Serum Neutralizing Antibody Titers 12 Months After Coronavirus Disease 2019 Messenger RNA Vaccination: Correlation to Clinical Variables in an Adult, US Population.

BACKGROUND: We studied whether comorbid conditions affect strength and duration of immune responses after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) messenger RNA vaccination in a US-based, adult population. METHODS: Sera (before and after BNT162b2 vaccination) were tested serially up to 12 months after 2 doses of vaccine for SARS-CoV-2-anti-Spike neutralizing capacity by pseudotyping assay in 124 individuals; neutralizing titers were correlated to clinical variables with multivariate regression. Postbooster (third dose) effect was measured at 1 and 3 months in 72 and 88 subjects, respectively. RESULTS: After completion of primary vaccine series, neutralizing antibody half maximal inhibitory concentration (IC50) values were high at 1 month (14-fold increase from prevaccination), declined at 6 months (3.3-fold increase), and increased at 1 month postbooster (41.5-fold increase). Three months postbooster, IC50 decreased in coronavirus disease (COVID)-naïve individuals (18-fold increase) and increased in prior COVID 2019 (COVID-19+) individuals (132-fold increase). Age >65 years (ß = -0.94, P = .001) and malignancy (ß = -0.88, P = .002) reduced strength of response at 1 month. Both neutralization strength and durability at 6 months, respectively, were negatively affected by end-stage renal disease ([ß = -1.10, P = .004]; [ß = -0.66, P = .014]), diabetes mellitus ([ß = -0.57, P = .032]; [ß = -0.44, P = .028]), and systemic steroid use ([ß = -0.066, P = .032]; [ß = -0.55, P = .037]). Postbooster IC50 was robust against WA-1 and B.1.617.2. Postbooster neutralization increased with prior COVID-19 (ß = 2.9, P < .0001), and malignancy reduced neutralization response (ß = -0.68, P = .03), regardless of infection status. CONCLUSIONS: Multiple clinical factors affect the strength and duration of neutralization response after primary series vaccination, but not the postbooster dose strength. Malignancy was associated with lower booster-dose response regardless of prior COVID infection, suggesting a need for clinically guided vaccine regimens.

Rapid, reliable, and reproducible cell fusion assay to quantify SARS-Cov-2 spike interaction with hACE2.

COVID-19 is a global crisis of unimagined dimensions. Currently, Remedesivir is only fully licensed FDA therapeutic. A major target of the vaccine effort is the SARS-CoV-2 spike-hACE2 interaction, and assessment of efficacy relies on time consuming neutralization assay. Here, we developed a cell fusion assay based upon spike-hACE2 interaction. The system was tested by transient co-transfection of 293T cells, which demonstrated good correlation with standard spike pseudotyping for inhibition by sera and biologics. Then established stable cell lines were very well behaved and gave even better correlation with pseudotyping results, after a short, overnight co-incubation. Results with the stable cell fusion assay also correlated well with those of a live virus assay. In summary we have established a rapid, reliable, and reproducible cell fusion assay that will serve to complement the other neutralization assays currently in use, is easy to implement in most laboratories, and may serve as the basis for high throughput screens to identify inhibitors of SARS-CoV-2 virus-cell binding and entry.

Neutralization Synergy between HIV-1 Attachment Inhibitor Fostemsavir and Anti-CD4 Binding Site Broadly Neutralizing Antibodies against HIV.

