutations involving G36D and other residues within the HR1 domain were also identified. In 5 of the 49 env clones, other mutations involving residues 32 (Q32R or Q32H) and 39 (Q39R) were found in combination with G36D. Cloned env sequences derived from the plasma virus of subject 30-3 also had single mutations in the GIV sequence (V38M and I37V) detectable following therapy with T-20. The plasma virus from subjects 30-2 and 30-4 did not contain changes within the GIV sequence. To analyze the biological resistance properties of these mutations, we developed a novel single-cycle HIV-1 entry assay using JC53BL cells which express β-galactosidase and luciferase under control of the HIV-1 long terminal repeat. Full-length env clones were derived from the plasma virus of patients 30-1 and 30-3 and used to generate pseudotyped virus stocks. The mean 50% inhibition concentrations (IC50s) for mutants G36D and V38A (patient 30-1) were 2.3 μg/ml and 11.2 μg/ml, respectively, statistically significant increases of 9.1- and 45-fold, respectively, compared with those of wild-type Env. The IC50 for the V38 M mutation (patient 30-3) was 7.6 μg/ml, an 8-fold increase compared with that of the wild type. The I37V mutation resulted in an IC50 3.2-fold greater than that of the wild type. Envs with double mutations (Q32R plus G36D and Q32H plus G36D) exhibited a level of resistance similar to that of G36D alone. These findings provide the first evidence for the rapid emergence of clinical resistance to a novel class of HIV-1 entry inhibitors and may be relevant to future treatment strategies involving these agents.

Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry.

T-20, a synthetic peptide corresponding to a region of the transmembrane subunit of the HIV-1 envelope protein, blocks cell fusion and viral entry at concentrations of less than 2 ng/ml in vitro. We administered intravenous T-20 (monotherapy) for 14 days to sixteen HIV-infected adults in four dose groups (3, 10, 30 and 100 mg twice daily). There were significant, dose-related declines in plasma HIV RNA in all subjects who received higher dose levels. All four subjects receiving 100 mg twice daily had a decline in plasma HIV RNA to less than 500 copies/ml, by bDNA assay. A sensitive RT–PCR assay (detection threshold 40 copies/ml) demonstrated that, although undetectable levels were not achieved in the 14-day dosing period, there was a 1.96 log10 median decline in plasma HIV RNA in these subjects. This study provides proof-of-concept that viral entry can be successfully blocked in vivo. Short-term administration of T-20 seems safe and provides potent inhibition of HIV replication comparable to anti-retroviral regimens approved at present.

Initial increase in blood CD4+ lymphocytes after HIV antiretroviral therapy reflects redistribution from lymphoid tissues

Previous studies proposed a dynamic, steady-state relationship between HIV-mediated cell killing and T-cell proliferation, whereby highly active antiretroviral therapy (HAART) blocks viral replication and tips the balance toward CD4(+) cell repopulation. In this report, we have analyzed blood and lymph node tissues obtained concurrently from HIV-infected patients before and after initiation of HAART. Activated T cells were significantly more frequent in lymph node tissue compared with blood at both time points. Ten weeks after HAART, the absolute number of lymphocytes per excised lymph node decreased, whereas the number of lymphocytes in the blood tended to increase. The relative proportions of lymphoid subsets were not significantly changed in tissue or blood by HAART. The expression levels of mRNA for several proinflammatory cytokines (IFN-gamma, IL-1beta, IL-6, and macrophage inflammatory protein-1alpha) were lower after HAART. After therapy, the expression of VCAM-1 and ICAM-1 -- adhesion molecules known to mediate lymphocyte sequestration in lymphoid tissue -- was also dramatically reduced. These data provide evidence suggesting that initial increases in blood CD4(+) cell counts on HAART are due to redistribution and that this redistribution is mediated by resolution of the immune activation that had sequestered T cells within lymphoid tissues.

