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We report a 23% asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) Omicron carriage rate in participants being enrolled into a clinical trial in South Africa, 15-fold higher than in trials before Omicron. We also found lower CD4 + T-cell counts in persons with human immunodeficiency virus (HIV) strongly correlated with increased odds of being SARS-CoV-2 polymerase chain reaction (PCR) positive.

Many new TB vaccine candidates in the development pipeline need to be studied in people with HIV. People with HIV are at high risk of developing Mycobacterium tuberculosis (Mtb) infection and TB disease and tend to develop less robust vaccine induced immune responses. Many questions remain unanswered regarding priority vaccine indications, clinical trial design, measures of safety, immunogenicity, and efficacy considerations for people with HIV. To address these gaps, a roadmap for developing TB vaccines for people with HIV was developed through a series of symposia that posed framing questions to a panel of international experts for discussion. Framing questions specific to people with HIV included: 1) What is the use case or rationale for developing TB vaccines?; 2) What is the landscape of TB vaccines?; 3) Which vaccine candidates should be prioritized ?; 4) What are the TB vaccine trial design considerations?; 5) What is the role of immunological correlates of protection?; and 6) What are the gaps in preclinical models for studying TB vaccines? We provide a summary of our roadmap with the intention of informing TB vaccine development and the prioritization of clinical trials for inclusion of people with HIV.

New tuberculosis vaccine candidates that are in the development pipeline need to be studied in people with HIV, who are at high risk of acquiring Mycobacterium tuberculosis infection and tuberculosis disease and tend to develop less robust vaccine-induced immune responses. To address the gaps in developing tuberculosis vaccines for people with HIV, a series of symposia was held that posed six framing questions to a panel of international experts: What is the use case or rationale for developing tuberculosis vaccines? What is the landscape of tuberculosis vaccines? Which vaccine candidates should be prioritised? What are the tuberculosis vaccine trial design considerations? What is the role of immunological correlates of protection? What are the gaps in preclinical models for studying tuberculosis vaccines? The international expert panel formulated consensus statements to each of the framing questions, with the intention of informing tuberculosis vaccine development and the prioritisation of clinical trials for inclusion of people with HIV.

The remarkable success of the US government-backed COVID-19 vaccine development in 2020 offers several lessons on how to effectively foster rapid vaccine discovery and development. Conceptually, the formation of a public-private partnership that included innovative government and academic involvement at all levels of the program was instrumental in promulgating and overseeing the effort. Decades of NIH-sponsored research on vaccine backbones, immunogen design, and clinical trial operations enabled evaluation of vaccine candidates within months of pathogen discovery. Operation Warp Speed fostered industry participation, permitted accelerated movement from preclinical/early phase to efficacy trials, and developed structured clinical trial testing that allowed independent assessment of, yet reasonable comparison between, each vaccine platform by harmonizing protocols, endpoints, laboratories, and statistical analytical criteria for efficacy. This coordinated effort by the US government, pharmaceutical companies, regulators, and academic research institutions resulted in the streamlined, safe, and transparent development and deployment of multiple COVID-19 vaccines in under a year. Lessons learned from this collaborative endeavor should be used to advance additional vaccines of public health importance.

