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Date of Acceptance: 29/04/2015 Unprecedented rates of deforestation on tropical islands are threatening high numbers of endemic species. A lack of empirical evidence for the impacts of land-use change on biodiversity is hindering conservation activities in these regions. We assessed the impacts of land-use change on the avifauna of the tropical island of Makira in the Solomon Islands. We examined species richness and community assemblages, with a particular focus on endemism and functional traits, to provide further insight into the 'conservation value' of the dominant land-use types present on Makira (i.e. intact forest, secondary forest, food gardens, mixed cocoa plantations, and monoculture cocoa plantations). We found species richness was similar across habitats, but endemic species richness decreased in more intensive land uses. There were significant differences in the occurrence of functional groups between habitats. Fifteen out of the 42 species observed (35%) showed significant variation in abundance across habitats. Of those species that varied, 80% were endemic to Makira or to Melanesia, with seven of these endemics being absent from the monoculture cocoa. As tropical islands have less functional redundancy than continental landmasses, protecting the remaining forest and improving habitat connectivity will be even more critical for conserving their endemic species and maintaining ecosystem functioning. Postprint

Pro-poor conservation strategies are touted as a panacea for achieving biodiversity conservation and poverty reduction, yet there remains limited evidence for success in achieving these dual objectives. Is this lack of evidence a sign that the approach is failing or an indicator that we are not capturing appropriate measures of success? In this review, we demonstrate that the lack of evidence for success in pro-poor conservation can be attributed to focusing on the bigger picture and overlooking the details. This has led to two fundamental flaws, (1) an ambiguous use of definitions and (2) inappropriate monitoring, both of which are compounded by limited understanding between traditional scientific disciplines and which render true conclusion of success impossible. Due to the current lack of evidence, pro-poor conservation risks basing decisions on belief rather than evidence, repeating mistakes and missing opportunities to replicate successes. We propose five key recommendations that will facilitate the identification of successful pro-poor conservation interventions and the conditions under which they work. Adoption of these recommendations will ensure pro-poor conservation is built on clear definitions and engages in meaningful participation with rigorous monitoring and reporting of outcomes, aiding replication and scaling up of pro-poor conservation successes in intelligent and evidence-based ways.

Aims and methodThe effectiveness of official support provided following a disaster has not been fully evaluated. This study aimed to ascertain whether there was an association between perceived support shortly after the 2004 Indian Ocean tsunami and later mental health symptoms in those affected, and to explore the factors associated with this. A survey, semi-structured interviews and focus group were used to explore the experiences and perceptions of 116 individuals severely affected by the tsunami.ResultsAgency or official support was perceived as poor overall. Perceived ineffectiveness of support available within a few days after the tsunami was associated with increased symptoms of post-traumatic stress disorder 15–19 months later. The strongest themes that emerged from our study were that support provided in a humane manner was perceived as effective and that uncoordinated support, poor communication and limited accessibility to support and information were perceived as ineffective.Clinical implicationsImproved planning of coordinated, flexible, multi-agency responses to traumatic events before they occur is required.

PurposeThis paper seeks to explore staff experiences of working in Crisis Resolution and Home Treatment (CRHT). There is a paucity of research in the area and a particular lack of in‐depth qualitative accounts of staff experiences with most research focused on output and outcomes of CRHTs, including inpatient admission and bed‐occupancy rates.Design/methodology/approachInterpretative phenomenological analysis (IPA) was used to investigate participants' experiences of working in this service context. Five CRHT workers were interviewed about their experiences, including the aspects of their work that they found enjoyable and those that they found stressful, and how they coped with challenges related to the work.FindingsThree master themes, each with corresponding subordinate themes, emerged from the analysis. These were: “motivating factors”, “stressors” and “coping”. These findings indicated these subjective experiences emphasised the opportunities to make a difference and help service users and build therapeutic relationships. Perceived stressors were understood in the context of a complex service context, operational issues, responsibility for service users, and supervision and training needs.Research limitations/implicationsThe implications of the research for CRHT staff are discussed as well as the limitations of this study.Originality/valueThis study offers a timely development in understanding the experiences of staff working in CRHTs.