The Safety, Plasma Pharmacokinetics, and Antiviral Activity of Subcutaneous Enfuvirtide (T-20), a Peptide Inhibitor of gp41-Mediated Virus Fusion, in HIV-Infected Adults

Enfuvirtide (T-20) is a novel antiretroviral agent that blocks HIV-1 cell fusion. A 28-day randomized dose-comparison study was conducted to determine the safety, pharmacokinetics, and antiviral activity of enfuvirtide in 78 HIV-infected adults, most with extensive treatment experience. Patients received enfuvirtide, added to a failing regimen, either by continuous subcutaneous infusion (CSI: 12.5, 25, 50 or 100 mg/day) or by subcutaneous (SC) injection (50 or 100 mg twice daily). Dose-related decreases in viral load were observed, with a maximum mean reduction from baseline of 1.6 log(10) copies/ml (p< 0.001) seen in the 100 mg bid SC group. Most responses diminished by 28 days. Plasma pharmacokinetics and antiviral responses were more consistent for SC injection than for CSI because of technical difficulties experienced with CSI. Injection site reactions were common but generally mild. These results indicate that enfuvirtide is a promising new therapeutic agent for HIV-infected patients, including those with prior antiretroviral treatment.

A Multicenter Observational Study of the Potential Benefits of Initiating Combination Antiretroviral Therapy during Acute HIV Infection

OBJECTIVE Uncontrolled studies have suggested a benefit, after treatment discontinuation, of initiating highly active antiretroviral therapy (HAART) during primary human immunodeficiency virus (HIV) infection. We assessed whether initiation of HAART within 2 weeks of (acute treatment) or between 2 weeks and 6 months after (early treatment) HIV seroconversion was associated with improvements in the viral load and the CD4+ T cell count after discontinuation of treatment in an observational cohort. METHODS Subjects from the multicenter Acute Infection and Early Disease Research Program cohort were enrolled in the present study within 6 months of HIV seroconversion and self-selected whether to initiate HAART. Subjects who received acute (n=13) or early (n=45) treatment received HAART for at least 12 weeks and then subsequently stopped treatment, whereas untreated subjects (n=337) declined treatment. HIV RNA levels and CD4+ T cell counts at 24, 48, and 72 weeks after treatment cessation in the 2 treatment groups were compared with those noted in the untreated group during the same periods of observation after enrollment. RESULTS The acute treatment group had lower mean HIV RNA levels at 24 weeks without therapy (-0.48 log(10) copies/mL [95% confidence interval {CI}, -0.82 to -0.13 log(10) copies/mL]) and higher mean CD4+ T cell counts (112 cells/ mu L [95% CI, 20-205 cells/ microL]), compared with the untreated group at 24 weeks. The differences in the laboratory values for the acute treatment group versus the untreated group at 72 weeks without therapy were as follows: for the HIV RNA level, -0.35 log(10) copies/mL (95% CI, -0.91 to 0.21 log(10) copies/mL) and, for the CD4 T+ cell count, 112 cells/ microL (95% CI, -15 to 213 cells/ microL). The early treatment group had lower HIV RNA levels at 24 weeks than did the untreated group, but differences were no longer apparent by week 48; CD4+ T cell counts were higher in the early treatment group at week 24 (116 cells/ microL [95% CI, 75-157 cells/ microL]) and week 72 (70 cells/ microL [95% CI, 2-138 cells/ microL]). CONCLUSIONS Initiation of HAART within 2 weeks of antibody seroconversion was associated with viral load and CD4+ T cell count benefits for 24 weeks after termination of HAART, with there being trends toward a longer-term benefit. Later initiation of HAART was associated with a persistent but decreasing CD4+ T cell count benefit and a loss of the viral load benefit by week 72 after discontinuation of treatment.

A phase II clinical study of the long-term safety and antiviral activity of enfuvirtide-based antiretroviral therapy.

