Use of DNAs Expressing HIV-1 Env and Noninfectious HIV-1 Particles to Raise Antibody Responses in Mice

AbstractTwo DNA constructs encoding portions of the human immunodeficiency virus type-1 (HIV-1) genome have been used to raise antibody responses in BABL/c mice. One DNA (pNL4-3.env) expresses the natural form of the envelope glycoprotein (Env) of HIV-1-NL4-3 (NL4-3). The second (pNL4-3.dpol) produces noninfectious NL4-3 particles. These two DNAs (alone or in combination) raised only transient titers of anti-Env IgG. In the same group in which pNL4-3.dpol DNA raised only transient antibody responses to Env, this DNA raised persistent antibody responses to the p24 virion capsid protein (CA). Antibody responses to Env and CA also showed different abilities to be boosted. The final boosts of pNL4-3.dpol DNA increased titers of anti-CA antibody, but failed to boost the falling titers of anti-Env antibody. At peak titers of anti-Env activity, sera with relatively low ELISA titers of anti-Env IgG (end points of 1:6250) had good titers of neutralizing antibody (∼ 1:3800 for 50 TCID50 of NL4-3). At the end of the experiment (a time when anti-Env antibodies had fallen to near background levels), in vitro-restimulated splenocytes from both pNL4-3.env and pNL4-3.dpol DNA vaccine groups exhibited similar cytotoxic activity

‘Give courage to the ladies’: expansive apprenticeship for women in rural Malawi

Apprenticeship in developed and industrialised nations is increasingly understood as a theory of learning which connects workplace activity and formal study. The concept of ‘expansive apprenticeship’ defines frameworks for workforce development where participants acquire knowledge and skills which will help them in the future as well as in their current roles, whilst ‘restrictive’ apprenticeships limit opportunities for wider, lifelong learning. In developing nations apprenticeship is a traditional route to learning and employment, but apprenticeships in these contexts tend to reflect a restrictive approach characterized by narrowly defined roles and weak educational outcomes. This paper examines a project in Malawi which uses concepts of expansive apprenticeship to address barriers to female continuing education and chronic teacher shortages. The Malawi Access to Teaching Scholarship recruited one thousand women to follow a year-long combined programme of academic distance study and practical work experience in rural primary schools. The aim is to increase the numbers of women teachers in Malawi, especially in rural areas. The Scholarship materials and support structures are designed to move participants from restrictive to expansive contexts for learning so that Scholars develop hybrid roles as students, community workers and apprentice pedagogues. The programme’s resources and approach offer an innovative model of expansive apprenticeship in Sub Saharan Africa

Evaluation of protection induced by a dengue virus serotype 2 envelope domain III protein scaffold/DNA vaccine in non-human primates

AbstractWe describe the preclinical development of a dengue virus vaccine targeting the dengue virus serotype 2 (DENV2) envelope domain III (EDIII). This study provides proof-of-principle that a dengue EDIII protein scaffold/DNA vaccine can protect against dengue challenge. The dengue vaccine (EDIII-E2) is composed of both a protein particle and a DNA expression plasmid delivered simultaneously via intramuscular injection (protein) and gene gun (DNA) into rhesus macaques. The protein component can contain a maximum of 60 copies of EDIII presented on a multimeric scaffold of Geobacillus stearothermophilus E2 proteins. The DNA component is composed of the EDIII portion of the envelope gene cloned into an expression plasmid. The EDIII-E2 vaccine elicited robust antibody responses to DENV2, with neutralizing antibody responses detectable following the first boost and reaching titers of greater than 1:100,000 following the second and final boost. Vaccinated and naïve groups of macaques were challenged with DENV2. All vaccinated macaques were protected from detectable viremia by infectious assay, while naïve animals had detectable viremia for 2–7 days post-challenge. All naïve macaques had detectable viral RNA from day 2–10 post-challenge. In the EDIII-E2 group, three macaques were negative for viral RNA and three were found to have detectable viral RNA post challenge. Viremia onset was delayed and the duration was shortened relative to naïve controls. The presence of viral RNA post-challenge corresponded to a 10–30-fold boost in neutralization titers 28 days post challenge, whereas no boost was observed in the fully protected animals. Based on these results, we determine that pre-challenge 50% neutralization titers of >1:6000 correlated with sterilizing protection against DENV2 challenge in EDIII-E2 vaccinated macaques. Identification of the critical correlate of protection for the EDIII-E2 platform in the robust non-human primate model lays the groundwork for further development of a tetravalent EDIII-E2 dengue vaccine

Therapeutic DNA vaccine induces broad T cell responses in the gut and sustained protection from viral rebound and AIDS in SIV-infected rhesus macaques.

