The Angolan Pandemic Rapid Response Team: An Assessment, Improvement, and Development Analysis of the First Self-sufficient African National Response Team Curriculum.

OBJECTIVE: The purpose of this study was to assess, through participant self-assessment, the effectiveness of a rapid response team curriculum based on the World Health Organization (WHO) Ebola Virus Disease Consolidated Preparedness Checklist, Revision 1. METHODS: A pre-and-post survey for the purpose of process improvement assessment involving 44 individuals was conducted in Angola. The survey was conducted before and after a 6-day training workshop held in Luanda, Angola, in December 2017. A paired t-test was used to identify any significant change on six 7-point Likert scale questions with α <.05 (95% CI). RESULTS: Two of the 6 questions, "I feel confident the team can effectively work together to accomplish its assigned goals and objectives during a suspected contagious hemorrhagic fever disease outbreak" and "I understand basic pandemic response concepts" changed significantly from the presurvey to the postsurvey. The 4 remaining questions had near statistical significant change or an upward trend. CONCLUSION: This Angolan rapid response team training curriculum based on WHO guidelines, After Action Reports, and internationally accepted standard operating procedures provides the nation of Angola with the confidence to rapidly respond at the national level to a highly infectious contagion in the region. (Disaster Med Public Health Preparedness. 2019;13:577-581).

Performance of the Xpert MTB/RIF assay for the diagnosis of pulmonary tuberculosis and rifampin resistance in a low-incidence, high-resource setting.

Performance of the Xpert MTB/RIF assay, designed to simultaneously detect Mycobacterium tuberculosis complex (MTBC) and rifampin (RIF) resistance, has been well documented in low-resource settings with high TB-incidence. However, few studies have assessed its accuracy in low TB incidence settings. We evaluated the performance of Xpert MTB/RIF using clinical sputum specimens routinely collected from suspect pulmonary TB patients over a 4-year time period in San Diego County, California. Xpert MTB/RIF results were compared to acid-fast bacilli (AFB) smear microscopy, mycobacterial culture, and phenotypic drug susceptibility testing (DST). Of 751 sputum specimens, 134 (17.8%) were MTBC culture-positive and 2 (1.5%) were multidrug-resistant (MDR). For the detection of MTBC, Xpert MTB/RIF sensitivity was 89.6% (97.7% and 74.5% in smear-positive and -negative sputa, respectively) and specificity was 97.2%; while AFB smear sensitivity and specificity were 64.9% and 77.8%, respectively. Xpert MTB/RIF detected 35 of 47 smear-negative culture-positive specimens, and excluded 124 of 137 smear-positive culture-negative specimens. Xpert MTB/RIF also correctly excluded 99.2% (121/122) of nontuberculous mycobacteria (NTM) specimens, including all 33 NTM false-positives by smear microscopy. For the detection of RIF resistance, Xpert MTB/RIF sensitivity and specificity were 100% and 98.3%, respectively. Our findings demonstrate that Xpert MTB/RIF is able to accurately detect MTBC and RIF resistance in routinely collected respiratory specimens in a low TB-incidence setting, with comparable performance to that achieved in high-incidence settings; and suggest that under these conditions the assay has particular utility in detecting smear-negative TB cases, excluding smear-positive patients without MTBC disease, and differentiating MTBC from NTM.

Kentucky Teen Institute: Results of a 1-Year, Health Advocacy Training Intervention for Youth.

The Kentucky Teen Institute trains youth throughout the state to advocate for policies that promote health in their communities. By evaluating two program summits held at universities, regularly scheduled community meetings, ongoing technical support, and an advocacy day at the state Capitol, the aims of this study were to assess the impact of the intervention on correlates of youths' advocacy intentions and behaviors and to assess youth participants' and other key stakeholders' perceptions of the intervention. An ecological model approach and the theory of planned behavior served as theoretical frameworks from which pre-post, one-group survey and qualitative data were collected (June 2013-June 2014). An equal number of low-income and non-low-income youth representing five counties participated in the Summer Summit pretest (n = 24) and Children's Advocacy Day at the Capitol posttest (n = 14). Survey data revealed that youths' attitude toward advocacy, intentions to advocate, and advocacy behaviors all improved over the intervention. Observations, interviews, a focus group, and other written evaluations identified that the youths', as well as their mentors' and advocacy coaches', confidence, communities' capacity, and mutually beneficial mentorship strengthened. Stronger public speaking skills, communication among the teams, and other recommendations for future advocacy interventions are described.