Introduction The last 12 years have seen remarkable progress in the isolation and characterization of at least five different epitope classes of HIV‐specific broadly neutralizing antibodies (bnAbs). Detailed analyses of these bnAb lineages, maturation pathways and epitopes have created new opportunities for vaccine development. In addition, interest exists in passive administration of monoclonal antibodies as a viable option for HIV prevention. Discussion Recently, two antibody‐mediated prevention (AMP) trials of a passively administered monoclonal antibody targeting the HIV envelope CD4 binding site, called VRC01, provided proof‐of‐concept that monoclonal antibody infusion could offer protection against HIV acquisition. While the trials failed to show overall protection against HIV acquisition, sub‐analyses revealed that VRC01 infusion provided a 75% prevention efficacy against HIV strains that were susceptible to the antibody. The study also demonstrated that in vitro neutralizing activity, measured by the TZM‐bl/pseudovirus assay, was able to predict HIV prevention efficacy in humans. In addition, the AMP trials defined a threshold protective concentration, or neutralization titer, for the VRC01 class of bnAbs, explaining the observed low overall efficacy and serving as a benchmark for the clinical testing of new bnAbs, bnAb cocktails and neutralizing antibody‐inducing vaccines. Newer bnAbs that exhibit greater potency and breadth of neutralization in vitro than VRC01 are available for clinical testing. Combinations of best‐in‐class bnAbs with complementary magnitude, breadth and extent of complete neutralization are predicted to far exceed the prevention efficacy of VRC01. Some engineered bi‐ and trispecific mAbs exhibit similar desirable neutralizing activity and afford advantages for manufacturing and delivery. Modifications that prolong the serum half‐life and improve genital tissue persistence offer additional advantages. Conclusions Iterative phase 1 trials are acquiring safety and pharmacokinetic data on dual and triple bnAbs and bi‐ and trispecific antibodies in preparation for future AMP studies that seek to translate findings from the VRC01 efficacy trials and achieve acceptable levels of overall prevention efficacy.

Determinants of protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection require the development of well-standardized, reproducible antibody assays. This need has led to the emergence of a variety of neutralization assays. Head-to-head evaluation of different SARS-CoV-2 neutralization platforms could facilitate comparisons across studies and laboratories. Five neutralization assays were compared using 40 plasma samples from convalescent individuals with mild to moderate coronavirus disease 2019 (COVID-19): four cell-based systems using either live recombinant SARS-CoV-2 or pseudotyped viral particles created with lentivirus (LV) or vesicular stomatitis virus (VSV) packaging and one surrogate enzyme-linked immunosorbent assay (ELISA)-based test that measures inhibition of the spike protein receptor binding domain (RBD) binding its receptor human angiotensin converting enzyme 2 (hACE2). Vero cells, Vero E6 cells, HEK293T cells expressing hACE2, and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 were tested. All cell-based assays showed 50% neutralizing dilution (ND50) geometric mean titers (GMTs) that were highly correlated (Pearson r = 0.81 to 0.89) and ranged within 3.4-fold. The live virus assay and LV pseudovirus assays with HEK293T/hACE2 cells showed very similar mean titers, 141 and 178, respectively. ND50 titers positively correlated with plasma IgG targeting SARS-CoV-2 spike protein and RBD (r = 0.63 to 0.89), but moderately correlated with nucleoprotein IgG (r = 0.46 to 0.73). ND80 GMTs mirrored ND50 data and showed similar correlation between assays and with IgG concentrations. The VSV pseudovirus assay and LV pseudovirus assay with HEK293T/hACE2 cells in low- and high-throughput versions were calibrated against the WHO SARS-CoV-2 IgG standard. High concordance between the outcomes of cell-based assays with live and pseudotyped virions enables valid cross-study comparison using these platforms.

