PhD - Pathology / The entry stage of the HIV-1 viral life cycle has become a prime target for preventing HIV-1 infection. This has led to the development of a new class of anti-retroviral agents termed entry inhibitors, which have proven effective in vitro and in the clinic. These new agents target three different stages of entry, namely CD4 binding, coreceptor interaction with either CCR5 or CXCR4 and the fusion process. Here we studied isolates from patients with HIV-1 subtype C infection and the effectiveness of different coreceptor and fusion inhibitors in vitro. Further we examined resistance profiles to the first licensed entry inhibitor, T-20. In Chapter 2 we examined coreceptor usage of HIV-1 subtype C isolates and their sensitivity to CCR5 and CXCR4 inhibitors. Twenty-nine viral isolates with different coreceptor usage profiles were isolated from patients with advanced AIDS. The CCR5-specific agents, PRO140 an anti-CCR5 monoclonal antibody and RANTES, the natural ligand for CCR5 inhibited all 24 R5 isolates, while the two X4 and the three R5X4 viruses were sensitive to the CXCR4-specific inhibitor, AMD3100. The five X4 or R5X4 viruses were all able to replicate in peripheral blood mononuclear cells (PBMC) that did not express CCR5 confirming their ability to use CXCR4 on primary cells. When tested using coreceptor-transfected cell lines, one R5 virus was also able to use CXCR6, and another R5X4 virus could use CCR3, Bob/GPR15 and CXCR6. The R5X4 and X4 viruses contained more diverse V3 loop sequences with a higher overall positive charge, compared to the R5 viruses. Hence, HIV-1 subtype C viruses are able to use CCR5, CXCR4 or both for entry, and they are sensitive to specific inhibitors of entry via these coreceptors. In Chapter 3 we analyzed isolates from 10 acutely infected individuals, who were followed longitudinally for up to three years. Two of these individuals (Du151 and Du179) underwent a coreceptor switch and were studied more intensively. The other eight individuals retained CCR5 usage throughout the duration of the study. The initial 4 isolates from Du151 were able to utilize CCR5 but the later isolates were able to use both CCR5 and CXCR4 (R5X4). Du179 used both CCR5 and CXCR4 (R5X4) initially, but the later isolate was found to be monotropic and used CXCR4 (X4) exclusively. Viral isolates were tested for their sensitivity to small molecule inhibitors of CCR5, CXCR4 and the fusion process in a PBMC assay. All of the R5 isolates were sensitive to RANTES and PRO140 and insensitive to the two CXCR4 coreceptor inhibitors AMD3100 and T-140. There was a tendency for later isolates to become slightly less sensitive to the CCR5 inhibitors and more sensitive to the CXCR4 entry inhibitors. None of the R5X4 Du179 isolates were effectively inhibited by PRO140 and RANTES, but the X4 isolate of Du179 became sensitive to CXCR4 entry inhibitors. Both Du151 and Du179 underwent amino acid changes in their V3 sequences that included an increased charge associated with CXCR4 usage. This indicates that coreceptor switching can occur in subtype C infections and is associated with changes in the V3 loop. However, both Du151 and Du179 were subsequently found to be dually infected with another subtype C strain, which may account for some of the phenotypic and genotypic changes seen in these individuals including the appearance of CXCR4-virus variants. In Chapter 4 we explored two HIV-1 isolates (CM4 and CM9) able to use alternate HIV-1 coreceptors for entry (i.e. coreceptors other than CCR5 or CXCR4) on transfected cell lines. These isolates were tested for their sensitivity to inhibitors of HIV-1 entry on primary cells. Both isolates were from patients with cryptococcal meningitis, a severe AIDS defining condition. CM4 was able to use CCR5 and Bob/GPR15 efficiently in transfected cells. This isolate grew in D32/D32 CCR5 PBMC in the presence of AMD3100, indicating that it used a receptor other than CCR5 or CXCR4 on primary cells. It was insensitive to the CCR5 entry inhibitors RANTES and PRO140, but was partially inhibited by vMIP-1, a chemokine that binds CCR3, CCR8, Bob/GPR15 and CXCR6. The coreceptor used by this isolate on primary cells is thus currently unknown. CM9 used CCR5, CXCR4, Bob/GPR15, CXCR6 and CCR3 on transfected cells and was able to replicate in the presence of AMD3100 in D32/D32 CCR5 PBMC. It was insensitive to vMIP-1, eotaxin and I309 used individually, but was inhibited completely