Oral administration of the nucleoside EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) provides rapid suppression of HIV viremia in humanized mice and favorable pharmacokinetic properties in mice and the rhesus macaque.

Like normal cellular nucleosides, the nucleoside reverse transcriptase (RT) inhibitor (NRTI) 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) has a 3'-hydroxyl moiety, and yet EFdA is a highly potent inhibitor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication with activity against a broad range of clinically important drug-resistant HIV isolates. We evaluated the anti-HIV activity of EFdA in primary human cells and in HIV-infected humanized mice. EFdA exhibited excellent potency against HIVJR-CSF in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs), with a 50% inhibitory concentration of 0.25 nM and a selectivity index of 184,000; similar antiviral potency was found against 12 different HIV clinical isolates from multiple clades (A, B, C, D, and CRF01_AE). EFdA was readily absorbed after oral dosing (5 mg/kg of body weight) in both mice and the rhesus macaque, with micromolar levels of the maximum concentration of drug in serum (Cmax) attained at 30 min and 90 min, respectively. Trough levels were at or above 90% inhibitory concentration (IC90) levels in the macaque at 24 h, suggesting once-daily dosing. EFdA showed reasonable penetration of the blood-brain barrier in the rhesus macaque, with cerebrospinal fluid levels at approximately 25% of plasma levels 8 h after single oral dosing. Rhesus PBMCs isolated 24 h following a single oral dose of 5 mg/kg EFdA were refractory to SIV infection due to sufficiently high intracellular EFdA-triphosphate levels. The intracellular half-life of EFdA-triphosphate in PBMCs was determined to be >72 h following a single exposure to EFdA. Daily oral administration of EFdA at low dosage levels (1 to 10 mg/kg/day) was highly effective in protecting humanized mice from HIV infection, and 10 mg/kg/day oral EFdA completely suppressed HIV RNA to undetectable levels within 2 weeks of treatment.

Impaired surfactant production by alveolar epithelial cells in a SCID-hu lung mouse model of congenital human cytomegalovirus infection.

Human cytomegalovirus (HCMV) is the leading viral cause of birth defects and life-threatening lung-associated diseases in premature infants and immunocompromised children. Although the fetal lung is a major target organ of the virus, HCMV lung pathogenesis has remained unexplored, possibly as a result of extreme host range restriction. To overcome this hurdle, we generated a SCID-hu lung mouse model that closely recapitulates the discrete stages of human lung development in utero. Human fetal lung tissue was implanted into severe combined immunodeficient (CB17-scid) mice and inoculated by direct injection with the VR1814 clinical isolate of HCMV. Virus replication in the fetal lung was assessed by the quantification of infectious virus titers and HCMV genome copies and the detection of HCMV proteins by immunohistochemistry and Western blotting. We show that HCMV efficiently replicated in the lung implants during a 2-week period, forming large viral lesions. The virus productively infected alveolar epithelial and mesenchymal cells, imitating congenital infection of the fetal lung. HCMV replication triggered apoptosis near and within the viral lesions and impaired the production of surfactant proteins in the alveolar epithelium. Our findings highlight that congenital and neonatal HCMV infection can adversely impact lung development, leading to pneumonia and acute lung injury. We have successfully developed a small-animal model that closely recapitulates fetal and neonatal lung development and provides a valuable, biologically relevant tool for an understanding of the lung pathogenesis of HCMV as well as other human respiratory viruses. Additionally, this model would greatly facilitate the development and testing of new antiviral therapies for HCMV along with select human pulmonary pathogens.

Immunotherapeutic Blockade of CD47 Inhibitory Signaling Enhances Innate and Adaptive Immune Responses to Viral Infection.

