Cryo-Electron Tomography Elucidates the Molecular Architecture of Treponema pallidum, the Syphilis Spirochete

Cryo-electron tomography (CET) was used to examine the native cellular organization of Treponema pallidum, the syphilis spirochete. T. pallidum cells appeared to form flat waves, did not contain an outer coat and, except for bulges over the basal bodies and widening in the vicinity of flagellar filaments, displayed a uniform periplasmic space. Although the outer membrane (OM) generally was smooth in contour, OM extrusions and blebs frequently were observed, highlighting the structure’s fluidity and lack of attachment to underlying periplasmic constituents. Cytoplasmic filaments converged from their attachment points opposite the basal bodies to form arrays that ran roughly parallel to the flagellar filaments along the inner surface of the cytoplasmic membrane (CM). Motile treponemes stably attached to rabbit epithelial cells predominantly via their tips. CET revealed that T. pallidum cell ends have a complex morphology and assume at least four distinct morphotypes. Images of dividing treponemes and organisms shedding cell envelope-derived blebs provided evidence for the spirochete’s complex membrane biology. In the regions without flagellar filaments, peptidoglycan (PG) was visualized as a thin layer that divided the periplasmic space into zones of higher and lower electron densities adjacent to the CM and OM, respectively. Flagellar filaments were observed overlying the PG layer, while image modeling placed the PG-basal body contact site in the vicinity of the stator–P-collar junction. Bioinformatics and homology modeling indicated that the MotB proteins of T. pallidum, Treponema denticola, and Borrelia burgdorferi have membrane topologies and PG binding sites highly similar to those of their well-characterized Escherichia coli and Helicobacter pylori orthologs. Collectively, our results help to clarify fundamental differences in cell envelope ultrastructure between spirochetes and gram-negative bacteria. They also confirm that PG stabilizes the flagellar motor and enable us to propose that in most spirochetes motility results from rotation of the flagellar filaments against the PG

Significance of Premature Stop Codons in \u3cem\u3eenv\u3c/em\u3e of Simian Immunodeficiency Virus

The location of the translational termination codon for the transmembrane protein (TMP) varies in three infectious molecular clones of simian immunodeficiency virus from macaques (SIVmac). The SIVmac251 and SIVmac142 infectious clones have premature stop signals that differ in location by one codon; transfection of these DNAs into human HUT-78 cells yielded virus with a truncated TMP (28 to 30 kilodaltons [kDa]). The SIVmac239 infectious clone does not have a premature stop codon in its TMP-coding region. Transfection of HUT-78 cells with this clone initially yielded virus with a full-length TMP (41 kDa). At 20 to 30 days posttransfection, SIVmac239 virus with a 41-kDa TMP gradually disappeared coincident with the emergence of a virus with a 28-kDa TMP. Virus production dramatically increased in parallel with the emergence of a virus with a 28-kDa TMP. Sequence analysis of viral DNAs from these cultures showed that premature stop codons arising by point mutation were responsible for the change in size of the TMP with time. A similar selective pressure for truncated forms of TMP was observed when the SIVmac239 clone was transfected into human peripheral blood lymphocytes (PBL). In contrast, no such selective pressure was observed in macaque PBL. When the SIVmac239 clone was transfected into macaque PBL and the resultant virus was serially passaged in macaque PBL, the virus replicated very well and maintained a 41-kDa TMP for 80 days in culture. Macaque monkeys were infected with SIVmac239 having a 28-kDa TMP; virus subsequently recovered from T4-enriched lymphocytes of peripheral blood showed only the 41-kDa form of TMP. These results indicate that the natural form of TMP in SIVmac is the full-length 41-kDa TMP, just as in human immunodeficiency virus type 1. Viruses with truncated forms of TMP appear to result from mutation and selection during propagation in unnatural human cells

Vaccine protection against simian immunodeficiency virus in monkeys using recombinant gamma-2 herpesvirus