when vMIP-1 or I309, the ligand for CCR8, were combined with AMD3100. These results strongly suggest that this isolate can use CCR8 on primary cells. Collectively these data suggest that some HIV-1 isolates can use alternate coreceptors on primary cells, which may have implications for strategies that aim to block viral entry using coreceptor inhibitors. In Chapter 5 we examined the effectiveness of T-20 to inhibit HIV-1 subtype C isolates. T-20 blocks the fusion stage of the viral entry cycle and it is the first entry inhibitor to be licensed for clinical use. T-20 consists of 36 amino acids and was designed based on the HR-2 region of HIV-1 subtype B. A total of 23 HIV-1 subtype C isolates were tested for their ability to replicate in the presence and absence of T-20. This included five isolates with multiple genotypic drug resistance mutations to reverse transcriptase and protease inhibitors. Among the 23 subtype C isolates there were 10-16 amino acid changes in the 36 amino acid region corresponding to T-20. However, all isolates were effectively inhibited by T-20 at 1 mg/ml, including the 5 isolates resistant to other anti-retroviral drugs. The gp41 region was sequenced and the HR-1 and HR-2 amino acids analyzed. All isolates had the amino acids GIV at positions 36-38 in gp41, which are associated with sensitivity to T-20. One X4 had a GVV motif but this did not affect its sensitivity. Thus, T-20 inhibited subtype C viruses despite significant genetic differences in the HR-2 regions of subtypes B and C. These data suggest that T-20 would be highly effective in patients with HIV-1 subtype C infection including those failing existing anti-retroviral drug regimens. In Chapter 6 we examined the in vitro resistance patterns of HIV-1 subtype C to T-20. Resistance to T-20 is a consequence of persistent exposure to the antiretroviral peptide. To establish if patterns of resitance to T-20 were similar to resistance mutations occurring in subtype B viruses, 11 subtype C and 4 subtype B viruses were passaged in the presence of increasing concentrations of T-20. The subtype C isolates showed varying levels of replication at 1 mg/ml T-20 by day 18, but by day 29 all replicated efficiciently at 10 mg/ml T-20. All isolates showed evidence of genotypic changes in gp41 HR-1 following exposure to T-20 that included G36S/E/D, I37T, V38M/A/L/E, N42D, N43K/S, L45R/M and A50T/V. Five viruses had compensatory changes in the HR-2 region, which corresponds to the T-20 sequence, and two isolates had changes in the V3 region. Mutational patterns among the 4 subtype B viruses were similar to those for subtype C and those previously published in the literature. These data indicate that in vitro resistance to T-20 develops rapidly among HIV-1 subtype C isolates. In general, mutational patterns for subtype C were similar to those described for subtype B, suggesting that the mechanism of action for T-20 is similar for HIV-1 subtype B and C isolates. Observations from these studies indicate that HIV-1 subtype C predominantly use the CCR5 coreceptor to enter cells. CXCR4 usage is rare compared to other subtypes, although such isolates are found in patients with advanced AIDS. The two cases of coreceptor switching reported here were dually infected. Subtype C isolates were sensitive to coreceptor and fusion inhibitors except for two isolates able to utilize alternate coreceptors. However, alternate coreceptor usage is very rare and unlikely to impact on the utility of coreceptor inhibitors. Given the propensity for CCR5 usage this may imply that CCR5 coreceptor inhibitors may be more effective in countries where HIV-1 subtype C predominate. Entry inhibitors may be useful for prevention and treatment strategies and have the potential to provide sterilizing immunity. These agents could be used as microbicides and as an adjunct to existing antiretroviral therapies for use in HIV-1 subtype C infected individuals. However resistance to entry inhibitors can emerge and should be used in combination with other antiretrovirals to minimize this outcome. While entry inhibitors provide a new line of defence against HIV-1, their cost may prevent their use in developing countries in the immediate future. Nevertheless, this is the first comprehensive study of the sensitivity of HIV-1 subtype C isolates to entry inhibitors providing a data-driven rationale for their use in individuals infected with HIV-1 subtype C.

No description available.