Attachment inhibitor (AI) BMS-626529 (fostemsavir) represents a novel class of antiretrovirals which target human immunodeficiency virus type 1 (HIV-1) gp120 and block CD4-induced conformational changes required for viral entry. It is now in phase III clinical trials and is expected to be approved by the U.S. Food and Drug Administration (FDA) in the near future. Although fostemsavir is very potent against HIV in vitro and in vivo, a number of resistant mutants have already been identified. Broadly neutralizing HIV antibodies (bNAbs) can potently inhibit a wide range of HIV-1 strains by binding to viral Env and are very promising candidates for HIV-1 prevention and therapy. Since both target viral Env to block viral entry, we decided to investigate the relationship between these two inhibitors. Our data show that Env mutants resistant to BMS-626529 retained susceptibility to bNAbs. A single treatment of bNAb NIH45-46G54W completely inhibited the replication of these escape mutants. Remarkable synergy was observed between BMS-626529 and CD4 binding site (CD4bs)-targeting bNAbs in neutralizing HIV-1 strains at low concentrations. This synergistic effect was enhanced against virus harboring mutations conferring resistance to BMS-626529. The mechanistic basis of the observed synergy is likely enhanced inhibition of CD4 binding to the HIV-1 Env trimer by the combination of BMS-626529 and CD4bs-targeting bNAbs. This work highlights the potential for positive interplay between small- and large-molecule therapeutics against HIV entry, which may prove useful as these agents enter clinical use.IMPORTANCE As the worldwide HIV pandemic continues, there is a continued need for novel drugs and therapies. A new class of drug, the attachment inhibitors, will soon be approved for the treatment of HIV. Broadly neutralizing antibodies are also promising candidates for HIV prevention and therapy. We investigated how this drug might work with these exciting antibodies that are very potent in blocking HIV infection of cells. These antibodies worked against virus known to be resistant to the new drug. In addition, a specific type of antibody worked really well with the new drug in blocking virus infection of cells. This work has implications for both the new drug and the antibodies that are poised to be used against HIV.

Multiple UBXN family members inhibit retrovirus and lentivirus production and canonical NFκΒ signaling by stabilizing IκBα.

UBXN proteins likely participate in the global regulation of protein turnover, and we have shown that UBXN1 interferes with RIG-I-like receptor (RLR) signaling by interacting with MAVS and impeding its downstream effector functions. Here we demonstrate that over-expression of multiple UBXN family members decreased lentivirus and retrovirus production by several orders-of-magnitude in single cycle assays, at the level of long terminal repeat-driven transcription, and three family members, UBXN1, N9, and N11 blocked the canonical NFκB pathway by binding to Cullin1 (Cul1), inhibiting IκBα degradation. Multiple regions of UBXN1, including its UBA domain, were critical for its activity. Elimination of UBXN1 resulted in early murine embryonic lethality. shRNA-mediated knockdown of UBXN1 enhanced human immunodeficiency virus type 1 (HIV) production up to 10-fold in single cycle assays. In primary human fibroblasts, knockdown of UBXN1 caused prolonged degradation of IκBα and enhanced NFκB signaling, which was also observed after CRISPR-mediated knockout of UBXN1 in mouse embryo fibroblasts. Knockout of UBXN1 significantly up- and down-regulated hundreds of genes, notably those of several cell adhesion and immune signaling pathways. Reduction in UBXN1 gene expression in Jurkat T cells latently infected with HIV resulted in enhanced HIV gene expression, consistent with the role of UBXN1 in modulating the NFκB pathway. Based upon co-immunoprecipitation studies with host factors known to bind Cul1, models are presented as to how UBXN1 could be inhibiting Cul1 activity. The ability of UBXN1 and other family members to negatively regulate the NFκB pathway may be important for dampening the host immune response in disease processes and also re-activating quiescent HIV from latent viral reservoirs in chronically infected individuals.

Mechanisms of Virologic Control and Clinical Characteristics of HIV+ Elite/Viremic Controllers.