Background Eliciting durable humoral immunity with sufficient breadth and magnitude is important for HIV-1 vaccine design. The HVTN 114 vaccine trial evaluated different boost regimens administered after a 7-year rest period in participants previously enrolled in HVTN 205, who received either three MVA/HIV62B (MMM) or two DNA and two MVA/HIV62B (DDMM) injections; both vaccines expressed multiple HIV-1 antigens in non-infectious virus-like-particles. The primary objective of HVTN 114 was to assess the impact of a heterologous gp120 protein AIDSVAX B/E boost on the magnitude, breadth and durability of vaccine-induced immune responses. Methods We enrolled 27 participants from HVTN 205 into five groups. Eight participants who previously received MMM were randomized and boosted with either MVA/HIV62B alone (T1; n = 4) or MVA/HIV62B and AIDSVAX B/E (T2; n = 4). Nineteen participants who received DDMM were randomized and boosted with MVA/HIV62B alone (T3; n = 6), MVA/HIV62B and AIDSVAX B/E (T4; n = 6), or AIDSVAX B/E alone (T5; n = 7). Boosts were at months 0 and 4. Participants were followed for safety and immunogenicity for 10 months and were pooled for analysis based on the regimen: MVA-only (T1 + T3), MVA + AIDSVAX (T2 + T4), and AIDSVAX-only (T5). Results All regimens were safe and well-tolerated. Prior to the boost vaccination, binding antibody and CD4+T-cell responses were observed 7 years after HVTN 205 vaccinations. Late boosting with AIDSVAX, with or without MVA, resulted in high binding antibody responses to gp120 and V1V2 epitopes, with increased magnitude and breadth compared to those observed in HVTN 205. Late boosting with MVA, with or without AIDSVAX, resulted in increased gp140 and gp41 antibody responses and higher CD4+T-cell responses to Env and Gag. Conclusions Late boosting with AIDSVAX, alone or in combination with MVA, can broaden binding antibody responses and increase T-cell responses even years following the original MVA/HIV62B with or without DNA-priming vaccine.

Background HIV Vaccine Trials Network 703/HIV Prevention Trials Network 081 is a phase 2b randomized, double-blind, placebo-controlled trial to assess the safety and efficacy of passively infused monoclonal antibody VRC01 in preventing HIV acquisition in heterosexual women between the ages of 18 and 50 years at risk of HIV. Participants were enrolled at 20 sites in Botswana, Kenya, Malawi, Mozambique, South Africa, Tanzania, and Zimbabwe. It is one of the 2 Antibody Mediated Prevention efficacy trials, with HIV Vaccine Trials Network 704/HIV Prevention Trials Network 085, evaluating VRC01 for HIV prevention. Methods Intense community engagement was used to optimize participant recruitment and retention. Participants were randomly assigned to receive intravenous VRC01 10 mg/kg, VRC01 30 mg/kg, or placebo in a 1:1:1 ratio. Infusions were given every 8 weeks with a total of 10 infusions and 104 weeks of follow-up after the first infusion. Results Between May 2016 and September 2018, 1924 women from sub-Saharan Africa were enrolled. The median age was 26 years (interquartile range: 22-30), and 98.9% were Black. Sexually transmitted infection prevalence at enrollment included chlamydia (16.9%), trichomonas (7.2%), gonorrhea (5.7%), and syphilis (2.2%). External condoms (83.2%) and injectable contraceptives (61.1%) were the methods of contraception most frequently used by participants. In total, through April 3, 2020, 38,490 clinic visits were completed with a retention rate of 96% and 16,807 infusions administered with an adherence rate of 98%. Conclusions This proof-of-concept, large-scale monoclonal antibody study demonstrates the feasibility of conducting complex trials involving intravenous infusions in high incidence populations in sub-Saharan Africa.

BACKGROUND The Antibody-Mediated Prevention trials (HVTN 704/HPTN 085 and HVTN 703/HPTN 081) are the first efficacy trials to evaluate whether VRC01, a broadly neutralizing monoclonal antibody targeting the CD4-binding site of the HIV envelope protein, prevents sexual transmission of HIV-1. HVTN 704/HPTN 085 enrolled 2701 cisgender men and transgender (TG) individuals who have sex with men at 26 sites in Brazil, Peru, Switzerland, and the United States. METHODS Participants were recruited and retained through early, extensive community engagement. Eligible participants were randomized 1:1:1 to 10 mg/kg or 30 mg/kg of VRC01 or saline placebo. Visits occurred monthly, with intravenous (IV) infusions every 8 weeks over 2 years, for a total of 10 infusions. Participants were followed for 104 weeks after first infusion. RESULTS The median HVTN 704/HPTN 085 participant age was 28 years; 99% were assigned male sex; 90% identified as cisgender men, 5% as TG women and the remaining as other genders. Thirty-two percent were White, 15% Black, and 57% Hispanic/Latinx. Twenty-eight percent had a sexually transmitted infection at enrollment. More than 23,000 infusions were administered with no serious IV administration complications. Overall, retention and adherence to the study schedule exceeded 90%, and the dropout rate was below 10% annually (7.3 per 100 person-years) through week 80, the last visit for the primary end point. CONCLUSIONS HVTN 704/HPTN 085 exceeded accrual and retention expectations. With exceptional safety of IV administration and operational feasibility, it paves the way for future large-scale monoclonal antibody trials for HIV prevention and/or treatment.