Effects of APC De-targeting and GAr modification on the duration of luciferase expression from plasmid DNA delivered to skeletal muscle.

Immune responses to expressed foreign transgenes continue to hamper progress of gene therapy development. Translated foreign proteins with intracellular location are generally less accessible to the immune system, nevertheless they can be presented to the immune system through both MHC Class I and Class II pathways. When the foreign protein luciferase was expressed following intramuscular delivery of plasmid DNA in outbred mice, expression rapidly declined over 4 weeks. Through modifications to the expression plasmid and the luciferase transgene we examined the effect of detargeting expression away from antigen-presenting cells (APCs), targeting expression to skeletal muscle and fusion with glycine-alanine repeats (GAr) that block MHC-Class I presentation on the duration of luciferase expression. De-targeting expression from APCs with miR142-3p target sequences incorporated into the luciferase 3'UTR reduced the humoral immune response to both native and luciferase modified with a short GAr sequence but did not prolong the duration of expression. When a skeletal muscle specific promoter was combined with the miR target sequences the humoral immune response was dampened and luciferase expression persisted at higher levels for longer. Interestingly, fusion of luciferase with a longer GAr sequence promoted the decline in luciferase expression and increased the humoral immune response to luciferase. These studies demonstrate that expression elements and transgene modifications can alter the duration of transgene expression but other factors will need to overcome before foreign transgenes expressed in skeletal muscle are immunologically silent.

An analogue peptide from the Cancer/Testis antigen PASD1 induces CD8+ T cell responses against naturally processed peptide.

We have previously identified the novel Cancer/Testis antigen PASD1 by immunoscreening a testis library with pooled acute myeloid leukemia (AML) patient sera. To develop a cytotoxic T lymphocyte (CTL)-inducing vaccine, we have now investigated the carboxy-terminal region, known to contain serological determinants, for MHC class I (HLA-A⋆0201)-binding peptides. Algorithm-selected natural peptides failed to show detectable HLA-A⋆0201 binding in T2 assays. However, anchor-modified analogue peptides showed enhanced binding, with decreased off-rates. Analogue peptide-loaded antigen-presenting cells (APCs) induced IFN-γ production by T cells from normal donors and patients. In addition, peptide-specific T cells could be expanded from cancer patients by stimulation with the PASD1 analogue peptide Pa14. For clinical application, a DNA fusion gene vaccine encoding Pa14 was designed and tested in "humanized" mice. Splenocytes from vaccinated mice showed in vitro cytotoxicity against tumour cells, either exogenously loaded with the corresponding wild-type peptide (Pw8) or expressing endogenously processed PASD1 protein. We show for the first time that a DNA vaccine encoding an altered PASD1 epitope can induce CTLs to target the natural peptide expressed by human tumour cells.

DNA fusion vaccine designs to induce tumor-lytic CD8+ T-cell attack via the immunodominant cysteine-containing epitope of NY-ESO 1.

The cancer/testis antigen NY-ESO-1 contains an immunodominant HLA-A2-binding peptide (SLLMWITQC), designated S9C, an attractive target for vaccination against several human cancers. As cysteine contains a reactive -SH, the oxidation status of exogenous synthetic peptide is uncertain. We have designed tolerance-breaking DNA fusion vaccines incorporating a domain of tetanus toxin fused to tumor-derived peptide sequences (p.DOM-peptide), placed at the C-terminus for optimal immunogenicity. In a "humanized" HLA-A2 preclinical model, p.DOM-S9C primed S9C-specific CD8+ T cells more effectively than adjuvanted synthetic peptide. A DNA vaccine encoding the full NY-ESO-1 sequence alone induced only weak S9C-specific responses, amplified by addition of DOM sequence. The analog peptide (SLLMWITQL) also primed peptide-specific CD8+ T cells, again increased by DNA delivery. Importantly, T cells induced by S9C-encoding DNA vaccines killed tumor cells expressing endogenous NY-ESO-1. Only a fraction of T cells induced by the S9L-encoding DNA vaccines was able to recognize S9C and kill tumor cells. These data indicate that DNA vaccines mimic posttranslational modifications of -SH-containing peptides expressed by tumor cells. Instability of synthetic peptides and the potential dangers of analog peptides contrast with the ability of DNA vaccines to induce high levels of tumor-lytic peptide-specific CD8+ T cells. These findings encourage clinical exploration of this vaccine strategy to target NY-ESO-1.