Hepatitis C virus (HCV) infects an estimated 170 million individuals worldwide and is associated with an increased incidence of liver fibrosis, cirrhosis, and hepatocellular carcinoma. Currently approved therapies to treat HCV infection consist of combinations of pegylated alpha interferon and ribavirin which result in a sustained viral response in 40 to 60% of patients. Efforts to develop improved therapies include the development of direct inhibitors of virally encoded enzymes such as the viral RNA-dependent RNA polymerase. A nucleoside analog, 2′- C -methyl-7-deaza-adenosine (MK-0608), has been shown to inhibit viral RNA replication in the subgenomic HCV genotype 1b replicon, with a 50% effective concentration (EC 50 ) of 0.3 μM (EC 90 = 1.3 μM). To determine efficacy in vivo, MK-0608 was administered to HCV-infected chimpanzees, resulting in dose- and time-dependent decreases in plasma viral loads. In separate experiments, chimpanzees dosed for 7 days with MK-0608 at 0.2 and 2 mg per kg of body weight per day by intravenous administration experienced average reductions in viral load of 1.0 and >5 log 10 IU/ml, respectively. Two other HCV-infected chimpanzees received daily doses of 1 mg MK-0608 per kg via oral administration. After 37 days of oral dosing, one chimpanzee with a high starting viral load experienced a reduction in viral load of 4.6 log 10 , and the viral load in the other chimpanzee fell below the limit of quantification (LOQ) of the HCV TaqMan assay (20 IU/ml). Importantly, viral load remained below the LOQ throughout the duration of dosing and for at least 12 days after dosing ended. The results demonstrate a robust antiviral effect on the administration of MK-0608 to HCV-infected chimpanzees.

Summary Background Adverse psychological reactions are relatively frequent in professional ambulance crews who attend traumatic events, yet appear unusual in lay persons who attempt resuscitation of victims of out of hospital cardiac arrest. Aim To investigate the psychological profile of first responders to gain insight into possible factors that might protect them against such reactions. Methods Qualitative study of first responders in a community scheme in Barry, South Wales. In depth semi-structured interviews with six subjects were analysed using Interpretive Phenomenological Analysis (IPA). Results The study identified a resilience phenomenon in first responders accounted for by certain enabling core beliefs about their role, their capacity, and about the meaning of negative and positive outcomes for themselves. A realistic appreciation of their own limitations, confidence in their ability to perform as trained and being able to handle positive and negative outcomes were prominent features. The ability to act with emotional detachment appears a further protective mechanism. This mindset, loosely described as ‘a philosophy’, protects against the development of adverse reactions to stress or from becoming unduly concerned about negative outcomes. The responders had altruistic motives for undertaking the role yet were capable of operating with a high degree of naturally occurring resilience to stress or undermining anxiety. It is the combination of being motivated by altruism coupled with an inherent resilience that appears to be the crucial protective mechanism. Conclusions The group demonstrated an apparently innate resilience to the adverse psychological effects of responding with an AED in a PAD scheme. This enables them to operate optimally in stressful situations without experiencing the negative psychological consequences that might otherwise arise. This information may be used to raise awareness about the psychological requirements for the role and to assist screening or selection processes.

The prophylactic efficacy of DNA and replication-incompetent adenovirus serotype 5 (Ad5) vaccine vectors expressing simian immunodeficiency virus (SIV) Gag was examined in rhesus macaques using an SIVmac239 challenge. Cohorts of either Mamu-A*01(+) or Mamu-A*01(−) macaques were immunized with a DNA prime-Ad5 boost regimen; for comparison, a third cohort consisting of Mamu-A*01(+) monkeys was immunized using the Ad5 vector alone for both prime and boost. All animals, along with unvaccinated control cohorts of Mamu-A*01(+) and Mamu-A*01(−) macaques, were challenged intrarectally with SIVmac239. Viral loads were measured in both peripheral and lymphoid compartments. Only the DNA prime-Ad5-boosted Mamu-A*01(+) cohort exhibited a notable reduction in peak plasma viral load (sevenfold) as well as in early set-point viral burdens in both plasma and lymphoid tissues (10-fold) relative to those observed in the control monkeys sharing the same Mamu-A*01 allele. The degree of control in each animal correlated with the levels of Gag-specific immunity before virus challenge. However, virus control was short-lived, and indications of viral escape were evident as early as 6 months postinfection. The implications of these results in vaccine design and clinical testing are discussed.

Small molecule mu agonists based on the 4-phenyl piperidine scaffold were designed and synthesized to further investigate the therapeutic potential of loperamide analogs. The resulting compounds show excellent agonistic activity towards the human mu receptor with interesting SAR trends within the series.