Immunotherapies that induce durable immune control of chronic HIV infection may eliminate the need for life-long dependence on drugs. We investigated a DNA vaccine formulated with a novel genetic adjuvant that stimulates immune responses in the blood and gut for the ability to improve therapy in rhesus macaques chronically infected with SIV. Using the SIV-macaque model for AIDS, we show that epidermal co-delivery of plasmids expressing SIV Gag, RT, Nef and Env, and the mucosal adjuvant, heat-labile E. coli enterotoxin (LT), during antiretroviral therapy (ART) induced a substantial 2-4-log fold reduction in mean virus burden in both the gut and blood when compared to unvaccinated controls and provided durable protection from viral rebound and disease progression after the drug was discontinued. This effect was associated with significant increases in IFN-γ T cell responses in both the blood and gut and SIV-specific CD8+ T cells with dual TNF-α and cytolytic effector functions in the blood. Importantly, a broader specificity in the T cell response seen in the gut, but not the blood, significantly correlated with a reduction in virus production in mucosal tissues and a lower virus burden in plasma. We conclude that immunizing with vaccines that induce immune responses in mucosal gut tissue could reduce residual viral reservoirs during drug therapy and improve long-term treatment of HIV infection in humans

GM-CSF Increases Mucosal and Systemic Immunogenicity of an H1N1 Influenza DNA Vaccine Administered into the Epidermis of Non-Human Primates

Background: The recent H5N1 avian and H1N1 swine-origin influenza virus outbreaks reaffirm that the threat of a worldwide influenza pandemic is both real and ever-present. Vaccination is still considered the best strategy for protection against influenza virus infection but a significant challenge is to identify new vaccine approaches that offer accelerated production, broader protection against drifted and shifted strains, and the capacity to elicit anti-viral immune responses in the respiratory tract at the site of viral entry. As a safe alternative to live attenuated vaccines, the mucosal and systemic immunogenicity of an H1N1 influenza (A/New Caledonia/20/99) HA DNA vaccine administered by particle-mediated epidermal delivery (PMED or gene gun) was analyzed in rhesus macaques. Methodology/Principal Findings: Macaques were immunized at weeks 0, 8, and 16 using a disposable single-shot particlemediated delivery device designed for clinical use that delivers plasmid DNA directly into cells of the epidermis. Significant levels of hemagglutination inhibiting (HI) antibodies and cytokine-secreting HA-specific T cells were observed in the periphery of macaques following 1-3 doses of the PMED HA DNA vaccine. In addition, HA DNA vaccination induced detectable levels of HA-specific mucosal antibodies and T cells in the lung and gut-associated lymphoid tissues of vaccinated macaques. Importantly, co-delivery of a DNA encoding the rhesus macaque GM-CSF gene was found to significantly enhance both the systemic and mucosal immunogenicity of the HA DNA vaccine. Conclusions/Significance: These results provide strong support for the development of a particle-mediated epidermal DNA vaccine for protection against respiratory pathogens such as influenza and demonstrate, for the first time, the ability of skindelivered GM-CSF to serve as an effective mucosal adjuvant for vaccine induction of immune responses in the gut and respiratory tract. © 2010 Loudon et al

Where do students in the health professions want to work?

<p>Abstract</p> <p>Background</p> <p>Rural and remote areas of Australia are facing serious health workforce shortages. While a number of schemes have been developed to improve recruitment to and retention of the rural health workforce, they will be effective only if appropriately targeted. This study examines the factors that most encourage students attending rural clinical placements to work in rural Australia, and the regions they prefer.</p> <p>Methods</p> <p>The Careers in Rural Health Tracking Survey was used to examine the factors that most influence medical, nursing and allied health students' preference for practice locations and the locations preferred.</p> <p>Results</p> <p>Students showed a preference for working in large urban centres within one year, but would consider moving to a more rural location later in life. Only 10% of students surveyed said they would never work in a rural community with a population of less than 10 000. Almost half the sample (45%) reported wanting to work overseas within five years. The type of work available in rural areas was found to be the factor most likely to encourage students to practice rurally, followed by career opportunities and challenge</p> <p>Conclusion</p> <p>The decision to practise rurally is the result of a complex interaction between a number of factors including ethnicity, discipline, age and sex, among others. Incentives that aim to entice all students to rural practice while considering only one of these variables are likely to be inadequate.</p

To whom does the law speak? Canvassing a neglected picture of law’s interpretive field

