The utility of adenoviral vectors for gene therapy is currently limited due, in part, to the widespread distribution of the cellular receptor for the adenovirus fiber that precludes the targeting of specific cell types. In order to develop a targeted adenovirus, it is therefore necessary both to ablate endogenous viral tropism and to introduce novel tropism. We hypothesized that these two goals could be achieved by employing a neutralizing anti-fiber antibody, or antibody fragment, chemically conjugated to a cell-specific ligand. To test this concept, we chose to target the folate receptor, which is overexpressed on the surface of a variety of malignant cells. Therefore, we conjugated folate to the neutralizing Fab fragment of an anti-fiber monoclonal antibody. This Fab-folate conjugate was complexed with an adenoviral vector carrying the luciferase reporter gene and was shown to redirect adenoviral infection of target cells via the folate receptor at a high efficiency. Furthermore, when complexed with an adenoviral vector carrying the gene for herpes simplex virus thymidine kinase, the Fab-folate conjugate mediated the specific killing of cells that overexpress the folate receptor. This work thus represents the first demonstration of the retargeting of a recombinant adenoviral vector via a non-adenoviral cellular receptor.

The conserved oligomannose epitope, Man9GlcNAc2, recognized by the broadly neutralizing human mAb 2G12 is an attractive prophylactic vaccine candidate for the prevention of HIV-1 infection. We recently reported total chemical synthesis of a series of glycopeptides incorporating one to three copies of Man 9GlcNAc2 coupled to a cyclic peptide scaffold. Surface plasmon resonance studies showed that divalent and trivalent, but not monovalent, compounds were capable of binding 2G12. To test the efficacy of the divalent glycopeptide as an immunogen capable of inducing a 2G12-like neutralizing antibody response, we covalently coupled the molecule to a powerful immune-stimulating protein carrier and evaluated immunogenicity of the conjugate in two animal species. We used a differential immunoassay to demonstrate induction of high levels of carbohydrate-specific antibodies; however, these antibodies showed poor recognition of recombinant gp160 and failed to neutralize a panel of viral isolates in entry-based neutralization assays. To ascertain whether antibodies produced during natural infection could recognize the mimetics, we screened a panel of HIV-1-positive and -negative sera for binding to gp120 and the synthetic antigens. We present evidence from both direct and competitive binding assays that no significant recognition of the glycopeptides was observed, although certain sera did contain antibodies that could compete with 2G12 for binding to recombinant gp120. molecular mimicry ͉ neutralizing antibody

Programmed Cell Death 1 (PD-1) plays a crucial role in immunomodulation. Binding of PD-1 to its ligand receptors down-regulates immune responses, and published reports suggest that this immune modulation is exploited in cases of tumor progression or chronic viral infection to evade immune surveillance. Thus, blockade of this signal could restore or enhance host immune functions. To test this hypothesis, we generated a panel of mAbs specific to human PD-1 that block PD ligand 1 and tested them for in vitro binding, blocking, and functional T cell responses, and evaluated a lead candidate in two in vivo rhesus macaque (Macaca mulatta) models. In the first therapeutic model, chronically SIV-infected macaques were treated with a single infusion of anti-PD-1 mAb; viral loads increased transiently before returning to, or falling below, pretreatment baselines. In the second prophylactic model, naive macaques were immunized with an SIV-gag adenovirus vector vaccine. Induced PD-1 blockade caused a statistically significant (p < 0.05) increase in the peak percentage of T cells specific for the CM9 Gag epitope. These new results on PD-1 blockade in nonhuman primates point to a broader role for PD-1 immunomodulation and to potential applications in humans.

bNonnucleoside reverse transcriptase inhibitors (NNRTIs) are a mainstay of therapy for treating human immunodeficiency type 1 virus (HIV-1)-infected patients. MK-1439 is a novel NNRTI with a 50% inhibitory concentration (IC 50 ) of 12, 9.7, and 9.7 nM against the wild type (WT) and K103N and Y181C reverse transcriptase (RT) mutants, respectively, in a biochemical assay. Selectivity and cytotoxicity studies confirmed that MK-1439 is a highly specific NNRTI with minimum off-target activities. In the presence of 50% normal human serum (

Gene therapy to correct defective genes requires efficient gene delivery and long-term gene expression. The available vector systems have not allowed the simultaneous achievement of both goals. We have developed a chimeric viral vector system that incorporates favorable aspects of both adenoviral and retroviral vectors. Adenoviral vectors induce target cells to function as transient retroviral producer cells in vivo. The progeny retroviral vector particles are then able to stably transduce neighboring cells. In this system, the nonintegrative adenoviral vector is rendered functionally integrative via the intermediate generation of a retroviral producer cell. The chimeric vectors may allow realization of the requisite goals for specific gene-therapy applications.

