Centers for AIDS Research (CFAR) Diversity, Equity, and Inclusion Pathway Initiative (CDEIPI): Developing Career Pathways for Early-Stage Scholars From Racial and Ethnic Groups Underrepresented in HIV Science and Medicine.

BACKGROUND: There is an urgent need to increase diversity among scientific investigators in the HIV research field to be more reflective of communities highly affected by the HIV epidemic. Thus, it is critical to promote the inclusion and advancement of early-stage scholars from racial and ethnic groups underrepresented in HIV science and medicine. METHODS: To widen the HIV research career pathway for early-stage scholars from underrepresented minority groups, the National Institutes of Health supported the development of the Centers for AIDS Research (CFAR) Diversity, Equity, and Inclusion Pathway Initiative (CDEIPI). This program was created through partnerships between CFARs and Historically Black Colleges and Universities and other Minority Serving Institutions throughout the United States. RESULTS: Seventeen CFARs and more than 20 Historically Black Colleges and Universities and Minority Serving Institutions have participated in this initiative to date. Programs were designed for the high school (8), undergraduate (13), post baccalaureate (2), graduate (12), and postdoctoral (4) levels. Various pedagogical approaches were used including didactic seminar series, intensive multiday workshops, summer residential programs, and mentored research internship opportunities. During the first 18 months of the initiative, 257 student scholars participated in CDEIPI programs including 150 high school, 73 undergraduate, 3 post baccalaureate, 27 graduate, and 4 postdoctoral students. CONCLUSION: Numerous student scholars from a wide range of educational levels, geographic backgrounds, and racial and ethnic minority groups have engaged in CDEIPI programs. Timely and comprehensive program evaluation data will be critical to support a long-term commitment to this unique training initiative.

Glycan-dependent HIV-specific neutralizing antibodies bind to cells of uninfected individuals.

A number of highly potent and broadly neutralizing antibodies (bNAbs) against the human immunodeficiency virus (HIV) have recently been shown to prevent transmission of the virus, suppress viral replication, and delay plasma viral rebound following discontinuation of antiretroviral therapy in animal models and infected humans. However, the degree and extent to which such bNAbs interact with primary lymphocytes have not been fully delineated. Here, we show that certain glycan-dependent bNAbs, such as PGT121 and PGT151, bind to B, activated T, and natural killer (NK) cells of HIV-infected and -uninfected individuals. Binding of these bNAbs, particularly PGT121 and PGT151, to activated CD4+ and CD8+ T cells was mediated by complex-type glycans and was abrogated by enzymatic inhibition of N-linked glycosylation. In addition, a short-term incubation of PGT151 and primary NK cells led to degranulation and cellular death. Our data suggest that the propensity of certain bNAbs to bind uninfected/bystander cells has the potential for unexpected outcomes in passive-transfer studies and underscore the importance of antibody screening against primary lymphocytes.

Highlights from the Fourth Biennial Strategies for an HIV Cure Meeting, 10-12 October 2018, Bethesda, MD, USA.

The National Institute of Allergy and Infectious Diseases (NIAID) organised the Strategies for an HIV Cure 2018 meeting focused on research to develop innovative strategies for eradicating or achieving long-term remission of HIV infection. The purpose was to bring together researchers studying HIV persistence and cure strategies, including the six National Institutes of Health (NIH)-funded Martin Delaney Collaboratories for HIV Cure Research (MDCs), as well as industry and community partners, to share scientific results and stimulate active discussion among all stakeholders about the merits of various approaches under investigation. These discussions were intended to stimulate new collaborations and ideas for future research. The meeting covered a comprehensive range of topics spanning basic and translational research, drug discovery and development, and clinical research. Aside from the oral presentations described here, the meeting also included 130 poster presentations. Each of the three days of presentations is available for viewing via the NIH VideoCast website at: https://videocast.nih.gov/PastEvents.asp.

Effect of analytical treatment interruption and reinitiation of antiretroviral therapy on HIV reservoirs and immunologic parameters in infected individuals.