We compared the human immunodeficiency virus type 1 (HIV-1)-specific cellular immune responses elicited in nonhuman primates by HIV-1 gag -expressing replication-defective adenovirus serotype 5 (Ad5) or poxvirus vectors, used either alone or in combination with each other. The responses arising from a heterologous Ad5 priming-poxvirus boosting regimen were significantly greater than those elicited by homologous regimens with the individual vectors or by a heterologous poxvirus priming-Ad5 boosting regimen. The heterologous Ad5 priming-poxvirus boosting approach may have potential utility in humans as a means of inducing high levels of cellular immunity.

A universal influenza virus vaccine that does not require frequent updates and/or annual immunizations will offer significant advantages over current seasonal flu vaccines. The highly conserved influenza virus A M2 membrane protein has been previously suggested as a potential antigen target for such a vaccine. Here, we report systematic evaluation of M2 peptide conjugate vaccines (synthetic peptides of M2 extracellular domain conjugated to keyhole limpet hemocyanin (KLH) or Neisseria meningitidis outer membrane protein complex (OMPC)) in mice, ferrets, and rhesus monkeys. The conjugate vaccines were highly immunogenic in all species tested and were able to confer both protection against lethal challenge of either H1N1 or H3N1 virus in mice and reduce viral shedding in the lower respiratory tracts of mice and ferrets. The protection against lethal challenge in mice could also be achieved by passive transfer of monkey sera containing high M2 antibody titers. In addition, we showed that M2 antisera were cross reactive with M2 peptides derived from a wide range of human influenza A strains, but they failed to react with M2 peptides of the pathogenic H5N1 virus (A/Hong Kong/97). The data presented here will permit better understanding of the potential of an M2-based vaccine approach.

Mu opioid receptors are expressed throughout the central and peripheral nervous systems. Peripheral inflammation leads to an increase in mu receptor present on the peripheral terminals of primary sensory neurons. Activation of peripheral mu receptors produces potent antihyperalgesic effects in both humans and animals. Here, we describe the in vivo pharmacological properties of the structurally novel, highly potent, systemically available yet peripherally restricted mu opioid agonist, [8-(3,3-diphenyl-propyl)-4-oxo-1-phenyl-1,3,8-triaza-spiro[4.5]dec-3-yl]-acetic acid (DiPOA). DiPOA administered i.p. produced naltrexone-sensitive, dose-dependent reversal of Freund's complete adjuvant-induced inflammatory mechanical hyperalgesia (1-10 mg/kg). Maximum percent reversal (67%) was seen 1 h postadministration at 10 mg/kg (the highest dose studied). DiPOA also proved antihyperalgesic in a model of postsurgical pain with a maximum percent reversal of 85% 1 h postadministration at 30 mg/kg i.p. (the highest dose studied). DiPOA administered i.p. had no effect in the tail flick assay of acute pain (0.1-10 mg/kg), produced no ataxia as measured by latency to fall from an accelerating rotarod (3-30 mg/kg), and was not antihyperalgesic in the Seltzer model of neuropathic pain (1-10 mg/kg). This is the first report of a peripherally restricted, small-molecule mu opioid agonist that is nonsedating, antihyperalgesic, and effective against inflammatory and postsurgical pain when administered systemically.

Mu opioid receptors are present throughout the central and peripheral nervous systems. Peripheral inflammation causes an increase in mu receptor levels on peripheral terminals of primary afferent neurons. Recent studies indicate that activation of peripheral mu receptors produces antihyperalgesic effects in animals and humans. Here, we describe the in vitro pharmacological and in vivo pharmacokinetic properties of a novel, highly potent, and peripherally restricted mu opioid agonist, [8-(3,3-diphenyl-propyl)-4-oxo-1-phenyl-1,3,8-triaza-spiro[4.5]dec-3-yl]-acetic acid (DiPOA). In a radioligand binding assay, DiPOA inhibited [(3)H]-diprenorphine binding to recombinant human mu receptors with a K(i) value of approximately 0.8 nM. The rank order of affinity for DiPOA binding to recombinant human opioid receptors was mukappa approximately ORL-1delta. DiPOA showed potent agonist effects in a human mu receptor guanosine 5'-O-(3-[(35)S]thio)triphosphate functional assay, with an EC(50) value of approximately 33 nM and efficacy of approximately 85% [normalized to the mu agonist, [d-Ala2,MePhe4,Gly(ol)5]enkephalin]. Low potency agonist activity was also seen at ORL-1 and kappa receptors. DiPOA bound competitively to the opioid binding site of human mu receptors as demonstrated by a parallel rightward shift in its concentration-response curve in the presence of increasing concentrations of naltrexone. High and sustained (or =5 h) plasma levels for DiPOA were achieved following intraperitoneal administration at 3 and 10 mg/kg; central nervous system penetration, however, wasor =4% of the plasma concentration, even at levels exceeding 1500 ng/ml. As such, DiPOA represents a systemically available, peripherally restricted small molecule mu opioid agonist that will aid in understanding the role played by mu opioid receptors in the periphery.