Among the most common strategies underlying the so-called indeterminacy thesis is the following two-step argument: (1) that law is an interpretive practice, and that evidently legal actors more generally hold different (and competing) theories of meaning, which lead to disagreements as to what the law says (that is, as to what the law is); (2) and that, as there is no way to establish the prevalence of one particular theory of meaning over the other, indeterminacy is pervasive in law. In this paper I offer some reflections to resist this trend. In particular I claim that a proper understanding of law as an authoritative communicative enterprise sheds new light on the relation between the functioning of the law and our theories of interpretation, leading to what can be considered a neglected conclusion: the centrality of the linguistic criterion of meaning in our juridical interpretive practices. In the first part of the chapter I discuss speech-act theory in the study of law, assessing its relevance between alternative options. Then I tackle the ‘to whom does the law speak?’ question, highlighting the centrality of lay-people for our juridical practices. Lastly, I examine the consequences of this neglected fact for our interpretive theories

De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo hACE2 decoys to neutralize SARS-CoV-2. The best decoy, CTC-445.2, binds with low nanomolar affinity and high specificity to the RBD of the spike protein. Cryo-EM shows that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, shows ~10-fold improvement in binding. CTC-445.2d potently neutralizes SARS-CoV-2 infection of cells in vitro and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge

Oral Immunization with a Live Coxsackievirus/HIV Recombinant Induces Gag p24-Specific T Cell Responses

The development of an HIV/AIDS vaccine has proven to be elusive. Because human vaccine trials have not yet demonstrated efficacy, new vaccine strategies are needed for the HIV vaccine pipeline. We have been developing a new HIV vaccine platform using a live enterovirus, coxsackievirus B4 (CVB4) vector. Enteroviruses are ideal candidates for development as a vaccine vector for oral delivery, because these viruses normally enter the body via the oral route and survive the acidic environment of the stomach.We constructed a live coxsackievirus B4 recombinant, CVB4/p24(73(3)), that expresses seventy-three amino acids of the gag p24 sequence (HXB2) and assessed T cell responses after immunization of mice. The CVB4 recombinant was physically stable, replication-competent, and genetically stable. Oral or intraperitoneal immunization with the recombinant resulted in strong systemic gag p24-specific T cell responses as determined by the IFN-gamma ELISPOT assay and by multiparameter flow cytometry. Oral immunization with CVB4/p24(73(3)) resulted in a short-lived, localized infection of the gut without systemic spread. Because coxsackieviruses are ubiquitous in the human population, we also evaluated whether the recombinant was able to induce gag p24-specific T cell responses in mice pre-immunized with the CVB4 vector. We showed that oral immunization with CVB4/p24(73(3)) induced gag p24-specific immune responses in vector-immune mice.The CVB4/p24(73(3)) recombinant retained the physical and biological characteristics of the parental CVB4 vector. Oral immunization with the CVB4 recombinant was safe and resulted in the induction of systemic HIV-specific T cell responses. Furthermore, pre-existing vector immunity did not preclude the development of gag p24-specific T cell responses. As the search continues for new vaccine strategies, the present study suggests that live CVB4/HIV recombinants are potential new vaccine candidates for HIV

Shiga Toxin Binding to Glycolipids and Glycans

Background: Immunologically distinct forms of Shiga toxin (Stx1 and Stx2) display different potencies and disease outcomes, likely due to differences in host cell binding. The glycolipid globotriaosylceramide (Gb3) has been reported to be the receptor for both toxins. While there is considerable data to suggest that Gb3 can bind Stx1, binding of Stx2 to Gb3 is variable. Methodology: We used isothermal titration calorimetry (ITC) and enzyme-linked immunosorbent assay (ELISA) to examine binding of Stx1 and Stx2 to various glycans, glycosphingolipids, and glycosphingolipid mixtures in the presence or absence of membrane components, phosphatidylcholine, and cholesterol. We have also assessed the ability of glycolipids mixtures to neutralize Stx-mediated inhibition of protein synthesis in Vero kidney cells. Results: By ITC, Stx1 bound both Pk (the trisaccharide on Gb3) and P (the tetrasaccharide on globotetraosylceramide, Gb4), while Stx2 did not bind to either glycan. Binding to neutral glycolipids individually and in combination was assessed by ELISA. Stx1 bound to glycolipids Gb3 and Gb4, and Gb3 mixed with other neural glycolipids, while Stx2 only bound to Gb3 mixtures. In the presence of phosphatidylcholine and cholesterol, both Stx1 and Stx2 bound well to Gb3 or Gb4 alone or mixed with other neutral glycolipids. Pre-incubation with Gb3 in the presence of phosphatidylcholine and cholesterol neutralized Stx1, but not Stx2 toxicity to Vero cells