Recombinant strains of replication-competent rhesus monkey rhadinovirus (RRV) were constructed in which strong promoter/enhancer elements were used to drive expression of simian immunodeficiency virus (SIV) Env or Gag or a Rev-Tat-Nef fusion protein. Cultured rhesus monkey fibroblasts infected with each recombinant strain were shown to express the expected protein. Three RRV-negative and two RRV-positive rhesus monkeys were inoculated intravenously with a mixture of these three recombinant RRVs. Expression of SIV Gag was readily detected in lymph node biopsy specimens taken at 3 weeks postimmunization. Impressive anti-SIV cellular immune responses were elicited on the basis of major histocompatibility complex (MHC) tetramer staining and gamma interferon enzyme-linked immunospot (ELISPOT) assays. Responses were much greater in magnitude in the monkeys that were initially RRV negative but were still readily detected in the two monkeys that were naturally infected with RRV at the time of immunization. By 3 weeks postimmunization, responses measured by MHC tetramer staining in the two Mamu-A*01(+) RRV-negative monkeys reached 9.3% and 13.1% of all CD8(+) T cells in peripheral blood to the Gag CM9 epitope and 2.3% and 7.3% of all CD8(+) T cells in peripheral blood to the Tat SL8 epitope. Virus-specific CD8(+) T cell responses persisted at high levels up to the time of challenge at 18 weeks postimmunization, and responding cells maintained an effector memory phenotype. Despite the ability of the RRVenv recombinant to express high levels of Env in cultured cells, and despite the appearance of strong anti-RRV antibody responses in immunized monkeys, anti-Env antibody responses were below our ability to detect them. Immunized monkeys, together with three unimmunized controls, were challenged intravenously with 10 monkey infectious doses of SIVmac239. All five immunized monkeys and all three controls became infected with SIV, but peak viral loads were 1.2 to 3.0 log(10) units lower and chronic-phase viral loads were 1.0 to 3.0 log(10) units lower in immunized animals than the geometric mean of unimmunized controls. These differences were statistically significant. Anti-Env antibody responses following challenge indicated an anamnestic response in the vaccinated monkeys. These findings further demonstrate the potential of recombinant herpesviruses as preventive vaccines for AIDS. We hypothesize that this live, replication-competent, persistent herpesvirus vector could match, or come close to matching, live attenuated strains of SIV in the degree of protection if the difficulty with elicitation of anti-Env antibody responses can be overcome

County and Demographic Differences in Drug Arrests and Controlled Substance Use in Maine

. Introduction: The Diversion Alert Program (DAP) was established to curb misuse of drugs and help identify people who may need treatment for substance use disorder (SUD). Law enforcement compiled arrest data into a database accessible by health care providers. Our objectives were to identify regional and demographic differences in drug use and misuse in Maine. Methods: All arrests (N = 11 234) reported to the DAP from 2013 to 2018 were examined by county and arrestee demographics, and classified into families (opioids, stimulants, sedatives). The Drug Enforcement Administration’s Automation of Reports and Consolidated Orders System (ARCOS) tracks the distribution of controlled pharmaceuticals (Schedule II-III). Opioids were converted to oral morphine milligram equivalents (MMEs). County and zip-code maps were constructed. Results: The most arrests per capita occurred in Androscoggin, Knox, and Cumberland Counties. Opioids were the most common drug class in arrests in all counties except Aroostook County, where stimulants were most common. Medical distribution of opioids varied. Although buprenorphine doubled, many prescription opioids (eg, hydrocodone, fentanyl, oxymorphone) exhibited large (\u3e 50%) reductions in distribution. Methadone was the predominant opioid statewide (56.4% of total MMEs), although there were sizable differences between regions (Presque Isle = 8.6%, Bangor = 78.9%). Amphetamine distribution increased by 67.9%. Discussion: The DAP, a unique pharmacoepidemiological resource, revealed a 6-fold difference in drug arrests by county. Regional differences in methadone may be due to heterogeneities in methadone clinic distribution. Conclusions: The decrease in most prescription opioids, but increase in prescription stimulants, may warrant continued monitoring to improve public health

Functional Cure of SIVagm Infection in Rhesus Macaques Results in Complete Recovery of CD4+ T Cells and Is Reverted by CD8+ Cell Depletion

Understanding the mechanism of infection control in elite controllers (EC) may shed light on the correlates of control of disease progression in HIV infection. However, limitations have prevented a clear understanding of the mechanisms of elite controlled infection, as these studies can only be performed at randomly selected late time points in infection, after control is achieved, and the access to tissues is limited. We report that SIVagm infection is elite-controlled in rhesus macaques (RMs) and therefore can be used as an animal model for EC HIV infection. A robust acute infection, with high levels of viral replication and dramatic mucosal CD4+ T cell depletion, similar to pathogenic HIV-1/SIV infections of humans and RMs, was followed by complete and durable control of SIVagm replication, defined as: undetectable VLs in blood and tissues beginning 72 to 90 days postinoculation (pi) and continuing at least 4 years; seroreversion; progressive recovery of mucosal CD4+ T cells, with complete recovery by 4 years pi; normal levels of T cell immune activation, proliferation, and apoptosis; and no disease progression. This “functional cure” of SIVagm infection in RMs could be reverted after 4 years of control of infection by depleting CD8 cells, which resulted in transient rebounds of VLs, thus suggesting that control may be at least in part immune mediated. Viral control was independent of MHC, partial APOBEC restriction was not involved in SIVagm control in RMs and Trim5 genotypes did not impact viral replication. This new animal model of EC lentiviral infection, in which complete control can be predicted in all cases, permits research on the early events of infection in blood and tissues, before the defining characteristics of EC are evident and when host factors are actively driving the infection towards the EC status