Set-point viral load is positively correlated with the extent of initial viral replication in pathogenic simian-human immunodeficiency virus (SHIV) infection. To elucidate the mechanisms underlying the correlation, we conducted a systematic investigation in rhesus monkeys infected with the highly pathogenic SHIV 89.6P. This model is widely used in the preclinical evaluation of AIDS vaccine candidates and a thorough understanding of the model's biology is important to the proper interpretation of these evaluations. We found that the levels of peak viremia were positively correlated not only with the levels of set-point viremia but, importantly, with the extent of initial overall immune destruction as indicated by the degree of CD4+ T cell depletion and lymph node germinal center (GC) formation. The extent of initial overall immune destruction was inversely correlated with subsequent development and maintenance of virus-specific cellular and humoral immune responses. Thus, these data suggest that the extent of early immune damage determines the development and durability of virus-specific immunity, thereby playing a critical role in establishing the levels of set-point viral replication in SHIV infection. Vaccines that limit both the initial viral replication and the extent of early immune damage will therefore mediate long-term virus replication control and mitigation of long-term immune destruction in this model of immunodeficiency virus infection.

The cellular immunogenicity of formulated plasmid DNA and replication-defective human adenovirus serotype 5 (Ad5) vaccine vectors expressing a codon-optimized human immunodeficiency virus type 1 gag gene was examined in baboons. The Ad5 vaccine was capable of inducing consistently strong, long-lived CD8 + -biased T-cell responses and in vitro cytotoxic activities. The DNA vaccine-elicited immune responses were weaker than those elicited by the Ad5 vaccine and highly variable; formulation with chemical adjuvants led to moderate increases in the levels of Gag-specific T cells. Increasing the DNA-primed responses with booster doses of either Ad5 or modified vaccinia virus Ankara vaccines suggests a difference in the relative levels of cytotoxic and helper responses. The implications of these results are discussed.

The tetracycline (Tc)-dependent system in its "on" version (rtTA system) displays a baseline activity in the uninduced state, severely limiting its potential applicability in human gene therapy. So far, two different strategies to circumvent this limitation have been described. On one side, co-expression of the tetracycline regulated repressor tTS kid has proved capable of substantially reducing the baseline activity of rtTA. On the other, novel versions of the activator, namely rtTA2 s -S2 and rtTA2 s -M2, with a lower basal activity have been engineered. We have combined these two approaches by co-expressing TS kid with the novel transactivators. Bicistronic vectors were constructed that co-express TS kid with rtTA, rtTA2 s -S2, or rtTA2 s M2, through an internal ribosome entry site (plasmids IRES-A, IRES-S2, and IRES-M2, respectively). IRES-M2 proved to be the most effective construct ex vivo: it displayed a negligible basal activity, > 1000 fold inducibility, and high responsiveness to doxycycline (Dox). Upon delivery as plasmid DNA in mouse muscles, IRES-M2 facilitated 1000-fold induction of serum alkaline phosphatase ( SEAP ) gene expression and long-term, stringent, and strictly Dox-dose-dependent regulation of erythropoietin ( Epo ) gene expression. Tight regulation of the gene encoding SEAP was demonstrated also in non-human primates. Notably, the system was induced in animals by Dox-dosing regimens comparable to those used in humans.

A synthetic gene consisting of the reverse transcriptase (RT) and integrase (IN) domains of human immunodeficiency virus type 1 (HIV-1) pol was constructed using codons most frequently used in humans. The humanized pol gave dramatically improved levels of Rev-independent, in vitro protein production in mammalian cells and elicited much stronger cellular immunity in rodents than did virus-derived gene. Specifically, BALB/c mice were immunized with plasmids and/or recombinant vaccinia virus constructs expressing the synthetic gene. High frequencies of Pol-specific T lymphocytes were detected in these animals by the gamma interferon enzyme-linked immunospot assay against pools of short overlapping peptides. Characterization of the stimulatory peptides from these pools indicates that the optimized gene constructs are able to effectively activate both CD4 + and CD8 + T cells. Immunization of rhesus macaques with DNA vaccines expressing the humanized pol coupled to a human tissue plasminogen activator leader sequence led to pronounced in vitro cytotoxic T-lymphocyte killing activities and enhanced levels of circulating Pol-specific T cells, comparable to those observed in HIV-1-infected human subjects. Thus, optimizing the immunogenic properties of HIV-1 Pol at the level of the gene sequence validates it as an antigen and provides an important step toward the construction of a potent pol -based HIV-1 vaccine component.