Background: Tuberculosis (TB) remains the leading cause of infectious disease-related death. Recently, a trial of BCG revaccination and vaccination with H4:IC31, a recombinant protein vaccine, in South African adolescents (Aeras C-040-404) showed efficacy in preventing sustained QuantiFERON (QFT) conversion, a proxy for Mycobacterium tuberculosis (M.tb) infection. A phase 1b trial of 84 South African adolescents was conducted, concurrent with Aeras C-040-404, to assess the safety and immunogenicity of H4:IC31, H56:IC31 and BCG revaccination, and to identify and optimize immune assays for identification of candidate correlates of protection in efficacy trials. Methods: Two doses of H4:IC31 and H56:IC31 vaccines were administered intramuscularly (IM) 56 days apart, and a single dose of BCG (2–8 × 105 CFU) was administered intradermally (ID). T-cell and antibody responses were measured using intracellular cytokine staining and binding antibody assays, respectively. Binding antibodies and CD4+/CD8+ T-cell responses to H4- and H56-matched antigens were measured in samples from all participants. The study was designed to characterize safety and immunogenicity and was not powered for group comparisons. (Clinicaltrials.gov NCT02378207). Findings: In total, 481 adolescents (mean age 13·9 years) were screened; 84 were enrolled (54% female). The vaccines were generally safe and well-tolerated, with no reported severe adverse events related to the study vaccines. H4:IC31 and H56:IC31 elicited CD4+ T cells recognizing vaccine-matched antigens and H4- and H56-specific IgG binding antibodies. The highest vaccine-induced CD4+ T-cell response rates were for those recognizing Ag85B in the H4:IC31 and H56:IC31 vaccinated groups. BCG revaccination elicited robust, polyfunctional BCG-specific CD4+ T cells, with no increase in H4- or H56-specific IgG binding antibodies. There were few antigen-specific CD8+ T-cell responses detected in any group. Interpretation: BCG revaccination administered as a single dose ID and both H4:IC31 and H56:IC31 administered as 2 doses IM had acceptable safety profiles in healthy, QFT-negative, previously BCG-vaccinated adolescents. Characterization of the assays and the immunogenicity of these vaccines may help to identify valuable markers of protection for upcoming immune correlates analyses of C-040-404 and future TB vaccine efficacy trials. Funding: NIAID and Aeras. Keywords: Mycobacterium tuberculosis, H4:IC31, H56:IC31, BCG