Paradoxically, early host responses to infection include the upregulation of the antiphagocytic molecule, CD47. This suggests that CD47 blockade could enhance antigen presentation and subsequent immune responses. Indeed, mice treated with anti-CD47 monoclonal antibody following lymphocytic choriomeningitis virus infections show increased activation of both macrophages and dendritic cells (DCs), enhancement of the kinetics and potency of CD8+ T cell responses, and significantly improved virus control. Treatment efficacy is critically dependent on both APCs and CD8+ T cells. In preliminary results from one of two cohorts of humanized mice infected with HIV-1 for 6 weeks, CD47 blockade reduces plasma p24 levels and restores CD4+ T cell counts. The results indicate that CD47 blockade not only enhances the function of innate immune cells but also links to adaptive immune responses through improved APC function. As such, immunotherapy by CD47 blockade may have broad applicability to treat a wide range of infectious diseases.

Cellular HIV Reservoirs and Viral Rebound from the Lymphoid Compartments of 4'-Ethynyl-2-Fluoro-2'-Deoxyadenosine (EFdA)-Suppressed Humanized Mice.

Although antiretroviral therapy (ART) greatly suppresses HIV replication, lymphoid tissues remain a sanctuary site where the virus may replicate. Tracking the earliest steps of HIV spread from these cellular reservoirs after drug cessation is pivotal for elucidating how infection can be prevented. In this study, we developed an in vivo model of HIV persistence in which viral replication in the lymphoid compartments of humanized mice was inhibited by the HIV reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) to very low levels, which recapitulated ART-suppression in HIV-infected individuals. Using a combination of RNAscope in situ hybridization (ISH) and immunohistochemistry (IHC), we quantitatively investigated the distribution of HIV in the lymphoid tissues of humanized mice during active infection, EFdA suppression, and after drug cessation. The lymphoid compartments of EFdA-suppressed humanized mice harbored very rare transcription/translation-competent HIV reservoirs that enable viral rebound. Our data provided the visualization and direct measurement of the early steps of HIV reservoir expansion within anatomically intact lymphoid tissues soon after EFdA cessation and suggest a strategy to enhance therapeutic approaches aimed at eliminating the HIV reservoir.

Efficacy of broadly neutralizing monoclonal antibody PG16 in HIV-infected humanized mice.

Highly potent broadly neutralizing human monoclonal antibodies hold promise for HIV prophylaxis and treatment. We used the SCID-hu Thy/Liv and BLT humanized mouse models to study the efficacy of these antibodies, primarily PG16, against HIV-1 clades A, B, and C. PG16 targets a conserved epitope in the V1/V2 region of gp120 common to 70-80% of HIV-1 isolates from multiple clades and has extremely potent in vitro activity against HIVJR-CSF. PG16 was highly efficacious in SCID-hu mice as a single intraperitoneal administration the day before inoculation of R5-tropic HIV directly into their Thy/Liv implants and demonstrated even greater efficacy if PG16 administration was continued after Thy/Liv implant HIV inoculation. However, PG16 as monotherapy had no activity in humanized mice with established R5-tropic HIV infection. These results provide evidence of tissue penetration of the antibodies, which could aid in their ability to prevent infection if virus crosses the mucosal barrier.

Superior human leukocyte reconstitution and susceptibility to vaginal HIV transmission in humanized NOD-scid IL-2Rγ(-/-) (NSG) BLT mice.

Humanized Bone marrow/Liver/Thymus (BLT) mice recapitulate the mucosal transmission of HIV, permitting study of early events in HIV pathogenesis and evaluation of preexposure prophylaxis methods to inhibit HIV transmission. Human hematopoiesis is reconstituted in NOD-scid mice by implantation of human fetal liver and thymus tissue to generate human T cells plus intravenous injection of autologous liver-derived CD34(+) hematopoietic stem cells to engraft the mouse bone marrow. In side-by-side comparisons, we show that NOD-scid mice homozygous for a deletion of the IL-2Rγ-chain (NOD-scid IL-2Rγ(-/-)) are far superior to NOD-scid mice in both their peripheral blood reconstitution with multiple classes of human leukocytes (e.g., a mean of 182 versus 14 CD4(+) T cells per µl 12 weeks after CD34(+) injection) and their susceptibility to intravaginal HIV exposure (84% versus 11% viremic mice at 4 weeks). These results should speed efforts to obtain preclinical animal efficacy data for new HIV drugs and microbicides.