Objectives: The primary objective was to determine the long-term safety of the subcutaneous self-administration of enfuvirtide. Secondary objectives included the determination of enfuvirtide pharmacokinetics and antiviral activity and the immunological response to the enfuvirtide-containing regimen. Methods: A multicenter 48-week uncontrolled open-label rollover study was conducted on 71 HIV-infected adults recruited from previous enfuvirtide clinical trials. Patients with extensive previous use of protease and reverse transcriptase inhibitors received a twice-daily dose of 50 mg enfuvirtide subcutaneously (45 mg deliverable) combined with two or more antiretroviral drugs selected for each individual, guided by resistance testing and previous treatment history. Results: The mean baseline plasma HIV-RNA level was 4.81 log10 copies/ml and the mean CD4 cell count was 134.8 cells/μl. The majority (86.9%) of treatment-emergent adverse events were grade 2 or less in severity. Injection site reactions were common, but no patients discontinued treatment. A mean HIV-RNA change of −1.33 log10 was achieved within 14 days of treatment initiation. At week 48, approximately one-third of all patients in the intent-to-treat population maintained significant suppression of plasma HIV RNA, with either less than 400 copies/ml or more than a 1.0 log10 decline from baseline. The mean gain in absolute CD4 cell counts at 48 weeks was 84.9 cells/μl. Trough plasma concentrations of enfuvirtide were consistently higher than target concentrations. Conclusion: Self-administration of enfuvirtide is not associated with unexpected toxicities for up to one year, and combined with oral antiretroviral drugs was associated with a significant decrease in HIV RNA and an increase in CD4 cell counts.

Modeling Sequence Evolution in Acute HIV-1 Infection

We describe a mathematical model and Monte Carlo (MC) simulation of viral evolution during acute infection. We consider both synchronous and asynchronous processes of viral infection of new target cells. The model enables an assessment of the expected sequence diversity in new HIV-1 infections originating from a single transmitted viral strain, estimation of the most recent common ancestor (MRCA) of the transmitted viral lineage, and estimation of the time to coalesce back to the MRCA. We also calculate the probability of the MRCA being the transmitted virus or an evolved variant. Excluding insertions and deletions, we assume HIV-1 evolves by base substitution without selection pressure during the earliest phase of HIV-1 infection prior to the immune response. Unlike phylogenetic methods that follow a lineage backwards to coalescence, we compare the observed data to a model of the diversification of a viral population forward in time. To illustrate the application of these methods, we provide detailed comparisons of the model and simulations results to 306 envelope sequences obtained from eight newly infected subjects at a single time point. The data from 68 patients were in good agreement with model predictions, and hence compatible with a single-strain infection evolving under no selection pressure. The diversity of the samples from the other two patients was too great to be explained by the model, suggesting multiple HIV-1-strains were transmitted. The model can also be applied to longitudinal patient data to estimate within-host viral evolutionary parameters.

Sex, Race, and Geographic Region Influence Clinical Outcomes Following Primary HIV-1 Infection

BACKGROUND It is unknown whether sex and race influence clinical outcomes following primary human immunodeficiency virus type 1 (HIV-1) infection. METHODS Data were evaluated from an observational, multicenter, primarily North American cohort of HIV-1 seroconverters. RESULTS Of 2277 seroconverters, 5.4% were women. At enrollment, women averaged .40 log₁₀ fewer copies/mL of HIV-1 RNA (P < .001) and 66 more CD4(+) T cells/μL (P = .006) than men, controlling for age and race. Antiretroviral therapy (ART) was less likely to be initiated at any time point by nonwhite women and men compared to white men (P < .005), and by individuals from the southern United States compared to others (P = .047). Sex and race did not affect responses to ART after 6 months (P > .73). Women were 2.17-fold more likely than men to experience >1 HIV/AIDS-related event (P < .001). Nonwhite women were most likely to experience an HIV/AIDS-related event compared to all others (P = .035), after adjusting for intravenous drug use and ART. Eight years after diagnosis, >1 HIV/AIDS-related event had occurred in 78% of nonwhites and 37% of whites from the southern United States, and 24% of whites and 17% of nonwhites from other regions (P < .001). CONCLUSIONS Despite more favorable clinical parameters initially, female HIV-1-seroconverters had worse outcomes than did male seroconverters. Elevated morbidity was associated with being nonwhite and residing in the southern United States.