TD was supported by a Natural Environment Research Council studentship (DTG: AGS6-NERC10). Additional funding for fieldwork was provided by Chester Zoo and Rufford Small Grants (11022-1) Date of Acceptance: 30/01/2014 Understanding the local socioeconomic context is important for the design of appropriate conservation initiatives and associated monitoring strategies, especially in areas with high degrees of inequality, to ensure conservation interventions do not inadvertently further disadvantage vulnerable people. Typical assessments of wealth inequality in remote rural areas are constrained by limited engagement with a cash economy, complex family and tribal ties, and an absence of basic infrastructure. This paper presents a simple participatory approach to measure wealth inequality that does not predefine indicators, such as income or assets, but allows the local people choose the most appropriate indicators. A case study from the Solomon Islands revealed poor households in Kahua were characterized by fewer members, fewer members of working age, and fewer male members than wealthier households. The poor also owned fewer of the locally defined indicators of wealth that were collectively correlated with limited land tenure, and, consequently, conservation or development initiatives that are tied to land in Kahua will be less likely to assist the poorest. Adopting this participatory approach could improve the effectiveness of community-based conservation, through facilitating opportunities to explore local poverty and routes for alleviation. Postprint

The potential utility of plasmid DNA as an HIV-1 vaccination modality currently is an area of active investigation. However, recent studies have raised doubts as to whether plasmid DNA alone will elicit immune responses of sufficient magnitude to protect against pathogenic AIDS virus challenges. We therefore investigated whether DNA vaccine-elicited immune responses in rhesus monkeys could be augmented by using either an IL-2/Ig fusion protein or a plasmid expressing IL-2/Ig. Sixteen monkeys, divided into four experimental groups, were immunized with (i) sham plasmid, (ii) HIV-1 Env 89.6P and simian immunodeficiency virus mac239 Gag DNA vaccines alone, (iii) these DNA vaccines and IL-2/Ig protein, or (iv) these DNA vaccines and IL-2/Ig plasmid. The administration of both IL-2/Ig protein and IL-2/Ig plasmid induced a significant and sustainedin vivoactivation of peripheral T cells in the vaccinated monkeys. The monkeys that received IL-2/Ig plasmid generated 30-fold higher Env-specific antibody titers and 5-fold higher Gag-specific, tetramer-positive CD8+ T cell levels than the monkeys receiving the DNA vaccines alone. IL-2/Ig protein also augmented the vaccine-elicited immune responses, but less effectively than IL-2/Ig plasmid. Augmentation of the immune responses by IL-2/Ig was evident after the primary immunization and increased with subsequent boost immunizations. These results demonstrate that the administration of IL-2/Ig plasmid can substantially augment vaccine-elicited humoral and cellular immune responses in higher primates.

The use of cytokines has shown promise as an approach for amplifying vaccine-elicited immune responses, but the application of these immunomodulatory molecules in this setting has not been systematically explored. In this report we investigate the use of protein- and plasmid-based cytokines to augment immune responses elicited by an HIV-1 gp120 plasmid DNA vaccine (pV1J-gp120) in mice. We demonstrate that immune responses elicited by pV1J-gp120 can be either augmented or suppressed by administration of plasmid cytokines. A dicistronic plasmid expressing both gp120 and IL-2 induced a surprisingly weaker gp120-specific immune response than did the monocistronic pV1J-gp120 plasmid. In contrast, systemic delivery of soluble IL-2/Ig fusion protein following pV1J-gp120 vaccination significantly amplified the gp120-specific immune response as measured by Ab, proliferative, and CTL levels. Administration of plasmid IL-2/Ig had different effects on the DNA vaccine-elicited immune response that depended on the temporal relationship between Ag and cytokine delivery. Injection of plasmid IL-2/Ig either before or coincident with pV1J-gp120 suppressed the gp120-specific immune response, whereas injection of plasmid IL-2/Ig after pV1J-gp120 amplified this immune response. To maximize immune responses elicited by a DNA vaccine, therefore, it appears that the immune system should first be primed with a specific Ag and then amplified with cytokines. The data also show that IL-2/Ig is more effective than native IL-2 as a DNA vaccine adjuvant.