Background DNA plasmids promise a pragmatic alternative to viral vectors for prime-boost HIV-1 vaccines. We evaluated DNA plasmid versus canarypox virus (ALVAC) primes in 2 randomized, double-blind, placebo-controlled trials in southern Africa with harmonized trial designs. HIV Vaccine Trials Network (HVTN) 111 tested DNA plasmid prime by needle or needleless injection device (Biojector) and DNA plasmid plus gp120 protein plus MF59 adjuvant boost. HVTN 100 tested ALVAC prime and ALVAC plus gp120 protein plus MF59 adjuvant boost (same protein/adjuvant as HVTN 111) by needle. Methods and findings The primary endpoints for this analysis were binding antibody (bAb) responses to HIV antigens (gp120 from strains ZM96, 1086, and TV1; variable 1 and 2 [V1V2] regions of gp120 from strains TV1, 1086, and B.CaseA, as 1086 V1V2 and B.CaseA were correlates of risk in the RV144 efficacy trial), neutralizing antibody (nAb) responses to pseudoviruses TV1c8.2 and MW925.26, and cellular responses to vaccine-matched antigens (envelope [Env] from strains ZM96, 1086, and TV1; and Gag from strains LAI and ZM96) at month 6.5, two weeks after the fourth vaccination. Per-protocol cohorts included vaccine recipients from HVTN 100 (n = 186, 60% male, median age 23 years) enrolled between February 9, 2015, and May 26, 2015 and from HVTN 111 (n = 56, 48% male, median age 24 years) enrolled between June 21, 2016, and July 13, 2017. IgG bAb response rates were 100% to 3 Env gp120 antigens in both trials. Response rates to V1V2 were lower and similar in both trials except to vaccine-matched 1086 V1V2, with rates significantly higher for the DNA-primed regimen than the ALVAC-primed regimen: 96.6% versus 72.7% (difference = 23.9%, 95% CI 15.6%–32.2%, p < 0.001). Among positive responders, bAb net mean fluorescence intensity (MFI) was significantly higher with the DNA-primed regimen than ALVAC-primed for 1086 V1V2 (geometric mean [GM] 2,833.3 versus 1,200.9; ratio = 2.36, 95% CI 1.42–3.92, p < 0.001) and B.CaseA V1V2 (GM 2314.0 versus 744.6, ratio = 3.11, 95% CI 1.51–6.38, p = 0.002). nAb response rates were >98% in both trials, with significantly higher 50% inhibitory dilution (ID50) among DNA-primed positive responders (n = 53) versus ALVAC-primed (n = 182) to tier 1A MW965.26 (GM 577.7 versus 265.7, ratio = 2.17, 95% CI 1.67–2.83, p < 0.001) and to TV1c8.2 (GM 187.3 versus 100.4, ratio = 1.87, 95% CI 1.48–2.35, p < 0.001). CD4+ T-cell response rates were significantly higher with DNA plasmid prime via Biojector than ALVAC prime (91.4% versus 52.8%, difference = 38.6%, 95% CI 20.5%–56.6%, p < 0.001 for ZM96.C; 88.0% versus 43.1%, difference = 44.9%, 95% CI 26.7%–63.1%, p < 0.001 for 1086.C; 55.5% versus 2.2%, difference = 53.3%, 95% CI 23.9%–82.7%, p < 0.001 for Gag LAI/ZM96). The study’s main limitations include the nonrandomized comparison of vaccines from 2 different trials, the lack of data on immune responses to other non–vaccine-matched antigens, and the uncertain clinical significance of the observed immunological effects. Conclusions In this study, we found that further investigation of DNA/protein regimens is warranted given enhanced immunogenicity to the V1V2 correlates of decreased HIV-1 acquisition risk identified in RV144, the only HIV vaccine trial to date to show any efficacy., Zoe Moodie and co-workers study immunological responses with DNA priming in trials of a candidate HIV vaccine., Author summary Why was this study done? HIV remains a major global health problem, especially in sub-Saharan Africa. Developing an efficacious HIV vaccine for subtype C infections, which predominate in southern Africa, is a high priority. Comparing immune responses after DNA plasmid versus viral vector priming in 2 similar subtype C HIV vaccine regimens could help inform the choice of priming strategy to use moving forward. DNA plasmid vaccines are cost-effective, easy to manufacture, and tolerable. What did the researchers do and find? We compared antibody and cellular immune responses to two phase I/II HIV-1 vaccines that differed in their priming mechanism (canarypox virus [ALVAC] vector versus DNA) but were matched in their protein boost and adjuvant. Specifically, we measured T-cell and binding and neutralizing antibody (nAb) responses to the vaccine-matched antigens from the HIV Vaccine Trials Network (HVTN) 100 and HVTN 111 trials. The vaccine regimens with DNA plasmid priming in general stimulated higher binding antibody (bAb) response magnitudes to the variable 1 and 2 (V1V2) correlates of decreased risk in RV144 and higher neutralizing responses to tier 1 viruses than the vaccine regimen with ALVAC priming. Both regimens stimulated high binding and neutralizing response rates, while DNA plasmid priming with MF59 adjuvanted protein boost induced significantly higher CD4+ T-cell response rates than ALVAC priming with MF59 adjuvanted protein boost. What do these findings mean? Our results suggest that DNA plasmid is a superior priming method for stimulating vaccine-matched humoral and cellular responses for HIV vaccine trials using this prime/boost approach. Additional investigations are warranted to optimize DNA plasmid delivery methods, such as electroporation for priming of DNA–protein combinations.