Therapeutic strategies aimed at achieving antiretroviral therapy (ART)-free HIV remission in infected individuals are under active investigation. Considering the vast majority of HIV-infected individuals experience plasma viral rebound upon cessation of therapy, clinical trials evaluating the efficacy of curative strategies would likely require inclusion of ART interruption. However, it is unclear what impact short-term analytical treatment interruption (ATI) and subsequent reinitiation of ART have on immunologic and virologic parameters of HIV-infected individuals. Here, we show a significant increase of HIV burden in the CD4+ T cells of infected individuals during ATI that was correlated with the level of plasma viral rebound. However, the size of the HIV reservoirs as well as immune parameters, including markers of exhaustion and activation, returned to pre-ATI levels 6-12 months after the study participants resumed ART. Of note, the proportions of near full-length, genome-intact and structurally defective HIV proviral DNA sequences were similar prior to ATI and following reinitiation of ART. In addition, there was no evidence of emergence of antiretroviral drug resistance mutations within intact HIV proviral DNA sequences following reinitiation of ART. These data demonstrate that short-term ATI does not necessarily lead to expansion of the persistent HIV reservoir nor irreparable damages to the immune system in the peripheral blood, warranting the inclusion of ATI in future clinical trials evaluating curative strategies.

A randomized controlled safety/efficacy trial of therapeutic vaccination in HIV-infected individuals who initiated antiretroviral therapy early in infection.

Despite substantial clinical benefits, complete eradication of HIV has not been possible using antiretroviral therapy (ART) alone. Strategies that can either eliminate persistent viral reservoirs or boost host immunity to prevent rebound of virus from these reservoirs after discontinuation of ART are needed; one possibility is therapeutic vaccination. We report the results of a randomized, placebo-controlled trial of a therapeutic vaccine regimen in patients in whom ART was initiated during the early stage of HIV infection and whose immune system was anticipated to be relatively intact. The objectives of our study were to determine whether the vaccine was safe and could induce an immune response that would maintain suppression of plasma viremia after discontinuation of ART. Vaccinations were well tolerated with no serious adverse events but produced only modest augmentation of existing HIV-specific CD4+ T cell responses, with little augmentation of CD8+ T cell responses. Compared with placebo, the vaccination regimen had no significant effect on the kinetics or magnitude of viral rebound after interruption of ART and no impact on the size of the HIV reservoir in the CD4+ T cell compartment. Notably, 26% of subjects in the placebo arm exhibited sustained suppression of viremia (<400 copies/ml) after treatment interruption, a rate of spontaneous suppression higher than previously reported. Our findings regarding the degree and kinetics of plasma viral rebound after ART interruption have potentially important implications for the design of future trials testing interventions aimed at achieving ART-free control of HIV infection.

Effect of HIV Antibody VRC01 on Viral Rebound after Treatment Interruption.

BACKGROUND: The discovery of potent and broadly neutralizing antibodies (bNAbs) against human immunodeficiency virus (HIV) has made passive immunization a potential strategy for the prevention and treatment of HIV infection. We sought to determine whether passive administration of VRC01, a bNAb targeting the HIV CD4-binding site, can safely prevent or delay plasma viral rebound after the discontinuation of antiretroviral therapy (ART). METHODS: We conducted two open-label trials (AIDS Clinical Trials Group [ACTG] A5340 and National Institutes of Health [NIH] 15-I-0140) of the safety, side-effect profile, pharmacokinetic properties, and antiviral activity of VRC01 in persons with HIV infection who were undergoing interruption of ART. RESULTS: A total of 24 participants were enrolled, and one serious alcohol-related adverse event occurred. Viral rebound occurred despite plasma VRC01 concentrations greater than 50 µg per milliliter. The median time to rebound was 4 weeks in the A5340 trial and 5.6 weeks in the NIH trial. Study participants were more likely than historical controls to have viral suppression at week 4 (38% vs. 13%, P=0.04 by a two-sided Fisher's exact test in the A5340 trial; and 80% vs. 13%, P<0.001 by a two-sided Fisher's exact test in the NIH trial) but the difference was not significant at week 8. Analyses of virus populations before ART as well as before and after ART interruption showed that VRC01 exerted pressure on rebounding virus, resulting in restriction of recrudescent viruses and selection for preexisting and emerging antibody neutralization-resistant virus. CONCLUSIONS: VRC01 slightly delayed plasma viral rebound in the trial participants, as compared with historical controls, but it did not maintain viral suppression by week 8. In the small number of participants enrolled in these trials, no safety concerns were identified with passive immunization with a single bNAb (VRC01). (Funded by the National Institute of Allergy and Infectious Diseases and others; ACTG A5340 and NIH 15-I-0140 ClinicalTrials.gov numbers, NCT02463227 and NCT02471326 .).