Human immunodeficiency virus type 1 (HIV-1) disease is pandemic, with approximately 36 million infected individuals world-wide. For the vast majority of these individuals, untreated HIV eventually causes CD4+ T cell depletion and profound immunodeficiency, resulting in morbidity and mortality. But for a remarkable few (0.2 to 0.5 percent), termed elite controllers (ECs), viral loads (VLs) remain suppressed to undetectable levels (< 50 copies/ml) and peripheral CD4+ T cell counts remain high (200 to 1000/µl), all in the absence of antiretroviral therapy (ART). Viremic controllers (VCs) are a similar but larger subset of HIV-1 infected individuals who have the ability to suppress their VLs to low levels. These patients have been intensively studied over the last 10 years in order to determine how they are able to naturally control HIV in the absence of medications, and a variety of mechanisms have been proposed. Defective HIV does not explain the clinical status of most ECs/VCs; rather these individuals appear to somehow control HIV infection, through immune or other unknown mechanisms. Over time, many ECs and VCs eventually lose the ability to control HIV, leading to CD4+ T cell depletion and immunologic dysfunction in the absence of ART. Elucidating novel mechanisms of HIV control in this group of patients will be an important step in understanding HIV infection. This will extend our knowledge of HIV-host interaction and may pave the way for the development of new therapeutic approaches and advance the cure agenda.

Increased Levels of Macrophage Inflammatory Proteins Result in Resistance to R5-Tropic HIV-1 in a Subset of Elite Controllers.

UNLABELLED: Elite controllers (ECs) are a rare group of HIV seropositive individuals who are able to control viral replication without antiretroviral therapy. The mechanisms responsible for this phenotype, however, have not been fully elucidated. In this study, we examined CD4(+) T cell resistance to HIV in a cohort of elite controllers and explored transcriptional signatures associated with cellular resistance. We demonstrate that a subgroup of elite controllers possess CD4(+) T cells that are specifically resistant to R5-tropic HIV while remaining fully susceptible to X4-tropic and vesicular stomatitis virus G (VSV-G)-pseudotyped viruses. Transcriptome analysis revealed 17 genes that were differentially regulated in resistant elite controllers relative to healthy controls. Notably, the genes encoding macrophage inflammatory protein 1α (MIP-1α), CCL3 and CCL3L1, were found to be upregulated. The MIP-1α, MIP-1ß, and RANTES chemokines are natural ligands of CCR5 and are known to interfere with HIV replication. For three elite controllers, we observed increased production of MIP-1α and/or MIP-1ß at the protein level. The supernatant from resistant EC cells contained MIP-1α and MIP-1ß and was sufficient to confer R5-tropic resistance to susceptible CD4(+) T cells. Additionally, this effect was reversed by using inhibitory anti-MIP antibodies. These results suggest that the T cells of these particular elite controllers may be naturally resistant to HIV infection by blocking R5-tropic viral entry. IMPORTANCE: HIV is a pandemic health problem, and the majority of seropositive individuals will eventually progress to AIDS unless antiretroviral therapy (ART) is administered. However, rare patients, termed elite controllers, have a natural ability to control HIV infection in the absence of ART, but the mechanisms by which they achieve this phenotype have not been fully explored. This paper identifies one mechanism that may contribute to this natural resistance: some elite controllers have CD4(+) T cells that produce high levels of MIP chemokines, which block R5-tropic HIV entry. This mechanism could potentially be exploited to achieve a therapeutic effect in other HIV-seropositive individuals.

JAK/STAT signaling pathway affects CCR5 expression in human CD4+ T cells.

CCR5 serves as R5-tropic HIV co-receptor. Knocking out CCR5 in HIV patients, which has occurred <10 times, is believed important for cure. JAK/STAT inhibitors tofacitinib and ruxolitinib inhibit CCR5 expression in HIV+ viremic patients. We investigated the association of JAK/STAT signaling pathway with CCR5/CCR2 expression in human primary CD4+ T cells and confirmed its importance. Six of nine JAK/STAT inhibitors that reduced CCR5/CCR2 expression were identified. Inhibitor-treated CD4+ T cells were relatively resistant, specifically to R5-tropic HIV infection. Furthermore, single JAK2, STAT3, STAT5A, and STAT5B knockout and different combinations of JAK/STAT knockout significantly reduced CCR2/CCR5 expression of both RNA and protein levels, indicating that CCR5/CCR2 expression was positively regulated by JAK-STAT pathway in CD4+ T cells. Serum and glucocorticoid-regulated kinase 1 (SGK1) knockout affected CCR2/CCR5 gene expression, suggesting that SGK1 is involved in CCR2/CCR5 regulation. If cell surface CCR5 levels can be specifically and markedly down-regulated without adverse effects, that may have a major impact on the HIV cure agenda.