A DNA vaccine strategy for effective antibody induction to pathogen-derived antigens.

DNA-based vaccines are currently being developed for treating a diversity of human diseases including cancers, autoimmune conditions, allergies, and microbial infections. In this chapter, we present a general protocol that can be used as a starting point for developing DNA vaccines to pathogen-derived antigens, using Neisseria meningitidis as an example. In addition, we describe a fusion gene-based vaccine protocol for increasing the potency of DNA vaccines that are based on poorly immunogenic antigens such as short pathogen-derived polypeptides. Finally, we provide a safe and effective protocol for delivery of DNA vaccines, based on intramuscular injection followed by electroporation.

DNA fusion gene vaccines induce cytotoxic T-cell attack on naturally processed peptides of human prostate-specific membrane antigen.

For long-term attack on tumor cells in patients with prostate cancer, induction of cytolytic T cells is desirable. Several lineage-specific target proteins are known and algorithms have identified candidate MHC class I-binding peptides, particularly for HLA-A*0201. We have designed tolerance-breaking DNA fusion vaccines incorporating a domain of tetanus toxin fused to candidate tumor-derived peptide sequences. Using three separate peptide sequences from prostate-specific membrane antigen (PSMA) (peptides PSMA(27) , PSMA(663) , and PSMA(711) ), this vaccine design induced high levels of CD8(+) T cells against each peptide in a HLA-A(*) 0201 preclinical model. In contrast, the full-length PSMA sequence containing all three epitopes was poorly immunogenic. Induced T cells were cytotoxic against peptide-loaded tumor cells, but only those against PSMA(27) or PSMA(663) peptides, and not PSMA(711) , were able to kill tumor cells expressing endogenous PSMA. Cytotoxicity was also evident in vivo. The preclinical model provides a powerful tool for generating CD8(+) T cells able to predict whether target cells can process and present peptides, essential for planning peptide vaccine-based clinical trials.

Spontaneous splenic rupture in an active duty Marine upon return from Iraq: a case report.

INTRODUCTION: Atraumatic splenic rupture is a rare event that has been associated with several infectious disease processes. In the active duty military population, potential exposure to these pathogens is significant. Here we discuss the case of an active duty Marine with spontaneous splenic rupture upon return from a six-month deployment in Iraq. CASE PRESENTATION: A previously healthy 30-year-old Caucasian male active duty Marine presented with abdominal pain, fever and diarrhea after deployment to Iraq in support of Operation Iraqi Freedom. Based on clinical and radiographic evidence, a diagnosis of spontaneous splenic rupture was ultimately suspected. After exploratory laparotomy with confirmation of rupture, splenectomy was performed, and the patient made a full, uneventful recovery. Histopathologic examination revealed mild splenomegaly with a ruptured capsule of undetermined cause. CONCLUSION: Spontaneous splenic rupture is a rare event that may lead to life-threatening hemorrhage if not diagnosed and treated quickly. Although the cause of this patient's case was unknown, atraumatic splenic rupture has been associated with a variety of infectious diseases and demonstrates some risks the active duty military population may face while on deployment. Having an awareness of these pathogens and their role in splenic rupture, clinicians caring for military personnel must be prepared to recognize and treat this potentially fatal complication.

DNA vaccines against cancer come of age.

Genetic technology allows construction of DNA vaccines encoding selected tumor antigens together with molecules to direct and amplify the desired effector pathways. Their enormous promise has been marred by a problem of scaling up to human subjects. This is now largely overcome by electroporation, which increases both antigen expression and the inflammatory milieu. While the principles of vaccine design can be developed in mouse models, the real operative test is in the clinic, using patients in temporary remission. Monitoring of induced immunity, although commonly limited to blood, is providing objective qualitative and quantitative data on T-cell and antibody responses. Prolongation of remission is the goal and an activated immune system should achieve this.