Natural polymorphisms in human APOBEC3H and HIV-1 Vif combine in primary T lymphocytes to affect viral G-to-A mutation levels and infectivity.

The Vif protein of HIV-1 allows virus replication by degrading several members of the host-encoded APOBEC3 family of DNA cytosine deaminases. Polymorphisms in both host APOBEC3 genes and the viral vif gene have the potential to impact the extent of virus replication among individuals. The most genetically diverse of the seven human APOBEC3 genes is APOBEC3H with seven known haplotypes. Overexpression studies have shown that a subset of these variants express stable and active proteins, whereas the others encode proteins with a short half-life and little, if any, antiviral activity. We demonstrate that these stable/unstable phenotypes are an intrinsic property of endogenous APOBEC3H proteins in primary CD4+ T lymphocytes and confer differential resistance to HIV-1 infection in a manner that depends on natural variation in the Vif protein of the infecting virus. HIV-1 with a Vif protein hypo-functional for APOBEC3H degradation, yet fully able to counteract APOBEC3D, APOBEC3F, and APOBEC3G, was susceptible to restriction and hypermutation in stable APOBEC3H expressing lymphocytes, but not in unstable APOBEC3H expressing lymphocytes. In contrast, HIV-1 with hyper-functional Vif counteracted stable APOBEC3H proteins as well as all other endogenous APOBEC3s and replicated to high levels. We also found that APOBEC3H protein levels are induced over 10-fold by infection. Finally, we found that the global distribution of stable/unstable APOBEC3H haplotypes correlates with the distribution a critical hyper/hypo-functional Vif amino acid residue. These data combine to strongly suggest that stable APOBEC3H haplotypes present as in vivo barriers to HIV-1 replication, that Vif is capable of adapting to these restrictive pressures, and that an evolutionary equilibrium has yet to be reached.

The APOBEC3 family of retroelement restriction factors.

The ability to regulate and even target mutagenesis is an extremely valuable cellular asset. Enzyme-catalyzed DNA cytosine deamination is a molecular strategy employed by vertebrates to promote antibody diversity and defend against foreign nucleic acids. Ten years ago, a family of cellular enzymes was first described with several proving capable of deaminating DNA and inhibiting HIV-1 replication. Ensuing studies on the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) restriction factors have uncovered a broad-spectrum innate defense network that suppresses the replication of numerous endogenous and exogenous DNA-based parasites. Although many viruses possess equally elaborate counter-defense mechanisms, the APOBEC3 enzymes offer a tantalizing possibility of leveraging innate immunity to fend off viral infection. Here, we focus on mechanisms of retroelement restriction by the APOBEC3 family of restriction enzymes, and we consider the therapeutic benefits, as well as the possible pathological consequences, of arming cells with active DNA deaminases.

APOBEC3B is an enzymatic source of mutation in breast cancer.

Several mutations are required for cancer development, and genome sequencing has revealed that many cancers, including breast cancer, have somatic mutation spectra dominated by C-to-T transitions. Most of these mutations occur at hydrolytically disfavoured non-methylated cytosines throughout the genome, and are sometimes clustered. Here we show that the DNA cytosine deaminase APOBEC3B is a probable source of these mutations. APOBEC3B messenger RNA is upregulated in most primary breast tumours and breast cancer cell lines. Tumours that express high levels of APOBEC3B have twice as many mutations as those that express low levels and are more likely to have mutations in TP53. Endogenous APOBEC3B protein is predominantly nuclear and the only detectable source of DNA C-to-U editing activity in breast cancer cell-line extracts. Knockdown experiments show that endogenous APOBEC3B correlates with increased levels of genomic uracil, increased mutation frequencies, and C-to-T transitions. Furthermore, induced APOBEC3B overexpression causes cell cycle deviations, cell death, DNA fragmentation, γ-H2AX accumulation and C-to-T mutations. Our data suggest a model in which APOBEC3B-catalysed deamination provides a chronic source of DNA damage in breast cancers that could select TP53 inactivation and explain how some tumours evolve rapidly and manifest heterogeneity.