Background The Pox-Protein Public-Private Partnership is performing a suite of trials to evaluate the bivalent subtype C envelope protein (TV1.C and 1086.C glycoprotein 120) vaccine in the context of different adjuvants and priming agents for human immunodeficiency virus (HIV) type 1 (HIV-1) prevention. Methods In the HIV Vaccine Trials Network 111 trial, we compared the safety and immunogenicity of DNA prime followed by DNA/protein boost with DNA/protein coadministration injected intramuscularly via either needle/syringe or a needle-free injection device (Biojector). One hundred thirty-two healthy, HIV-1–uninfected adults were enrolled from Zambia, South Africa, and Tanzania and were randomized to 1 of 6 arms: DNA prime, protein boost by needle/syringe; DNA and protein coadministration by needle/syringe; placebo by needle/syringe; DNA prime, protein boost with DNA given by Biojector; DNA and protein coadministration with DNA given by Biojector; and placebo by Biojector. Results All vaccinations were safe and well tolerated. DNA and protein coadministration was associated with increased HIV-1 V1/V2 antibody response rate, a known correlate of decreased HIV-1 infection risk. DNA administration by Biojector elicited significantly higher CD4+ T-cell response rates to HIV envelope protein than administration by needle/syringe in the prime/boost regimen (85.7% vs 55.6%; P = .02), but not in the coadministration regimen (43.3% vs 48.3%; P = .61). Conclusions Both the prime/boost and coadministration regimens are safe and may be promising for advancement into efficacy trials depending on whether cellular or humoral responses are desired. Clinical Trials Registration South African National Clinical Trials Registry (application 3947; Department of Health [DoH] no. DOH-27–0715–4917) and ClinicalTrials.gov (NCT02997969)., DNA and protein coadministration was associated with increased human immunodeficiency virus (HIV) type 1 V1/V2 antibody response. Biojector DNA administration elicited significantly greater CD4+ T-cell responses to HIV envelope protein than needle/syringe in the prime/boost but not the coadministration regimen.

The innate immune response plays an important but unknown role in host defense against Mycobacterium tuberculosis . To define the function of innate immunity during tuberculosis, we evaluated M. tuberculosis replication dynamics during murine infection. Our data show that the early pulmonary innate immune response limits M. tuberculosis replication in a MyD88-dependent manner. Strikingly, we found that little M. tuberculosis cell death occurs during the first 2 weeks of infection. In contrast, M. tuberculosis cells deficient in the surface lipid phthiocerol dimycocerosate (PDIM) exhibited significant death rates, and consequently, total bacterial numbers were reduced. Host restriction of PDIM-deficient M. tuberculosis was not alleviated by the absence of interferon gamma (IFN-γ), inducible nitric oxide synthase (iNOS), or the phagocyte oxidase subunit p47. Taken together, these data indicate that PDIM protects M. tuberculosis from an early innate host response that is independent of IFN-γ, reactive nitrogen intermediates, and reactive oxygen species. By employing a pathogen replication tracking tool to evaluate M. tuberculosis replication and death during infection, we identify both host and pathogen factors affecting the outcome of infection.