Yes / ABSTRACT Entry inhibitors represent a potent class of antiretroviral drugs that target a host cell protein, CCR5, an HIV-1 entry coreceptor, and not viral protein. Lack of sensitivity can occur due to preexisting virus that uses the CXCR4 coreceptor, while true resistance occurs through viral adaptation to use a drug-bound CCR5 coreceptor. To understand this R5 resistance pathway, we analyzed >500 envelope protein sequences and phenotypes from viruses of 20 patients from the clinical trials MOTIVATE 1 and 2, in which treatment-experienced patients received maraviroc plus optimized background therapy. The resistant viral population was phylogenetically distinct and associated with a genetic bottleneck in each patient, consistent with de novo emergence of resistance. Recombination analysis showed that the C2-V3-C3 region tends to genotypically correspond to the recombinant’s phenotype, indicating its primary importance in conferring resistance. Between patients, there was a notable lack of commonality in the specific sites conferring resistance, confirming the unusual nature of R5-tropic resistance. We used coevolutionary and positive-selection analyses to characterize the genotypic determinants of resistance and found that (i) there are complicated covariation networks, indicating frequent coevolutionary/compensatory changes in the context of protein structure; (ii) covarying sites under positive selection are enriched in resistant viruses; (iii) CD4 binding sites form part of a unique covariation network independent of the V3 loop; and (iv) the covariation network formed between the V3 loop and other regions of gp120 and gp41 intersects sites involved in glycosylation and protein secretion. These results demonstrate that while envelope sequence mutations are the key to conferring maraviroc resistance, the specific changes involved are context dependent and thus inherently unpredictable. IMPORTANCE The entry inhibitor drug maraviroc makes the cell coreceptor CCR5 unavailable for use by HIV-1 and is now used in combination antiretroviral therapy. Treatment failure with drug-resistant virus is particularly interesting because it tends to be rare, with lack of sensitivity usually associated with the presence of CXCR4-using virus (CXCR4 is the main alternative coreceptor HIV-1 uses, in addition to CD4). We analyzed envelope sequences from HIV-1, obtained from 20 patients who enrolled in maraviroc clinical trials and experienced treatment failure, without detection of CXCR4-using virus. Evolutionary analysis was employed to identify molecular changes that confer maraviroc resistance. We found that in these individuals, resistant viruses form a distinct population that evolved once and was successful as a result of drug pressure. Further evolutionary analysis placed the complex network of interdependent mutational changes into functional groups that help explain the impediments to the emergence of maraviroc-associated R5 drug resistance. / X.J. was supported by Medical Research Council (G1001806/1) and Wellcome Trust (097820/Z/11/A) funding and F.F. by a Biotechnology and Biological Sciences Research Council studentship to D.L.R.

Chemokines are small immune system proteins mediating leukocyte migration and activation, and are important in many aspects of health and diseases. Some chemokines also have the ability to block HIV-1 infection by binding to the HIV-1 co-receptors CCR5 (CC chemokine receptor 5) and CXCR4 (CXC chemokine receptor 4). The first part of this work is to determine the mechanism of action of a human herpesvirus-8 encoded viral chemokine analog vMIP-II (viral macrophage inflammatory protein-II) by characterizing its interactions with endothelial surface glycosaminoglycans (GAGs) and cell surface receptors. Nuclear magnetic resonance (NMR), mutagenesis and molecular-docking were conducted and results show that vMIP-II tightly binds glycosaminoglycans using residues distributed along one face of the protein, such as R18, R46 and R48, and that there is a shift in the GAG binding site between the monomer and dimer form of vMIP-II where the N-terminus is involved in GAG binding for the dimer. This study, for the first time, provides a model that explains the mechanism of how quaternary structure affects chemokine-GAG binding. Mutagenesis and competition binding assays were conducted to study the receptor-binding mechanism of vMIP-II. Preliminary results suggest that vMIP-II uses the same positively charged binding surface comprising R18, K45, R46 and R48 to interact with the negatively charged N-termini of CCR5 and CXCR4. NMR studies on how vMIP-II interacts with N-terminal peptides of CCR5 and CXCR4 is on-going. The second part of this work was to rationally design HIV-1 entry inhibitors based on our knowledge of the mechanisms of chemokine-receptor binding and HIV-1 cell entry. We successfully designed two chimeric HIV entry inhibitors composed of CCR5-targeting RANTES variants (5P12-RANTES and 5P14-RANTES) linked to a gp41 targeting C-peptide, C37. In in vitro assays, chimeric inhibitors 5P12-linker-C37 and 5P14-linker-C37 showed the highest anti-viral potency yet published with IC50 values as low as 0.001 nM against certain virus strains. On human peripheral blood mononuclear cells, the chimeric inhibitors also exhibited very strong inhibition against R5-tropic and X4-tropic viruses, with IC50 values as low as 0.015 nM and 0.44 nM, respectively. A clear delivery mechanism was observed and characterized. These fully recombinant inhibitors can be easily produced at low cost and are excellent candidates for HIV microbicides.