Endogenous origins of HIV-1 G-to-A hypermutation and restriction in the nonpermissive T cell line CEM2n.

The DNA deaminase APOBEC3G converts cytosines to uracils in retroviral cDNA, which are immortalized as genomic strand G-to-A hypermutations by reverse transcription. A single round of APOBEC3G-dependent mutagenesis can be catastrophic, but evidence suggests that sublethal levels contribute to viral genetic diversity and the associated problems of drug resistance and immune escape. APOBEC3G exhibits an intrinsic preference for the second cytosine in a 5'CC dinucleotide motif leading to 5'GG-to-AG mutations. However, an additional hypermutation signature is commonly observed in proviral sequences from HIV-1 infected patients, 5'GA-to-AA, and it has been attributed controversially to one or more of the six other APOBEC3 deaminases. An unambiguous resolution of this problem has been difficult to achieve, in part due to dominant effects of protein over-expression. Here, we employ gene targeting to dissect the endogenous APOBEC3 contribution to Vif-deficient HIV-1 restriction and hypermutation in a nonpermissive T cell line CEM2n. We report that APOBEC3G-null cells, as predicted from previous studies, lose the capacity to inflict 5'GG-to-AG mutations. In contrast, APOBEC3F-null cells produced viruses with near-normal mutational patterns. Systematic knockdown of other APOBEC3 genes in an APOBEC3F-null background revealed a significant contribution from APOBEC3D in promoting 5'GA-to-AA hypermutations. Furthermore, Vif-deficient HIV-1 restriction was strong in parental CEM2n and APOBEC3D-knockdown cells, partially alleviated in APOBEC3G- or APOBEC3F-null cells, further alleviated in APOBEC3F-null/APOBEC3D-knockdown cells, and alleviated to the greatest extent in APOBEC3F-null/APOBEC3G-knockdown cells revealing clear redundancy in the HIV-1 restriction mechanism. We conclude that endogenous levels of APOBEC3D, APOBEC3F, and APOBEC3G combine to restrict Vif-deficient HIV-1 and cause the hallmark dinucleotide hypermutation patterns in CEM2n. Primary T lymphocytes express a similar set of APOBEC3 genes suggesting that the same repertoire may be important in vivo.

Human and rhesus APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H demonstrate a conserved capacity to restrict Vif-deficient HIV-1.

Successful intracellular pathogens must evade or neutralize the innate immune defenses of their host cells and render the cellular environment permissive for replication. For example, to replicate efficiently in CD4(+) T lymphocytes, human immunodeficiency virus type 1 (HIV-1) encodes a protein called viral infectivity factor (Vif) that promotes pathogenesis by triggering the degradation of the retrovirus restriction factor APOBEC3G. Other APOBEC3 proteins have been implicated in HIV-1 restriction, but the relevant repertoire remains ambiguous. Here we present the first comprehensive analysis of the complete, seven-member human and rhesus APOBEC3 families in HIV-1 restriction. In addition to APOBEC3G, we find that three other human APOBEC3 proteins, APOBEC3D, APOBEC3F, and APOBEC3H, are all potent HIV-1 restriction factors. These four proteins are expressed in CD4(+) T lymphocytes, are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, mutate proviral DNA, and are counteracted by HIV-1 Vif. Furthermore, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H of the rhesus macaque also are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, and they are all neutralized by the simian immunodeficiency virus Vif protein. On the other hand, neither human nor rhesus APOBEC3A, APOBEC3B, nor APOBEC3C had a significant impact on HIV-1 replication. These data strongly implicate a combination of four APOBEC3 proteins--APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H--in HIV-1 restriction.

Quantitative proteomics reveals myosin and actin as promising saliva biomarkers for distinguishing pre-malignant and malignant oral lesions.