Recently, cholesterol was identified as a physiologically important nutrient for Mycobacterium tuberculosis survival in chronically infected mice. However, it remained unclear precisely when cholesterol is available to the bacterium and what additional bacterial functions are required for its metabolism. Here, we show that the igr locus, which we previously found to be essential for intracellular growth and virulence of M . tuberculosis , is required for cholesterol metabolism. While igr -deficient strains grow identically to the wild type in the presence of short- and long-chain fatty acids, the growth of these bacteria is completely inhibited in the presence of cholesterol. Interestingly, this mutant is still able to respire under cholesterol-dependent growth inhibition, suggesting that the bacteria can metabolize other carbon sources during cholesterol toxicity. Consistent with this hypothesis, we found that the growth-inhibitory effect of cholesterol in vitro depends on cholesterol import, as mutation of the mce4 sterol uptake system partially suppresses this effect. In addition, the Δ igr mutant growth defect during the early phase of disease is completely suppressed by mutating mce4 , implicating cholesterol intoxication as the primary mechanism of attenuation. We conclude that M . tuberculosis metabolizes cholesterol throughout infection.

The addition of a methyl group (CH3) to specific cytosines in mammalian DNA controls early development by regulating gene activity (reviewed in 1–12). DNA methylation is defined as an “epigenetic” modification that results in a heritable change in gene function without altering the primary DNA sequence itself. The requirement for a properly functioning DNA methylation system in mammals is best appreciated when observing the severe consequences that result when this system fails. Mutations in the enzymes controlling methyl group addition, the DNA methyltransferases (DNMTs), cause deficient or nonphysiologic DNA methylation patterns that interrupt normal development. For example, targeted deletion of DNMT1 in mice is lethal to developing embryos, while naturally occurring DNMT3B mutations may cause the ICF (immunodeficiency, centromere instability, and facial anomalies) syndrome in humans (13, 14). Also, deletion of MeCP2, a protein that binds to methylated DNA, results in a Rett syndrome-like neurodevelopmental disorder in mice (15, 16). These findings highlight the importance of DNA methylation in epigenetic control of early mammalian development. The role of DNA methylation following embryogenesis is not resolved. However, DNA methylation patterns have been determined for numerous genes in multiple types of human malignancy. One rationale for this effort has been that nonphysiologic methylation patterns could promote transformation of normal cells to malignant cells through disruptions in physiologic gene regulatory processes. For example, hypermethylation could lead to aberrant tumor suppressor gene silencing, while hypomethylation could lead to inap

Bacteria need to adjust as they move between different environments. In this Progress article, Freitag, Port and Miner describe howListeria monocytogenesregulates the transition from saprophyte to human pathogen. Listeria monocytogenes is a bacterium that lives in the soil as a saprophyte but is capable of making the transition into a pathogen following its ingestion by susceptible humans or animals. Recent studies suggest that L. monocytogenes mediates its saprophyte-to-cytosolic-parasite transition through the careful modulation of the activity of a virulence regulatory protein known as PrfA, using a range of environmental cues that include available carbon sources. In this Progress article we describe the regulation of PrfA and its role in the L. monocytogenes transition from the saprophytic stage to the virulent intracellular stage.

The transcriptional activator PrfA is required for the expression of virulence factors necessary for Listeria monocytogenes pathogenesis. PrfA is believed to become activated following L. monocytogenes entry into the cytosol of infected host cells resulting in the induction of target genes whose products are required for bacterial intracellular growth and cell-to-cell spread. Several mutations have been identified that appear to lock PrfA into its highly activated cytosolic form (known as prfA* mutations). In this study PrfA and five PrfA* mutant proteins exhibiting differing degrees of activity were purified and analyzed to define the influences of the mutations on distinct aspects of PrfA activity. Based on limited proteolytic digestion conformational changes were detected for the PrfA* mutant proteins in comparison to wild type PrfA. For all but one mutant (PrfA Y63C), the DNA binding affinity as measured by electophoretic mobility shift assay (EMSA) appeared to directly correlate with levels of PrfA mutational activation such that the high activity mutants exhibited the largest increases in DNA binding affinity and moderately activated mutants exhibited more moderate increases. Surprisingly, the ability of PrfA and PrfA* mutants to form dimers in solution appeared to inversely correlate with levels of PrfA-dependent gene expression. Based on comparisons of protein activity and structural similarities with PrfA family members Crp and CooA, the prfA* mutations modify distinct aspects of PrfA activity that include DNA binding and protein-protein interactions.