Reovirus is internalized after interaction of the outer proteins μ1, σ1 and σ3 with the host cell. Proteolysis of σ3 and cleavage of μ1 (into δ and φ) eventually leads to the formation of a more infectious subviral particle named “ISVP”. The infectious entry of viruses, but not of ISVPs, can be blocked using various entry inhibitors and therefore, suggests that there is a threshold of σ3 digestion required to allow particle to bypass entry blockers. By combining protease and detergent to the digestion of virions, data from this work showed distinct particles generated along the transition pathway. In addition, studies involving flow cytometry and specific antibodies (anti-μ1) showed that between virus and ISVP there is a gradual yet heterogeneous particle proteolysis that is directly related to the virus infectivity. The findings and approaches taken for this thesis work can possibly be extended for studying other non-enveloped viruses. Moreover, it may help to shed some light on the development of safe and effective oncolytic agents.

Reovirus is internalized after interaction of the outer proteins μ1, σ1 and σ3 with the host cell. Proteolysis of σ3 and cleavage of μ1 (into δ and φ) eventually leads to the formation of a more infectious subviral particle named “ISVP”. The infectious entry of viruses, but not of ISVPs, can be blocked using various entry inhibitors and therefore, suggests that there is a threshold of σ3 digestion required to allow particle to bypass entry blockers. By combining protease and detergent to the digestion of virions, data from this work showed distinct particles generated along the transition pathway. In addition, studies involving flow cytometry and specific antibodies (anti-μ1) showed that between virus and ISVP there is a gradual yet heterogeneous particle proteolysis that is directly related to the virus infectivity. The findings and approaches taken for this thesis work can possibly be extended for studying other non-enveloped viruses. Moreover, it may help to shed some light on the development of safe and effective oncolytic agents.

L’entrée virale du VIH-1 dans ses cellules cibles constitue une cible thérapeutique de choix. À l’heure actuelle, un inhibiteur de fusion et un inhibiteur ciblant le corécepteur CCR5 sont utilisés en thérapie. Dans notre étude, notre objectif était d’évaluer le profil antiviral et fonctionnel de plusieurs types moléculaires envisagés comme inhibiteurs d’entrée du corécepteur CXCR4, soient : 1) de dérivés peptidiques mimant des portions du récepteur 2) de dérivés peptidiques issus du ligand naturel de CXCR4, le SDF-1 et 3) de dérivés du puissant inhibiteur de faible poids moléculaire, le T140. Notre recherche se concentrait sur la mise au point de molécules capables d’inhiber l’entrée du VIH en ciblant sélectivement le corécepteur CXCR4 tout en conservant les fonctions naturelles de ce dernier. Parmi les molécules testées, certaines possèdent un grand potentiel dans le développement de nouveaux médicaments antirétroviraux. / HIV entry into target cell is one of the main therapeutic goals in AIDS research. One fusion inhibitor is actually used as therapeutics and one more entry inhibitor targeting the CCR5 coreceptor was approved recently. The discovery of inhibitors targeting the second HIV coreceptor CXCR4 with high antiviral power versus low interference with natural functions of CXCR4 represents a crucial issue in AIDS research. In our study, antiviral properties and functional effect of different kinds of molecules were tested: 1) peptides derivative mimicking CXCR4 domains 2) SDF-1 derivative peptides and 3) T140 (a low molecular weight inhibitor) derivative molecules. These molecules were investigated in HIV infection and cellular migration. The main goal of our study was to determine molecules having great antiviral properties versus low interference on chemotactic response induced by the natural ligand of CXCR4 (SDF-1). Within the panel of molecules tested, some have great potential to become lead compounds for inhibition of viral entry through CXCR4.

No description available.