Transmembrane protein aptamers that inhibit CCR5 expression and HIV coreceptor function.

We have exploited the ability of transmembrane domains to engage in highly specific protein-protein interactions to construct a new class of small proteins that inhibit HIV infection. By screening a library encoding hundreds of thousands of artificial transmembrane proteins with randomized transmembrane domains (termed "traptamers," for transmembrane aptamers), we isolated six 44- or 45-amino-acid proteins with completely different transmembrane sequences that inhibited cell surface and total expression of the HIV coreceptor CCR5. The traptamers inhibited transduction of human T cells by HIV reporter viruses pseudotyped with R5-tropic gp120 envelope proteins but had minimal effects on reporter viruses with X4-tropic gp120. Optimization of two traptamers significantly increased their activity and resulted in greater than 95% inhibition of R5-tropic reporter virus transduction without inhibiting expression of CD4, the primary HIV receptor, or CXCR4, another HIV coreceptor. In addition, traptamers inhibited transduction mediated by a mutant R5-tropic gp120 protein resistant to maraviroc, a small-molecule CCR5 inhibitor, and they dramatically inhibited replication of an R5-tropic laboratory strain of HIV in a multicycle infection assay. Genetic experiments suggested that the active traptamers specifically interacted with the transmembrane domains of CCR5 and that some of the traptamers interacted with different portions of CCR5. Thus, we have constructed multiple proteins not found in nature that interfere with CCR5 expression and inhibit HIV infection. These proteins may be valuable tools to probe the organization of the transmembrane domains of CCR5 and their relationship to its biological activities, and they may serve as starting points to develop new strategies to inhibit HIV infection.

Correlation of a commercial platform's results with post-vaccination SARS-CoV-2 neutralizing antibody response and clinical host factors.

INTRODUCTION: The objective of this study was to describe the correlation between the commercially available assay for anti-S1/RBD IgG and protective serum neutralizing antibodies (nAb) against SARS-CoV-2 in an adult population after SARS-CoV-2 vaccination, and determine if clinical variables impact this correlation. METHODS: We measured IgG anti-S1/RBD using the IgG-II CMIA assay and nAb IC50 values against SARS-CoV-2 WA-1 in sera serially collected post-mRNA vaccination in veterans and healthcare workers of the Veterans Affairs Connecticut Healthcare System (VACHS) between December 2020 and January 2022. The correlation between IgG and IC50 was measured using Pearson correlation. Clinical variables (age, sex, race, ethnicity, prior COVID infection defined by RT-PCR, history of malignancy, estimated glomerular filtration rate (GFR calculated using CKD-EPI equation) were collected by manual chart review. The impact of these clinical variables on the IgG-nAb correlation was analyzed first with univariable regression. Variables with a significance of p < 0.15 were analyzed with forward stepwise regression analysis. RESULTS: From 127 sera samples in 100 unique subjects (age 20-93 years; mean 63.83; SD 15.63; 29% female; 67% White), we found a robust correlation between IgG anti-S1/RBD and nAb IC50 (R2 = 0.83, R2adj = 0.70, p < 0.0001). Race, ethnicity, and a history of malignancy were not significant on univariable analysis. GFR (p < 0.05) and prior COVID infection (p < 0.001) had a significant impact on the correlation between IgG anti-S1/RBD and nAb IC50. Age (p = 0.06) and sex (p = 0.07) trended towards significance on univariable analysis, but were not significant on multivariable regression. CONCLUSIONS: There was a strong correlation between IgG anti-S1/RBD and nAb IC50 after SARS-CoV-2 vaccination. Clinical comorbidities, such as prior COVID infection and renal function, impacted this correlation. These results may assist the prediction of post-vaccination immune protection in clinical settings using cost-effective commercial platforms.