DNA fusion gene vaccination mobilizes effective anti-leukemic cytotoxic T lymphocytes from a tolerized repertoire.

The majority of known human tumor-associated antigens derive from non-mutated self proteins. T cell tolerance, essential to prevent autoimmunity, must therefore be cautiously circumvented to generate cytotoxic T cell responses against these targets. Our strategy uses DNA fusion vaccines to activate high levels of peptide-specific CTL. Key foreign sequences from tetanus toxin activate tolerance-breaking CD4(+) T cell help. Candidate MHC class I-binding tumor peptide sequences are fused to the C terminus for optimal processing and presentation. To model performance against a leukemia-associated antigen in a tolerized setting, we constructed a fusion vaccine encoding an immunodominant CTL epitope derived from Friend murine leukemia virus gag protein (FMuLV(gag)) and vaccinated tolerant FMuLV(gag)-transgenic (gag-Tg) mice. Vaccination with the construct induced epitope-specific IFN-gamma-producing CD8(+) T cells in normal and gag-Tg mice. The frequency and avidity of activated cells were reduced in gag-Tg mice, and no autoimmune injury resulted. However, these CD8(+) T cells did exhibit gag-specific cytotoxicity in vitro and in vivo. Also, epitope-specific CTL killed FBL-3 leukemia cells expressing endogenous FMuLV(gag) antigen and protected against leukemia challenge in vivo. These results demonstrate a simple strategy to engage anti-microbial T cell help to activate epitope-specific polyclonal CD8(+) T cell responses from a residual tolerized repertoire.

DNA vaccination induces WT1-specific T-cell responses with potential clinical relevance.

The Wilms tumor antigen, WT1, is associated with several human cancers, including leukemia. We evaluated WT1 as an immunotherapeutic target using our proven DNA fusion vaccine design, p.DOM-peptide, encoding a minimal tumor-derived major histocompatibility complex (MHC) class I-binding epitope downstream of a foreign sequence of tetanus toxin. Three p.DOM-peptide vaccines, each encoding a different WT1-derived, HLA-A2-restricted epitope, induced cytotoxic T lymphocytes (CTLs) in humanized transgenic mice expressing chimeric HLA-A2, without affecting hematopoietic stem cells. Mouse CTLs killed human leukemia cells in vitro, indicating peptide processing/presentation. Low numbers of T cells specific for these epitopes have been described in cancer patients. Expanded human T cells specific for each epitope were lytic in vitro. Focusing on human WT1(37-45)-specific cells, the most avid of the murine responses, we demonstrated lysis of primary leukemias, underscoring their clinical relevance. Finally, we showed that these human CTL kill target cells transfected with the relevant p.DOM-peptide DNA vaccine, confirming that WT1-derived epitopes are presented to T cells similarly by tumors and following DNA vaccination. Together, these data link mouse and human studies to suggest that rationally designed DNA vaccines encoding WT1-derived epitopes, particularly WT1(37-45), have the potential to induce/expand functional tumor-specific cytotoxic responses in cancer patients.

The L1-type cell adhesion molecule Neuroglian is necessary for maintenance of sensory axon advance in the Drosophila embryo.

BACKGROUND: Cell adhesion molecules have long been implicated in the regulation of axon growth, but the precise cellular roles played by individual cell adhesion molecules and the molecular basis for their action are still not well understood. We have used the sensory system of the Drosophila embryo to shed light on the mechanism by which the L1-type cell adhesion molecule Neuroglian regulates axon growth. RESULTS: We have found a highly penetrant sensory axon stalling phenotype in neuroglian mutant embryos. Axons stalled at a variety of positions along their normal trajectory, but most commonly in the periphery some distance along the peripheral nerve. All lateral and dorsal cluster sensory neurons examined, except for the dorsal cluster neuron dbd, showed stalling. Sensory axons were never seen to project along inappropriate pathways in neuroglian mutants and stalled axons showed normal patterns of fasciculation within nerves. The growth cones of stalled axons possessed a simple morphology, similar to their appearance in wild-type embryos when advancing along nerves. Driving expression of the wild-type form of Neuroglian in sensory neurons alone rescued the neuroglian mutant phenotype of both pioneering and follower neurons. A partial rescue was achieved by expressing the Neuroglian extracellular domain. Over/mis-expression of Neuroglian in all neurons, oenocytes or trachea had no apparent effect on sensory axon growth. CONCLUSION: We conclude that Neuroglian is necessary to maintain axon advance along axonal substrates, but is not required for initiation of axon outgrowth, axon fasciculation or recognition of correct growth substrates. Expression of Neuroglian in sensory neurons alone is sufficient to promote axon advance and the intracellular region of the molecule is largely dispensable for this function. It is unlikely, therefore, that Nrg acts as a molecular 'clutch' to couple adhesion of F-actin within the growth cone to the extracellular substrate. Rather, we suggest that Neuroglian mediates sensory axon advance by promoting adhesion of the surface of the growth cone to its substrate. Our finding that stalling of a pioneer sensory neuron is rescued by driving Neuroglian in sensory neurons alone may suggest that Neuroglian can act in a heterophilic fashion.