El programa de la síndrome de la immunodeficiència adquirida (SIDA o sida) de les Nacions Unides va estimar a finals de 2008 que més de 33 milions de persones arreu del món vivien amb el virus de la immunodeficiència humana (VIH). En l’estudi de les dianes terapèutiques contra l’agent etiològic de la sida s’han validat com a tals els coreceptors CXCR4 i CCR5 d’unió i fusió del virus a la cèl•lula hoste. Actualment hi ha un inhibidor d’entrada i un de fusió per al tractament de la infecció per aquest retrovirus, tot i que cap d’ells inhibeix el coreceptor CXCR4. Això, unit a la capacitat del virus de desenvolupar resistència als fàrmacs actuals, fa necessària la cerca de noves famílies de compostos que hi facin front. El Grup d’Enginyeria Molecular (GEM) de l’IQS ha descrit prèviament una quimioteca combinatòria d’anàlegs de l’AMD3100, el cap de sèrie més actiu, formats per unitats estructurals polinitrogenades (que consisteixen en un heterocicle nitrogenat, un seguit de baules metilèniques i un grup amina primària terminal) unides a un espaiador p xililènic. La selecció d’aquesta quimioteca va dur a la identificació d’un nou candidat amb dos centres quirals amb activitat submicromolar contra el VIH. Aquest treball presenta diverses modificacions estructurals sobre ambdós caps de sèrie, en la cerca de nous inhibidors potencials del coreceptor d’entrada CXCR4. En primer lloc, per tal de rebaixar la basicitat de l’AMD3100, s’han substituït els seus anells ciclàmics per sistemes pirido[2,3 d]pirimidínics, en la síntesi dels quals es té molta experiència en el GEM. La baixa solubilitat en dissolvents comuns d’alguns dels compostos obtinguts es presenta com un greu inconvenient per al seu desenvolupament com a fàrmac. D’altra banda, tenint en compte el disseny racional descrit anteriorment al grup de recerca, s’amplia la quimioteca amb l’objectiu d’avaluar l’efecte de la distància entre àtoms de nitrogen i la presència de major impediment estèric entre l’espaiador aromàtic i el nitrogen que hi està directament unit. Tot i que no s’ha aconseguit millorar l’activitat del cap de sèrie, els compostos obtinguts presenten activitats prou satisfactòries. Igualment, s’obtenen els tres estereoisòmers del cap de sèrie amb activitat submicromolar i se n’avalua l’activitat antiviral per separat. Finalment, es duu a terme un disseny de novo de fàrmacs basat en l’estructura per buscar nous candidats que permetin obrir la porta a altres famílies de compostos amb activitat anti-VIH, a través de l’enumeració de la quimioteca virtual, la selecció i la posterior síntesi. / El programa del síndrome de la inmunodeficiencia adquirida (SIDA o sida) de las Naciones Unidas estimó a finales de 2008 que más de 33 millones de personas en todo el mundo vivían con el virus de la inmunodeficiencia humana (VIH). En el estudio de las dianas terapéuticas contra el agente etiológico del sida se han validado como tales los correceptores CXCR4 y CCR5 de unión y fusión del virus a la célula huésped. Actualmente existe un inhibidor de entrada y uno de fusión para el tratamiento de la infección por este retrovirus, aunque ninguno de ellos inhibe el correceptor CXCR4. Ello, unido a la capacidad del virus de desarrollar resistencia a los fármacos actuales, hace necesaria la búsqueda de nuevas familias de compuestos que le hagan frente. El Grup d’Enginyeria Molecular (GEM) del IQS ha descrito previamente una quimioteca combinatoria de análogos del AMD3100, el candidato más activo, formados por unidades estructurales polinitrogenadas (que consisten en un heterociclo nitrogenado, una serie de eslabones metilénicos y un grupo amina primaria terminal) unidas a un espaciador p xililénico. La selección de esta quimioteca llevó a la identificación de un nuevo candidato con dos centros quirales con actividad submicromolar contra el VIH. Este trabajo presenta diversas modificaciones estructurales sobre ambos candidatos, en la búsqueda de nuevos inhibidores potenciales del correceptor de entrada CXCR4. En primer lugar, para rebajar la basicidad del AMD3100, se han substituido sus anillos ciclámicos por sistemas pirido[2,3 d]pirimidínicos, en la síntesis de los cuales se tiene mucha experiencia en el GEM. La baja solubilidad en disolventes comunes de algunos de los compuestos obtenidos se presenta como un grave inconveniente para su desarrollo como fármaco. Por otra parte, teniendo en cuenta el diseño racional descrito anteriormente en el grupo de investigación, se amplía la quimioteca con el objetivo de evaluar el efecto de la distancia entre átomos de nitrógeno y la presencia de mayor impedimento estérico entre el espaciador aromático y el nitrógeno que está directamente unido a él. Aunque no se ha conseguido mejorar la actividad de la estructura de referencia, los compuestos obtenidos presentan actividades satisfactorias. Asimismo, se obtienen los tres