Cell-based and cell-free firefly luciferase complementation assay to quantify Human Immunodeficiency Virus type 1 Rev-Rev interaction.

Rev is an essential regulatory protein of Human Immunodeficiency Virus type 1 (HIV) that is found in the nucleus of infected cells. Rev multimerizes on the Rev-response element (RRE) of HIV RNA to facilitate the export of intron-containing HIV mRNAs from the nucleus to the cytoplasm, and, as such, HIV cannot replicate in the absence of Rev. We have developed cell-intact and cell-free assays based upon a robust firefly split-luciferase complementation system, both of which quantify Rev-Rev interaction. Using the cell-based system we show that additional Crm1 did not impact the interaction, whereas excess Rev reduced it. Furthermore, when a series of mutant Revs were tested, there was a strong correlation between the results of the cell-based assay and the results of a functional Rev trans-complementation infectivity assay. Of interest, a camelid nanobody (NB) that was known to inhibit Rev function enhanced Rev-Rev interaction in the cell-based system. We observed a similar increase in Rev-Rev interaction in a cell-free system, when cell lysates expressing Rev-NLUC or CLUC-Rev were simply mixed. In the cell-free system Rev-Rev interaction occurred within minutes and was inhibited by excess Rev. The levels of interaction between the mutant Revs tested varied by mutant type. Treatment of Rev lysates with RNAse minimally reduced the degree of interaction whereas addition of HIV RRE RNA enhanced the interaction. Purified GST-Rev protein inhibited the interaction. The Z-factor (Z') for the cell-free system was ∼0.85 when tested in 96-well format, and the anti-Rev NB enhanced the interaction in the cell-free system. Thus, we have developed both cell-intact and cell-free systems that can reliably, rapidly, and reproducibly quantify Rev-Rev interaction. These assays, particularly the cell-free one, may be useful in screening and identifying compounds that inhibit Rev function on a high throughput basis.

Development of an efficient reproducible cell-cell transmission assay for rapid quantification of SARS-CoV-2 spike interaction with hACE2.

Efficient quantitative assays for measurement of viral replication and infectivity are indispensable for future endeavors to develop prophylactic or therapeutic antiviral drugs or vaccines against SARS-CoV-2. We developed a SARS-CoV-2 cell-cell transmission assay that provides a rapid and quantitative readout to assess SARS-CoV-2 spike hACE2 interaction in the absence of pseudotyped particles or live virus. We established two well-behaved stable cell lines, which demonstrated a remarkable correlation with standard cell-free viral pseudotyping for inhibition by convalescent sera, small-molecule drugs, and murine anti-spike monoclonal antibodies. The assay is rapid, reliable, and highly reproducible, without a requirement for any specialized research reagents or laboratory equipment and should be easy to adapt for use in most investigative and clinical settings. It can be effectively used or modified for high-throughput screening for compounds and biologics that interfere with virus-cell binding and entry to complement other neutralization assays currently in use.

Clinical Variables Correlate with Serum Neutralizing Antibody Titers after COVID-19 mRNA Vaccination in an Adult, US-based Population.