EC 95 in the presence of 10% fetal bovine serum. In the resistance selection of subtype B virus with DOR, a V106A mutant virus led to two mutation pathways, followed by the emergence separately of either F227L or L234I. In the resistance selection of subtype A and C viruses, similar mutation development pathways were detected, in which a V106A or V106M mutant was also the starting virus in the pathways. Mutations that are commonly associated with RPV and EFV in clinical settings were also identified in subtype B viruses such as the E138K and K103N mutants, respectively, in this in vitro resistance selection study. The susceptibility of subtype B mutant viruses selected by DOR, RPV, and EFV to NNRTIs was evaluated. Results suggest that mutant viruses selected by DOR are susceptible to RPV and EFV and mutants selected by RPV and EFV are susceptible to DOR. When the replication capacity of the V106A mutant was compared with that of the wild-type (WT) virus, the mutant virus was 4-fold less fit than the WT virus.

Doravirine (DOR), which is currently in a phase 3 clinical trial, is a novel human immunodeficiency type 1 virus (HIV-1) nonnucleoside reverse transcriptase inhibitor (NNRTI). DOR exhibits potent antiviral activity against wild-type virus and K103N, Y181C, and K103N/Y181C mutant viruses, with 50% inhibitory concentrations (IC 50 s) of 12, 21, 31, and 33 nM, respectively, when measured in 100% normal human serum (NHS). To assess the potential for DOR to suppress NNRTI-associated and rilpivirine (RPV)-specific mutants at concentrations achieved in the clinic setting, inhibitory quotients (IQs) were calculated by determining the ratio of the clinical trough concentration over the antiviral IC 50 for each virus with DOR and RPV and efavirenz (EFV). DOR displayed IQs of 39, 27, and 25 against the K103N, Y181C, and K103N/Y181C mutants, respectively. In contrast, RPV exhibited IQs of 4.6, 1.4, and 0.8, and EFV showed IQs of 2.5, 60, and 1.9 against these viruses, respectively. DOR also displayed higher IQs than those of RPV and EFV against other prevalent NNRTI-associated mutants, with the exception of Y188L. Both DOR and EFV exhibited higher IQs than RPV when analyzed with RPV-associated mutants. Resistance selections were conducted with K103N, Y181C, G190A, and K103N/Y181C mutants at clinically relevant concentrations of DOR, RPV, and EFV. No viral breakthrough was observed with DOR, whereas breakthrough viruses were readily detected with RPV and EFV against Y181C and K103N viruses, respectively. These data suggest that DOR should impose a higher barrier to the development of resistance than RPV and EFV at the concentrations achieved in the clinic setting.T he introduction of highly active antiretroviral therapy (HAART) in 1996 has significantly reduced the morbidity and mortality associated with HIV-1 infection (1, 2). However, the clinical and immunologic benefits of antiretroviral therapy can be compromised by the emergence of drug-resistant viruses due to suboptimal treatment or adherence.Drug-resistant mutants can be transmitted to an uninfected individual. Transmitted drug-resistant (TDR) mutants limit the choice of first-line combination antiretroviral therapy, decrease the efficacy of subsequent antiretroviral regimens, and increase the risk of treatment failure (3-5). TDR mutants have been documented among treatment-naive patients, with prevalences ranging from 3% to 24%, depending on the cohort and geographic characteristics (6). Importantly, transmitted nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant mutants are of particular concern, as they have the ability to persist for years after initial infection, suggesting the low impact of NNRTI-resistant mutations on viral fitness (7-9). The persistence of NNRTI-associated mutants after the discontinuation of an NNRTI-containing regimen may contribute to the prevalence of NNRTI-associated TDR mutants.Three mutants, K103N, Y181C, and G190A, account for Ͼ90% of the NNRTI-associated TDR mutants in the United States (10). K103N, Y181C, and K103N/Y181...

Specific killing of erbB-2-overexpressing tumor cells can be achieved using expression of an intracellular antibody directed against the erbB-2 oncoprotein. We have developed a strategy using a recombinant adenovirus encoding an anti-erbB-2 single chain antibody to achieve targeted tumor cell killing in vivo and can show significantly prolonged survival of animals carrying a human ovarian carcinoma tumor burden within their peritoneal cavities. This strategy of gene therapy for ovarian carcinoma offers the potential to achieve highly specific, targeted killing of human tumor cells and thus establishes the rationale to undertake human clinical trials on this basis. (J. Clin. Invest. 1995. 96:2980-2989