It is generally thought that an effective vaccine to prevent HIV-1 infection should elicit both strong neutralizing antibody and cytotoxic T lymphocyte responses. We recently demonstrated that potent, boostable, long-lived HIV-1 envelope (Env)-specific cytotoxic T lymphocyte responses can be elicited in rhesus monkeys using plasmid-encoded HIV-1envDNA as the immunogen. In the present study, we show that the addition of HIV-1 Env protein to this regimen as a boosting immunogen generates a high titer neutralizing antibody response in this nonhuman primate species. Moreover, we demonstrate in a pilot study that immunization with HIV-1envDNA (multiple doses) followed by a final immunization with HIV-1envDNA plus HIV-1 Env protein (envgene from HXBc2 clone of HIV IIIB; Env protein from parental HIV IIIB) completely protects monkeys from infection after i.v. challenge with a chimeric virus expressing HIV-1env(HXBc2) on a simian immmunodeficiency virusmacbackbone (SHIV-HXBc2). The potent immunity and protection seen in these pilot experiments suggest that a DNA prime/DNA plus protein boost regimen warrants active investigation as a vaccine strategy to prevent HIV-1 infection.

Plasmid DNA vaccines encoding HIV-1 env were used to immunize mice and nonhuman primates. Plasmids were prepared that produced either secreted gp120 or full-length gp160. Mice immunized with gp120 DNA developed strong antigen-specific antibody responses, CD8+ cytotoxic T lymphocytes (CTL) (following in vitro restimulation with gp120-derived peptide), and showed in vitro proliferation and Th1-like cytokine secretion [γ-interferon, interleukin (IL)-2 with little or no IL-4] by lymphocytes obtained from all lymphatic compartments tested (spleen, blood, and inguinal, iliac, and mesenteric lymph nodes). This indicated that systemic anti-gp120 cell-mediated immunity was induced by this DNA vaccine. Although similar antibody responses were observed in mice immunized by either intramuscular or intradermal routes, T cell responses were significantly stronger in mice injected intramuscularly. Rhesus monkeys immunized with both gp120 and gp160 DNAs exhibited significant CD8+ CTL responses, following in vitro restimulation of peripheral blood lymphocytes with antigen. These experiments demonstrate that DNA immunization elicits potent immune responses against HIV env in both a rodent and a nonhuman primate species.

Expression vectors encoding either HIV-1 gp160/rev, gp120, or rev alone were used for direct vaccination of mice and nonhuman primates. Each vaccine elicited long-lived (> 7 months) helper T cell responses in mice and monkeys as measured by in vitro proliferation of splenocytes following recombinant antigen treatment. Cytokine assays of the cell supernatants showed that approximately 100-fold more gamma-interferon than IL-4 was secreted during culture indicating that these vaccines elicited TH1-like responses. CD8+ CTL activities were also observed both in mice and rhesus. The gp120 and gp160/rev vaccines elicited antigen-specific antibodies, although these responses were more variable and lower magnitude for gp160/rev, and gp120 DNA-vaccinated African green monkeys had moderate levels of neutralizing antibodies. No antibodies were found against rev (an intracellular protein) with either rev vaccine. Similar antibody titers were obtained for gp120 by either intramuscular or intradermal injection although T cell responses were generally lower by intradermal route. These results indicate that DNA vaccines may provide a powerful means to elicit cellular and humoral immune responses against HIV.

Efforts to develop novel, interferon-sparing therapies for treatment of chronic hepatitis C (HCV) infection are contingent on the ability of combination therapies consisting of direct antiviral inhibitors to achieve a sustained virologic response. This work demonstrates a proof of concept that coadministration of the nucleoside analogue MK-0608 with the protease inhibitor MK-7009, both of which produced robust viral load declines as monotherapy, to an HCV-infected chimpanzee can achieve a cure of infection.

The prophylactic efficacies of several multivalent replication-incompetent adenovirus serotype 5 (Ad5) vaccines were examined in rhesus macaques using an intrarectal high-dose simian immunodeficiency virus SIVmac239 challenge model. Cohorts of Mamu-A*01 + /B*17 − Indian rhesus macaques were immunized with one of several combinations of Ad5 vectors expressing Gag, Pol, Nef, and Env gp140; for comparison, a Mamu-A*01 + cohort was immunized using the Ad5 vector alone. There was no sign of immunological interference between antigens in the immunized animals. In general, expansion of the antigen breadth resulted in more favorable virological outcomes. In particular, the order of efficacy trended as follows: Gag/Pol/Nef/Env ≈ Gag/Pol > Gag ≈ Gag/Pol/Nef > Nef. However, the precision in ranking the vaccines based on the study results may be limited by the cohort size, and as such, may warrant additional testing. The implications of these results in light of the recent discouraging results of the phase IIb study of the trivalent Ad5 HIV-1 vaccine are discussed.