Background: We studied whether comorbid conditions impact strength and duration of immune responses after SARS-CoV-2 mRNA vaccination in a US-based, adult population. Methods: Sera (pre-and-post-BNT162b2 vaccination) were tested serially up to 12 months after two doses of vaccine for SARS-CoV-2-anti-Spike neutralizing capacity by pseudotyping assay in 124 individuals; neutralizing titers were correlated to clinical variables with multivariate regression. Post-booster (third dose) effect was measured at 1 and 3 months in 72 and 88 subjects respectively. Results: After completion of primary vaccine series, neutralizing antibody IC50 values were high at one month (14-fold increase from pre-vaccination), declined at six months (3.3-fold increase), and increased at one month post-booster (41.5-fold increase). Three months post-booster, IC50 decreased in COVID-naïve individuals (18-fold increase) and increased in prior COVID-19+ individuals (132-fold increase). Age >65 years (ß=-0.94, p=0.001) and malignancy (ß=-0.88, p=0.002) reduced strength of response at 1 month. Both strength and durability of response at 6 months, respectively, were negatively impacted by end-stage renal disease [(ß=-1.10, p=0.004); (ß=-0.66, p=0.014)], diabetes mellitus [(ß=-0.57, p=0.032); (ß=-0.44, p=0.028)], and systemic steroid use [(ß=-0.066, p=0.032); (ß=-0.55, p=0.037)]. Post-booster IC50 was robust against WA-1 and B.1.617.2, but the immune response decreased with malignancy (ß =-0.68, p=0.03) and increased with prior COVID-19 (p-value < 0.0001). Conclusion: Multiple clinical factors impact the strength and duration of neutralization response post-primary series vaccination, but not the post-booster dose strength. Prior COVID-19 infection enhances the booster-dose response except in individuals with malignancy, suggesting a need for clinically guiding vaccine dosing regimens. Summary: Multiple clinical factors impact the strength and duration of neutralization response post-primary series vaccination. All subjects, irrespective of prior COVID infection, benefited from a third dose. Malignancy decreased response following third dose, suggesting the importance of clinically guided vaccine regimens.

The FDA-Approved Drug Cobicistat Synergizes with Remdesivir To Inhibit SARS-CoV-2 Replication In Vitro and Decreases Viral Titers and Disease Progression in Syrian Hamsters.

Combinations of direct-acting antivirals are needed to minimize drug resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and testing of a number of drug regimens has led to conflicting results. Here, we show that cobicistat, which is an FDA-approved drug booster that blocks the activity of the drug-metabolizing proteins cytochrome P450-3As (CYP3As) and P-glycoprotein (P-gp), inhibits SARS-CoV-2 replication. Two independent cell-to-cell membrane fusion assays showed that the antiviral effect of cobicistat is exerted through inhibition of spike protein-mediated membrane fusion. In line with this, incubation with low-micromolar concentrations of cobicistat decreased viral replication in three different cell lines including cells of lung and gut origin. When cobicistat was used in combination with remdesivir, a synergistic effect on the inhibition of viral replication was observed in cell lines and in a primary human colon organoid. This was consistent with the effects of cobicistat on two of its known targets, CYP3A4 and P-gp, the silencing of which boosted the in vitro antiviral activity of remdesivir in a cobicistat-like manner. When administered in vivo to Syrian hamsters at a high dose, cobicistat decreased viral load and mitigated clinical progression. These data highlight cobicistat as a therapeutic candidate for treating SARS-CoV-2 infection and as a potential building block of combination therapies for COVID-19. IMPORTANCE The lack of effective antiviral treatments against SARS-CoV-2 is a significant limitation in the fight against the COVID-19 pandemic. Single-drug regimens have so far yielded limited results, indicating that combinations of antivirals might be required, as previously seen for other RNA viruses. Our work introduces the drug booster cobicistat, which is approved by the FDA and typically used to potentiate the effect of anti-HIV protease inhibitors, as a candidate inhibitor of SARS-CoV-2 replication. Beyond its direct activity as an antiviral, we show that cobicistat can enhance the effect of remdesivir, which was one of the first drugs proposed for treatment of SARS-CoV-2. Overall, the dual action of cobicistat as a direct antiviral and a drug booster can provide a new approach to design combination therapies and rescue the activity of compounds that are only partially effective in monotherapy.

Notch signaling contributes to the pathogenesis of human osteosarcomas.