estereoisómeros del candidato con actividad submicromolar y se evalúa la actividad antiviral por separado. Finalmente, se lleva a cabo un diseño de novo de fármacos basado en la estructura para buscar nuevos candidatos que permitan abrir la puerta a otras familias de compuestos con actividad anti-VIH, a través de la enumeración de la quimioteca virtual, la selección y la posterior síntesis. / The Joint United Nations Programme on Acquired Immunodeficiency Syndrome (AIDS) estimated in 2008 that more than 33 million people around the world lived with the human immunodeficiency virus (HIV). HIV cell fusion and entry coreceptors CXCR4 and CCR5 have been validated as targets for therapeutic intervention. Nowadays, there is one entry and one fusion inhibitor available for the treatment of this retrovirus infection, although none of them inhibits CXCR4 coreceptor. Due to this fact, added to the virus’ capacity of developing resistance to actual drugs, the search of new families of compounds facing up to these situations becomes necessary. The Grup d’Enginyeria Molecular (GEM) at IQS previously described a combinatorial library of analogues of the currently most active lead AMD3100, whose polinitrogenated building block consisted of a nitrogen-containing heterocycle, a saturated chain spacer and a terminal primary amine group, bonded to a p xylylene spacer. Library selection led to the identification of a new lead with two chiral centres and submicromolar activity against HIV. This work presents various structural modifications on both leads, looking for new potential CXCR4 inhibitors. First of all, in order to reduce basicity of AMD3100, cyclam rings were substituted by pyrido[2,3 d]pyrimidinic systems, whose synthesis has been widely studied at GEM. Low solubility in common solvents of some of the obtained compounds remains as a great inconvenience for their development as a drug. Furthermore, regarding the rational design previously described by the research group, the library has been expanded with the aim of evaluating the effect of the distance between nitrogen atoms and the steric effects between the linker and the nitrogen atom directly connected to it. Although biological activities do not improve the lead’s one, the obtained compounds present interesting activities. Moreover, the three stereoisomers of the lead compound with submicromolar activity have been synthesised and their activity has been evaluated separately. Finally, a de novo structure based drug design has been carried out looking for new hits allowing to identify new families of compounds with anti-HIV activity, by enumeration of the virtual library, selection of candidates and their synthesis.

Animal models have greatly contributed to the understanding of different aspects of human biology, as well as a variety of human-related pathogens and diseases. In order to study them, humanized mice susceptible to pathogens that replicate in human immune cells have been developed (e.g., humanized Rag2-/-γc-/- mice). These animals are engrafted with human hematopoietic stem cells (HSCs), resulting in the de novo development and maturation of the major functional components of the human adaptive immune system and the production of a variety of human cell types. Primary and secondary lymphoid organs in the mouse are populated with human cells, and animals have long term engraftment. These features make humanized mice an excellent in vivo model to study pathogenesis of human-specific viruses in the context of a human antiviral immune response. In addition, humanized mice have been shown to be useful preclinical models for the development and validation of antiviral therapeutics. In the present study, we aimed to successfully re-establish the humanized Rag2-/-γc-/- mouse model using cord blood-derived HSCs in our laboratory. We have shown that these mice sustain long term engraftment and systemic expansion of human cells, including the major targets of Kaposi's sarcoma Herpesvirus (KSHV) and Human immunodeficiency virus type 1 (HIV-1), in peripheral blood and different lymphoid organs. Further, we have begun to evaluate the susceptibility of the humanized Rag2-/-γc-/- mouse model to infection with KSHV. We demonstrate that human lymphocytes differentiated in reconstituted Rag2-/-γc-/- mice are permissive to KSHV infection ex vivo. This finding was corroborated by detection of KSHV mRNA expression in the spleen of a humanized mouse at 6 months post infection. In a different study, we tested the in vivo antiviral efficacy of a novel HIV-1 fusion inhibitor (PIE-12-trimer) in humanized Rag2-/-γc-/- mice. We have determined the half life of PIE-12-trimer in mouse plasma. Furthermore, the administration of PIE-12-trimer to HIV-1 infected humanized Rag2-/-γc-/- mice prevents depletion of CD4+ T cells in blood, thus it may be useful to prevent AIDS in human patients.