Gp120 on HIV-1 Virions Lacks O-Linked Carbohydrate

As HIV-1-encoded envelope protein traverses the secretory pathway, it may be modified with N- and O-linked carbohydrate. When the gp120s of HIV-1 NL4-3, HIV-1 YU2, HIV-1 Bal, HIV-1 JRFL, and HIV-1 JRCSF were expressed as secreted proteins, the threonine at consensus position 499 was found to be O-glycosylated. For SIVmac239, the corresponding threonine was also glycosylated when gp120 was recombinantly expressed. Similarly-positioned, highly-conserved threonines in the influenza A virus H1N1 HA1 and H5N1 HA1 envelope proteins were also found to carry O-glycans when expressed as secreted proteins. In all cases, the threonines were modified predominantly with disialylated core 1 glycans, together with related core 1 and core 2 structures. Secreted HIV-1 gp140 was modified to a lesser extent with mainly monosialylated core 1 O-glycans, suggesting that the ectodomain of the gp41 transmembrane component may limit the accessibility of Thr499 to glycosyltransferases. In striking contrast to these findings, gp120 on purified virions of HIV-1 Bal and SIV CP-MAC lacked any detectable O-glycosylation of the C-terminal threonine. Our results indicate the absence of O-linked carbohydrates on Thr499 as it exists on the surface of virions and suggest caution in the interpretation of analyses of post-translational modifications that utilize recombinant forms of envelope protein

Role of Occult and Post-acute Phase Replication in Protective Immunity Induced with a Novel Live Attenuated SIV Vaccine

In order to evaluate the role of persisting virus replication during occult phase immunisation in the live attenuated SIV vaccine model, a novel SIVmac239Δnef variant (SIVrtTA) genetically engineered to replicate in the presence of doxycycline was evaluated for its ability to protect against wild-type SIVmac239. Indian rhesus macaques were vaccinated either with SIVrtTA or with SIVmac239Δnef. Doxycycline was withdrawn from 4 of 8 SIVrtTA vaccinates before challenge with wild-type virus. Unvaccinated challenge controls exhibited ~107 peak plasma viral RNA copies/ml persisting beyond the acute phase. Six vaccinates, four SIVmac239Δnef and two SIVrtTA vaccinates exhibited complete protection, defined by lack of wild-type viraemia post-challenge and virus-specific PCR analysis of tissues recovered post-mortem, whereas six SIVrtTA vaccinates were protected from high levels of viraemia. Critically, the complete protection in two SIVrtTA vaccinates was associated with enhanced SIVrtTA replication in the immediate post-acute vaccination period but was independent of doxycycline status at the time of challenge. Mutations were identified in the LTR promoter region and rtTA gene that do not affect doxycycline-control but were associated with enhanced post-acute phase replication in protected vaccinates. High frequencies of total circulating CD8+T effector memory cells and a higher total frequency of SIV-specific CD8+ mono and polyfunctional T cells on the day of wild-type challenge were associated with complete protection but these parameters were not predictive of outcome when assessed 130 days after challenge. Moreover, challenge virus-specific Nef CD8+ polyfunctional T cell responses and antigen were detected in tissues post mortem in completely-protected macaques indicating post-challenge control of infection. Within the parameters of the study design, on-going occult-phase replication may not be absolutely required for protective immunity

Trivalent Adenovirus Type 5 HIV Recombinant Vaccine Primes for Modest Cytotoxic Capacity That Is Greatest in Humans with Protective HLA Class I Alleles

If future HIV vaccine design strategies are to succeed, improved understanding of the mechanisms underlying protection from infection or immune control over HIV replication remains essential. Increased cytotoxic capacity of HIV-specific CD8+ T-cells associated with efficient elimination of HIV-infected CD4+ T-cell targets has been shown to distinguish long-term nonprogressors (LTNP), patients with durable control over HIV replication, from those experiencing progressive disease. Here, measurements of granzyme B target cell activity and HIV-1-infected CD4+ T-cell elimination were applied for the first time to identify antiviral activities in recipients of a replication incompetent adenovirus serotype 5 (Ad5) HIV-1 recombinant vaccine and were compared with HIV-negative individuals and chronically infected patients, including a group of LTNP. We observed readily detectable HIV-specific CD8+ T-cell recall cytotoxic responses in vaccinees at a median of 331 days following the last immunization. The magnitude of these responses was not related to the number of vaccinations, nor did it correlate with the percentages of cytokine-secreting T-cells determined by ICS assays. Although the recall cytotoxic capacity of the CD8+ T-cells of the vaccinee group was significantly less than that of LTNP and overlapped with that of progressors, we observed significantly higher cytotoxic responses in vaccine recipients carrying the HLA class I alleles B*27, B*57 or B*58, which have been associated with immune control over HIV replication in chronic infection. These findings suggest protective HLA class I alleles might lead to better outcomes in both chronic infection and following immunization due to more efficient priming of HIV-specific CD8+ T-cell cytotoxic responses