Listeria monocytogenes is an environmental bacterium that becomes a pathogen following ingestion by a mammalian host. The transition from environmental organism to pathogen requires significant changes in gene expression, including the increased expression of gene products that contribute to bacterial growth within host cells. PrfA is an L. monocytogenes transcriptional regulator that becomes activated upon bacterial entry into mammalian cells and induces the expression of gene products required for virulence. How PrfA activation occurs is not known, however several mutations have been identified that increase PrfA activity in strains grown in vitro (prfA mutations). Here we describe a novel prfA mutation that enhances extracellular PrfA-dependent gene expression but in contrast to prfA mutants inhibits the cytosol-mediated induction of virulence genes. prfA Y154C strains entered cells and escaped from phagosomes with an efficiency similar to wild type bacteria, however the mutation prevented efficient L. monocytogenes actin polymerization and reduced spread of bacteria to adjacent cells. The prfA Y154C mutation severely attenuated bacterial virulence in mice but the mutant strains did generate target antigen specific CD8(+) effector cells. Interestingly, the prfA Y154C mutant was significantly less cytotoxic for host cells than wild type L. monocytogenes. The prfA Y154C mutant strain may therefore represent a novel attenuated strain of L. monocytogenes for antigen delivery with reduced host cell toxicity.

Cis-regions and trans-factors controlling TCL1 oncogene expression are not known. We identified the functional TCL1 promoter by mapping four transcriptional start sites 24-30 bp downstream of a TATA box. A 424-bp fragment upstream of the major start site showed robust promoter activity comparable with SV40 in both TCL1 expressing and non-expressing cell lines. Additional constructs spanning 10 kb upstream and 20 kb downstream of the start site showed only modest increases in reporter activity indicating that TCL1 expression is primarily controlled by the promoter. Ten putative Sp1-binding sites were identified within 300 bp of the start site, and three of these specifically bound Sp1. A dose-dependent transactivation of the TCL1 promoter with Sp1 addition in Sp1-negative Drosophila SL2 cells was observed, and mutation of the three identified Sp1-binding sites significantly repressed reporter gene expression in 293T cells, confirming a key role for Sp1 in activating the TCL1 promoter in vivo. In TCL1 silent cell lines, CpG DNA methylation was rarely observed at functional Sp1 sites, and methylation of a previously reported NotI restriction site was associated with dense CpG methylation rather than endogenous TCL1 gene silencing. Together, these results indicate that Sp1 mediates transactivation of the TCL1 core promoter and that TCL1 gene silencing is not dependent on mechanisms involving Sp1 and NotI site methylation.

DNA methylation has been linked to gene silencing in cancer. Primary effusion lymphoma (PEL) and myeloma are lymphoid malignancies that arise from terminally differentiated B cells. Interestingly, PEL do not express immunoglobulins or most B lineage-specific genes. The B cell-specific B29 (Igβ/CD79b) gene is silenced in PEL and some myelomas but is expressed in other normal and malignant B cells. B29 expression was reactivated in PEL by demethylating and histone deacetylase inhibiting treatments. Bisulfite sequencing revealed two types of DNA methylation in silenced B29 promoters: at conventional CpG and at CC(A/T)GG B29 promoter sites. The pattern of methylated CpG ( m CpG) and C m C(A/T)GG B29 promoter methylation observed was similar to that recently reported for epigenetic silencing of an integrated retrovirus. Methylation of C m C(A/T)GG sites in the B29 promoter significantly repressed in vivo transcriptional activity. Also, methylation of a central conserved C m CTGG B29 promoter site blocked the binding of early B cell factor. This methylated motif formed DNA–protein complexes with nuclear extracts from all cell types examined. Therefore, C m C(A/T)GG methylation may represent an important type of epigenetic marker on mammalian DNA that impacts transcription by altering DNA–protein complex formation.