Notch signaling plays an important role in developmental processes and adult tissue homeostasis. Altered Notch signaling has been associated with various diseases including cancer. While the importance of altered Notch signaling in cancers of hematopoietic and epithelial origins has been established, its role in tumors of mesenchymal origin is less clear. Here, we report that human osteosarcoma cell lines and primary human osteosarcoma tumor samples show significant up-regulation of Notch, its target genes and Osterix. Notch inhibition by gamma-secretase inhibitors or by using lentiviral mediated expression of dominant negative Mastermind-like protein (DN-MAML) decreases osteosarcoma cell proliferation in vitro. In vivo, established human tumor xenografts in nude mice show decreased tumor growth after chemical or genetic inhibition of Notch signaling. Finally, transcriptional profiling of osteosarcomas from p53 mutant mice confirmed up-regulation of Notch1 target genes Hes1, Hey1 and its ligand Dll4. Our data suggest that activation of Notch signaling contributes to the pathogenesis of human osteosarcomas and its inhibition may be a therapeutic approach for the treatment of this mesenchymal tumor.

Functional pseudotyping of human immunodeficiency virus type 1 vectors by Western equine encephalitis virus envelope glycoprotein.

We investigated the ability of western equine encephalitis virus envelope glycoproteins (WEEV GP) to pseudotype lentiviral vectors. The titers of WEEV GP-pseudotyped human immunodeficiency virus type 1 (HIV) ranged as high as 8.0 x 10(4) IU/ml on permissive cells. Sera from WEEV-infected mice specifically neutralized these pseudotypes; cell transduction was also sensitive to changes in pH. The host range of the pseudotyped particles in vitro was somewhat limited, which is atypical for most alphaviruses. HIV vectors pseudotyped by WEEV GP may be a useful tool for characterizing WEEV cell binding and entry and screening for small-molecule inhibitors.

Titers of HIV-based vectors encoding shRNAs are reduced by a dicer-dependent mechanism.

Gene transfer vectors encoding short hairpin RNAs (shRNAs) are useful in deciphering gene function, and are being considered for therapeutic knockdown of target genes in humans. We constructed HIV-based vectors encoding shRNA against HIV coreceptor chemokine (C-C motif) receptor 5 (CCR5). Initially we noted that vectors encoding CCR5 shRNA showed >30-fold lower viral titers than those of the empty vector. Co-transfection of expression plasmids encoding CCR5 in the producer cells yielded a tenfold increase in viral titer, thereby indicating that CCR5 mRNA, rather than HIV vector mRNA, could be the target of CCR5 shRNA. Similar increases in vector titer were observed after the H1 promoter was deleted. When Nodamura-virus B2 protein or Adenovirus VA.1 RNA (inhibitors of the Dicer-dependent siRNA pathway) were added to the producer cells, the vector titer rose almost to the level of that of the empty vector. Near identical increases in titer were observed with siRNA specifically directed against Dicer. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) suggested that the effects were in part caused by reduction in vector RNA in the producer cells. Similar results were observed with a retroviral vector. These results suggest that retrovirally-encoded shRNAs reduce vector titer in the producer cells through a Dicer-dependent mechanism which, to a large extent, can be reversed by inhibiting that pathway. This may have important implications for large-scale production of RNA vectors encoding shRNAs.

Transcriptional down-regulation of ccr5 in a subset of HIV+ controllers and their family members.

HIV +Elite and Viremic controllers (EC/VCs) are able to control virus infection, perhaps because of host genetic determinants. We identified 16% (21 of 131) EC/VCs with CD4 +T cells with resistance specific to R5-tropic HIV, reversed after introduction of ccr5. R5 resistance was not observed in macrophages and depended upon the method of T cell activation. CD4 +T cells of these EC/VCs had lower ccr2 and ccr5 RNA levels, reduced CCR2 and CCR5 cell-surface expression, and decreased levels of secreted chemokines. T cells had no changes in chemokine receptor mRNA half-life but instead had lower levels of active transcription of ccr2 and ccr5, despite having more accessible chromatin by ATAC-seq. Other nearby genes were also down-regulated, over a region of ~500 kb on chromosome 3p21. This same R5 resistance phenotype was observed in family members of an index VC, also associated with ccr2/ccr5 down-regulation, suggesting that the phenotype is heritable.