Transmission in Heteronymous Spinal Pathways Is Modified after Stroke and Related to Motor Incoordination

Changes in reflex spinal pathways after stroke have been shown to affect motor activity in agonist and antagonist muscles acting at the same joint. However, only a few studies have evaluated the heteronymous reflex pathways modulating motoneuronal activity at different joints. This study investigates whether there are changes in the spinal facilitatory and inhibitory pathways linking knee to ankle extensors and if such changes may be related to motor deficits after stroke. The early facilitation and later inhibition of soleus H reflex evoked by the stimulation of femoral nerve at 2 times the motor threshold of the quadriceps were assessed in 15 healthy participants and on the paretic and the non-paretic sides of 15 stroke participants. The relationships between this reflex modulation and the levels of motor recovery, coordination and spasticity were then studied. Results show a significant (Mann-Whitney U; P<0.05) increase in both the peak amplitude (mean±SEM: 80±22% enhancement of the control H reflex) and duration (4.2±0.5 ms) of the facilitation on the paretic side of the stroke individuals compared to their non-paretic side (36±6% and 2.9±0.4 ms) and to the values of the control subjects (33±4% and 2.8±0.4 ms, respectively). Moreover, the later strong inhibition observed in all control subjects was decreased in the stroke subjects. Both the peak amplitude and the duration of the increased facilitation were inversely correlated (Spearman r = −0.65; P = 0.009 and r = −0.67; P = 0.007, respectively) with the level of coordination (LEMOCOT) of the paretic leg. Duration of this facilitation was also correlated (r = −0.58, P = 0.024) with the level of motor recovery (CMSA). These results confirm changes in transmission in heteronymous spinal pathways that are related to motor deficits after stroke

Generation and characterization of a defective HIV-1 Virus as an immunogen for a therapeutic vaccine

BACKGROUND: The generation of new immunogens able to elicit strong specific immune responses remains a major challenge in the attempts to obtain a prophylactic or therapeutic vaccine against HIV/AIDS. We designed and constructed a defective recombinant virus based on the HIV-1 genome generating infective but non-replicative virions able to elicit broad and strong cellular immune responses in HIV-1 seropositive individuals. RESULTS: Viral particles were generated through transient transfection in producer cells (293-T) of a full length HIV-1 DNA carrying a deletion of 892 base pairs (bp) in the pol gene encompassing the sequence that codes for the reverse transcriptase (NL4-3/ΔRT clone). The viral particles generated were able to enter target cells, but due to the absence of reverse transcriptase no replication was detected. The immunogenic capacity of these particles was assessed by ELISPOT to determine γ-interferon production in a cohort of 69 chronic asymptomatic HIV-1 seropositive individuals. Surprisingly, defective particles produced from NL4-3/ΔRT triggered stronger cellular responses than wild-type HIV-1 viruses inactivated with Aldrithiol-2 (AT-2) and in a larger proportion of individuals (55% versus 23% seropositive individuals tested). Electron microscopy showed that NL4-3/ΔRT virions display immature morphology. Interestingly, wild-type viruses treated with Amprenavir (APV) to induce defective core maturation also induced stronger responses than the same viral particles generated in the absence of protease inhibitors. CONCLUSIONS: We propose that immature HIV-1 virions generated from NL4-3/ΔRT viral clones may represent new prototypes of immunogens with a safer profile and stronger capacity to induce cellular immune responses than wild-type inactivated viral particles.This study was supported by grants FIS PI050265, FIS PI040503, FIS PI070291, FIS Intrasalud 080752, FIS PS09/01297, FIS PI10/02984, SAF2006-26667-E, FIT 09-010-205-9, FIPSE 36780/08, Fundación Mútua Madrileña, TRA-094, EC10-153, ISCIII-RETIC RD06/0006, HIVACAT–HIV Development Program in Catalonia, FIPSE 36630/07, UE Program Health 2009 CHAARM. Spanish Health Institute Carlos III (ISCIII) and the Health Department of the Catalan Government (Generalitat de Catalunya). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S