Public T cell receptor beta-chains are not advantaged during positive selection.

Studies of human and murine T cells have shown that public TCR beta-chain rearrangements can dominate the Ag-specific and naive repertoires of distinct individuals. We show that mouse T cells responding to the minor histocompatibility Ag HYDbSmcy share an invariant Vbeta8.2-Jbeta2.3 TCR gene rearrangement. The dominance of this rearrangement shows that it successfully negotiated thymic selection and was highly favored during clonal expansion in all animals examined. We hypothesized that such beta-chains are advantaged during thymic and/or peripheral selection and, as a result, may be over-represented in the naive repertoire. A sequencing study was undertaken to examine the diversity of Vbeta8.2-Jbeta2.3 CDR3 loops from naive T cell repertoires of multiple mice. Public TCR beta-chain sequences were identified across different repertoires and MHC haplotypes. To determine whether such public beta-chains are advantaged during thymic selection, individual chains were followed through T cell development in a series of novel bone marrow competition chimeras. We demonstrate that beta-chains were positively selected with similar efficiency regardless of CDR3 loop sequence. Therefore, the establishment and maintenance of public beta-chains in the periphery is predominantly controlled by post-thymic events through modification of the primary, thymus-derived TCR repertoire.

DNA vaccines: precision tools for activating effective immunity against cancer.

DNA vaccination has suddenly become a favoured strategy for inducing immunity. The molecular precision offered by gene-based vaccines, together with the facility to include additional genes to direct and amplify immunity, has always been attractive. However, the apparent failure to translate operational success in preclinical models to the clinic, for reasons that are now rather obvious, reduced initial enthusiasm. Recently, novel delivery systems, especially electroporation, have overcome this translational block. Here, we assess the development, current performance and potential of DNA vaccines for the treatment of cancer.

A DNA fusion vaccine induces bactericidal antibodies to a peptide epitope from the PorA porin of Neisseria meningitidis.

An experimental DNA plasmid vaccine was developed based on a well-characterized and protective peptide epitope derived from a bacterial porin protein. For this study, we used the P1.16b serosubtype epitope, located in variable region (VR)2 in loop 4 of the PorA outer membrane (OM) porin from Neisseria meningitidis serogroup B strain MC58. A plasmid that encoded the entire loop (pPorAloop4) was prepared, as well as a fusion plasmid that encoded the loop in tandem with the fragment C (FrC) immunostimulatory sequence from tetanus toxin (pPorAloop4-FrC). The constructs were used for intramuscular immunization without exogenous adjuvant. Murine antisera raised to the pPorAloop4-FrC DNA fusion plasmid reacted significantly with OMs in enzyme-linked immunosorbent assay and with whole bacteria by immunofluorescence, whereas antisera raised to the pPorAloop4 DNA plasmid and to control plasmid showed little or no reactivity. Significantly, only the pPorALoop4-FrC plasmid induced bactericidal antibodies, demonstrating that the intrinsic immunostimulatory sequence was essential for inducing a protective immune response. The antibodies raised to the P1.16b pPorALoop4-FrC plasmid were serosubtype specific, showing no significant immunofluorescence reactivity or bactericidal activity against other PorA variants. These data provide proof of principle for a DNA fusion plasmid strategy as a novel approach to preparing vaccines based on defined, protective epitopes.