BACKGROUND: Oral cancer survival rates increase significantly when it is detected and treated early. Unfortunately, clinicians now lack tests which easily and reliably distinguish pre-malignant oral lesions from those already transitioned to malignancy. A test for proteins, ones found in non-invasively-collected whole saliva and whose abundances distinguish these lesion types, would meet this critical need. METHODOLOGY/PRINCIPAL FINDINGS: To discover such proteins, in a first-of-its-kind study we used advanced mass spectrometry-based quantitative proteomics analysis of the pooled soluble fraction of whole saliva from four subjects with pre-malignant lesions and four with malignant lesions. We prioritized candidate biomarkers via bioinformatics and validated selected proteins by western blotting. Bioinformatic analysis of differentially abundant proteins and initial western blotting revealed increased abundance of myosin and actin in patients with malignant lesions. We validated those results by additional western blotting of individual whole saliva samples from twelve other subjects with pre-malignant oral lesions and twelve with malignant oral lesions. Sensitivity/specificity values for distinguishing between different lesion types were 100%/75% (p = 0.002) for actin, and 67%/83% (p<0.00001) for myosin in soluble saliva. Exfoliated epithelial cells from subjects' saliva also showed increased myosin and actin abundance in those with malignant lesions, linking our observations in soluble saliva to abundance differences between pre-malignant and malignant cells. CONCLUSIONS/SIGNIFICANCE: Salivary actin and myosin abundances distinguish oral lesion types with sensitivity and specificity rivaling other non-invasive oral cancer tests. Our findings provide a promising starting point for the development of non-invasive and inexpensive salivary tests to reliably detect oral cancer early.

Quantitative profiling of the full APOBEC3 mRNA repertoire in lymphocytes and tissues: implications for HIV-1 restriction.

The human APOBEC3 proteins are DNA cytidine deaminases that impede the replication of many different transposons and viruses. The genes that encode APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G and APOBEC3H were generated through relatively recent recombination events. The resulting high degree of inter-relatedness has complicated the development of specific quantitative PCR assays for these genes despite considerable interest in understanding their expression profiles. Here, we describe a set of quantitative PCR assays that specifically measures the mRNA levels of each APOBEC3 gene. The specificity and sensitivity of each assay was validated using a full matrix of APOBEC3 cDNA templates. The assays were used to quantify the APOBEC3 repertoire in multiple human T-cell lines, bulk leukocytes and leukocyte subsets, and 20 different human tissues. The data demonstrate that multiple APOBEC3 genes are expressed constitutively in most types of cells and tissues, and that distinct APOBEC3 genes are induced upon T-cell activation and interferon treatment. These data help define the APOBEC3 repertoire relevant to HIV-1 restriction in T cells, and they suggest a general model in which multiple APOBEC3 proteins function together to provide a constitutive barrier to foreign genetic elements, which can be fortified by transcriptional induction.

Interactions among DNA ligase I, the flap endonuclease and proliferating cell nuclear antigen in the expansion and contraction of CAG repeat tracts in yeast.

Among replication mutations that destabilize CAG repeat tracts, mutations of RAD27, encoding the flap endonuclease, and CDC9, encoding DNA ligase I, increase the incidence of repeat tract expansions to the greatest extent. Both enzymes bind to proliferating cell nuclear antigen (PCNA). To understand whether weakening their interactions leads to CAG repeat tract expansions, we have employed alleles named rad27-p and cdc9-p that have orthologous alterations in their respective PCNA interaction peptide (PIP) box. Also, we employed the PCNA allele pol30-90, which has changes within its hydrophobic pocket that interact with the PIP box. All three alleles destabilize a long CAG repeat tract and yield more tract contractions than expansions. Combining rad27-p with cdc9-p increases the expansion frequency above the sum of the numbers recorded in the individual mutants. A similar additive increase in tract expansions occurs in the rad27-p pol30-90 double mutant but not in the cdc9-p pol30-90 double mutant. The frequency of contractions rises in all three double mutants to nearly the same extent. These results suggest that PCNA mediates the entry of the flap endonuclease and DNA ligase I into the process of Okazaki fragment joining, and this ordered entry is necessary to prevent CAG repeat tract expansions.