Differential interaction between human and murine Crm1 and lentiviral Rev proteins.

Mice have multiple obstacles to HIV replication, including a block of unspliced and partially spliced viral mRNA nuclear export. In human, Rev binds to the Rev-response element and human (h) Crm1, facilitating nuclear export of RRE-containing viral RNAs. Murine (m) Crm1 is less functional than hCrm1 in this regard. Here we demonstrated that in biochemical experiments mCrm1 failed to interact with HIV Rev whereas hCrm1 did. In genetic experiments in human cells, we observed a modest but significant differential effect between mCrm1 and hCrm1, which was also true of other lentiviral Revs tested. Triple mutant hCrm1 P411T-M412V-F414S behaved similarly to mCrm1, whereas mCrm1 with T411P-V412M-S414F regained some activity, although contribution of additional residues to its function can not be excluded. Similar results were observed in murine cells. This suggests a differential interaction between hCrm1 and mCrm1 and many lentiviral Revs, which may partially explain the HIV replicative defect in mice.

Brief Report: Identification of Elite and Viremic Controllers From a Large Urban HIV Ambulatory Center in Kampala, Uganda.

BACKGROUND: Throughout the world, there are antiretroviral therapy-naive HIV+ individuals who maintain elevated peripheral CD4 T-cell counts, historically referred to as long-term nonprogressors (LTNPs). With recent improvements in viral load (VL) detection methods to levels as low as 20 copies per milliliter, 2 subsets of LTNPs have been defined: elite controllers (ECs), with undetectable VLs for at least 6-12 months, and viremic controllers (VCs), with VLs between 200 and 2000 copies per milliliter. ECs and VCs have been extensively studied in the developed world to determine underlying mechanisms responsible for virologic control. In sub-Saharan Africa, most studies have characterized LTNPs based on immunologic criteria making it difficult to compare findings with the Western cohorts, which use virologic criteria. Here, we describe a cohort of Uganda ECs and VCs attending a large HIV ambulatory center in Kampala, Uganda, based initially on CD4 counts and confirmed by repeated VL measurements. METHODS: A cross-sectional study was conducted among 14,492 HIV-infected, antiretroviral therapy-naive individuals aged 18 years and older under care for at least 5 years with serial peripheral CD4 counts ≥500 cells/µL. Among those, we determined the frequency of individuals with VLs <2000 copies per milliliter for at least 6 months. RESULTS: We report a prevalence of 0.26% (38/14,492) of HIV controllers in the clinic. We identified 36 ECs and 2 VCs. These individuals were middle-aged with an average CD4 count of 858 ± 172 (mean ± SD, 95% confidence interval: 795 to 921). Their average duration in HIV care was 7.4 ± 2.1 years (mean ± SD, 95% confidence interval: 6.6 to 8.1). The majority of EC/VCs were women (87%, 33/38), reflecting the demographics of the urban clinic. CONCLUSIONS: For the first time, this study demonstrates the frequency of EC/VCs in a large urban clinic in Uganda. Further study of these East African subjects may provide insights into how some individuals are able to control HIV in the absence of medications.

Aspergillus fumigatus native valve infective endocarditis in an otherwise healthy adult.

INTRODUCTION: Fungal endocarditis is a rare cause of infective endocarditis, and Aspergillus spp. account for up to 30 % of all cases. Risk factors include intravenous drug use, immunosuppression, malignancy and the presence of prosthetic valves. CASE PRESENTATION: We present a case of A. fumigatus endocarditis in a patient without any known or described risk factors. CONCLUSION: Diagnosis of Aspergillus endocarditis requires a high clinical index of suspicion, given the initial non-specific presentation, and treatment may require both medical and surgical therapies to ensure improved outcomes, but mortality rates still approach 80 %. Voriconazole remains the antifungal agent of choice.