Priming protective CD8 T cell immunity by DNA vaccines encoding chimeric, stress protein-capturing tumor-associated antigen.

DNA vaccines encoding heat shock protein (hsp)-capturing, chimeric peptides containing antigenic determinants of the tumor-associated Ag (TAA) gp70 (an envelope protein of endogenous retrovirus) primed stable, specific, and tumor-protective CD8 T cell immunity. Expression of gp70 transcripts was detectable in most normal tissues but was particularly striking in some (but not all) tumor cell lines tested (including the adenocarcinoma cell line CT26). An approximately 200 residue gp70 fragment or its L(d)-binding antigenic AH1 peptide cloned in-frame behind an hsp-capturing (cT(272)) or noncapturing (T(60)) N-terminal large SV40 tumor Ag sequence was expressed as either hsp-binding or -nonbinding chimeric Ags. Only hsp-capturing, chimeric fusion proteins were expressed efficiently in transfected cell lines and primed TAA-specific CD8 T cell immunity. This immunity mediated protection in the CT26 and mKSA models. A vaccination strategy based on delivering antigenic, hsp-associated TAA fragments can thus prime protective CD8 T cell immunity even if these TAA are of low intrinsic immunogenicity.

DNA fusion vaccines induce epitope-specific cytotoxic CD8(+) T cells against human leukemia-associated minor histocompatibility antigens.

The graft-versus-leukemia effect of allogeneic stem-cell transplantation is believed to be mediated by T-cell recognition of minor histocompatibility antigens on recipient cells. For minor histocompatibility antigens HA-1 and HA-2, normal cell expression is restricted to hemopoietic cells, and boosting the immune response to these antigens may potentiate graft-versus-leukemia effect without accompanying graft-versus-host disease. To increase efficacy, expansion of HA-1- or HA-2-specific CTL before transplantation is desirable. However, primary HA-1- or HA-2-specific CTL expanded in vitro are often of low avidity. An alternative approach is to prime specific CTL responses in vivo by vaccination. Clearly, donor vaccination must be safe and specific. We have developed DNA fusion vaccines able to induce high levels of epitope-specific CTL using linked CD4(+) T-cell help. The vaccines incorporate a domain of tetanus toxin (DOM) fused to a sequence encoding a candidate MHC class I binding peptide. This design generates antitumor CD8(+) T-cell responses and protective immunity in preclinical models. For clinical application, we constructed vaccines encoding HLA-A*0201-restricted peptides from human HA-1 and HA-2, which were fused to DOM, and tested their performance in HLA-A*0201-transgenic mice. Priming induced epitope-specific, IFNgamma-producing CD8(+) T cells with cytotoxic function boosted to high levels with electroporation. Strikingly, these mouse T cells efficiently killed human lymphoblastoid cell lines expressing endogenous HA-1 or HA-2. High avidity is indicated by the independence of cytolysis from CD8/MHC class I interaction. These safe epitope-specific vaccines offer a potential strategy to prime HA-1- or HA-2-specific CTL in transplant donors before adoptive transfer.

Optimizing cancer immunotherapy trials: back to basics.

Attempts to raise effective immunity against cancer are benefiting from information on the nature of the immunity involved and its regulation and, perhaps, now it is time to step back and define our approach in molecular terms prior to clinical testing. Although there are immunological differences between mice and patients, results from murine studies are encouraging early 'translation' of concepts to the clinic and it is vital to take immunological principles emerging from mice into clinical vaccine design. One is the requirement to break tolerance against over-expressed self-antigens, a potentially risky procedure but necessary for several cancer targets. A study in this issue of the European Journal of Immunology attempts to do this by using xenogeneic antigens, albeit with variable outcome. The unstated goal is to activate T-cell help but this can be achieved more effectively by harnessing a predictable anti-microbial repertoire. The second issue lies in the delivery of antigen. One strategy is "prime/boost" using DNA priming and boosting with a viral vector; however, this induces blocking immunity against viral proteins, and must be used judiciously. There are other physical methods to increase immunity such as electroporation, which can itself be used in 'prime/boost' sequence. These twin problems of engagement of T-cell help and delivery of adequate antigen can now be addressed by applying immunological logic to cancer vaccines.