Safe and efficient methods for in vivo delivery of transgenes of interest must be developed so that the promise of these therapies can be practically used in the clinic. In this work, we describe the use of electrostimulation to enhance the in vivo efficiency of plasmid DNA delivery. The method was optimized to work over a range of moderate frequencies, utilizing low field strengths and simple symmetrical waveforms. After studying several parameters of delivery in mice, we demonstrate how this methodology can be employed to significantly improve both gene expression (over 16-fold) and the immunogenicity of HIV-1 vaccines (over 28-fold) compared to naked DNA in non-human primates. Compared to an efficient viral Ad5 vector system, the gene expression levels of DNA + electrostimulation were surprisingly within a factor of four of the viral delivery system.

Lack of virus specific antibody response is commonly observed in both HIV-1-infected humans and SIV-infected monkeys with rapid diseaseprogression. However, the mechanisms underlying this important observation still remain unclear. In a titration study of a SIVmac239 viral stock,three out of six animals with viral inoculation rapidly progressed to AIDS within 5 months. Unexpectedly, there was no obvious depletion ofCD4 + T cells in both peripheral and lymph node (LN) compartments in these animals. Instead, progressive depletion of proliferating B cells anddisruption of the follicular dendritic cell (FDC) network in germinal centers (GC) was evident in the samples collected at as early as 20 days afterviral challenge. This coincided with undetectable, or weak and transient, virus-specific antibody responses over the course of infection. In situhybridization of SIV RNA in the LN samples revealed a high frequency of SIV productively infected cells and large amounts of accumulated viralRNA in the GCs in these animals. Early severe depletion of GC proliferating B cells and disruption of the FDC network may thus result in aninability to mount a virus-specific antibody response in rapid progressors, which has been shown to contribute to accelerated disease progressionof SIV infection.© 2006 Elsevier Inc. All rights reserved.

Success in resolving hepatitis C virus (HCV) infection has been correlated to vigorous, multispecific, and sustained CD8 + T-cell response in humans and chimpanzees. The efficacy of inducing T-cell-mediated immunity by recombinant serotype 5 adenovirus vector has been proven in many animal models of infectious diseases, but its immunogenicity can be negatively influenced by preexisting immunity against the vector itself. To evaluate the less prevalent adenovirus serotype 6 (Ad6) as an alternative vector for and HCV vaccine development, we have generated serotype 5 and 6 adenoviral vectors directing expression of the nonstructural region of HCV (MRKAd5-NSmut and MRKAd6-NSmut). Immunogenicity studies in mice showed that the two vectors induced comparable T-cell responses but that only MRKAd6-NSmut was not suppressed in the presence of anti-Ad5 immunity. In contrast, preexisting anti-Ad5 immunity dramatically blunted the immunogenicity of the serotype 5-based HCV vector. Furthermore, MRKAd6-NSmut showed equivalent potency, breadth, and longevity of HCV-specific T-cell responses in rhesus macaques as the corresponding Ad5-based vector over a wide range of doses and was capable of boosting DNA-primed animals even if administered at low doses. These data support the use of the MRKAd6-NSmut for anti-HCV immunotherapy and, more generally, for the Ad6 serotype as a better genetic vaccine vehicle than Ad5.

Simian-human immunodeficiency virus (SHIV) challenge studies in rhesus macaques were conducted to evaluate the efficacy of adenovirus-based vaccines in the context of different major histocompatibility complex class I genetic backgrounds and different vaccine compositions. Mamu-A * 01 allele-negative rhesus monkeys were immunized with one of the following vaccine constructs: (i) replication-defective recombinant adenovirus type 5 (Ad5) expressing human immunodeficiency virus type 1 (HIV-1) Tat (Ad5/HIVTat); (ii) Ad5 vector expressing simian immunodeficiency virus (SIV) Gag (Ad5/SIVGag); (iii) Ad5 vector expressing the truncated HIV-1 jrfl Env, gp140 (Ad5/gp140_jrfl); (iv) Ad5 vector expressing the SHIV-89.6P gp140 (Ad5/gp140_89.6P); or (v) the combination of Ad5/SIVGag and Ad5/gp140_jrfl. Following intravenous challenge with SHIV-89.6P, only those cohorts that received vaccines expressing Gag or Env exhibited an attenuation of the acute viremia and associated CD4-cell lymphopenia. While no prechallenge neutralizing antibody titers were detectable in either Ad5/gp140-vaccinated group, an accelerated neutralizing antibody response was observed in the Ad5/gp140_89.6P-vaccinated group upon viral challenge. The set-point viral loads in the Ad5/SIVGag- and Ad5/gp140_jrfl-vaccinated groups were associated with the overall strength of the induced cellular immune responses. To examine the contribution of Mamu-A * 01 allele in vaccine efficacy against SHIV-89.6P challenge, Mamu-A * 01-positive monkeys were immunized with Ad5/SIVGag. Vaccine-mediated protection was significantly more pronounced in the Mamu-A * 01-positive monkeys than in Mamu-A * 01-negative monkeys, suggesting the strong contributions of T-cell epitopes restricted by the Mamu-A * 01 molecule. The implications of these results in the development of an HIV-1 vaccine will be discussed.

Replication-defective recombinant adenoviruses (rAd) are used as vectors for vaccines as well as for gene therapy. To determine type-specific antibodies to adenovirus (Ad) serotypes 2, 5, 24, 34, and 35, we developed quantitative neutralization assays using recombinant adenoviruses with the secreted alkaline phosphatase (SEAP) reporter gene. Among the standardized parameters, the concentration of infectious and noninfectious adenoviral particles used in the assay is critical for a reliable comparison of data from different studies. The usefulness of this assay was demonstrated in a pilot epidemiologic study of 40 healthy individuals. In this study, the highest prevalence of antiadenovirus antibodies was found for the Ad2 serotype (82.5%), followed by Ad5 (35%). The prevalence of antiadenovirus antibodies for the serotypes 24, 34, and 35 was low (7.5%, 2.5%, and 0%, respectively). In addition, epidemiologic parameters such as gender and age were statistically evaluated. A positive association was found between age and the presence of anti-Ad5 antibodies. The assay was also useful for evaluating the presence of antiadenovirus antibodies in the design of vaccines using a rhesus monkey model. In this animal model, it was possible to determine differential dose and time responses, and the specificity for the detection of neutralizing antibodies was assessed. The evaluation of serotype-specific neutralizing antibodies can be of both clinical and epidemiologic importance as a means of selecting the appropriate serotype adenovector(s).

Cellular immune responses, particularly those associated with CD3 + CD8 + cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defective adenovirus serotype 5 (Ad5) vector, each expressing the same codon-optimized HIV-1 gag gene for immunogenicity in rhesus monkeys. The DNA vaccines were formulated with and without one of two chemical adjuvants (aluminum phosphate and CRL1005). The Ad5- gag vector was the most effective in eliciting anti-Gag CTL. The vaccine produced both CD4 + and CD8 + T-cell responses, with the latter consistently being the dominant component. To determine the effect of existing antiadenovirus immunity on Ad5- gag -induced immune responses, monkeys were exposed to adenovirus subtype 5 that did not encode antigen prior to immunization with Ad5- gag . The resulting anti-Gag T-cell responses were attenuated but not abolished. Regimens that involved priming with different DNA vaccine formulations followed by boosting with the adenovirus vector were also compared. Of the formulations tested, the DNA-CRL1005 vaccine primed T-cell responses most effectively and provided the best overall immune responses after boosting with Ad5- gag . These results are suggestive of an immunization strategy for humans that are centered on use of the adenovirus vector and in which existing adenovirus immunity may be overcome by combined immunization with adjuvanted DNA and adenovirus vector boosting.

Numerous observations have shown that host genetic factors play important roles in disease progression in human immunodeficiency virus type 1 (HIV-1) infection (24, 28). The genetics of the major histocompatibility complex (MHC) appears to be a particularly critical factor (5). Rapid disease progression has been demonstrated to be associated with certain class I HLA molecules, such as the HLA-B35-Cw∗04 haplotype (3, 11). Slow disease progression is linked with several class I HLA molecules, notably HLA-B∗27 and HLA-B∗57 (13, 17, 23). Recently, this protective effect was demonstrated in a mother-to-child HIV-1 transmission setting, where it was found that HLA-B∗27-restricted immunodominant cytotoxic T-lymphocyte (CTL) responses play a crucial role in delaying disease progression (12). Similar protective effects were also observed in simian immunodeficiency virus (SIV) infection, particularly in association with expression of the Mamu-A∗01 MHC allele. For instance, expression of Mamu-A∗01 significantly delayed disease progression in SIVmac 251 infection (8, 9, 25). Since this allele has been consistently associated with the CTL response to an immunodominant epitope derived from the SIV Gag region (SIV Gag residues 181 to 189, designated p11CM), detectable by sensitive tetramer and ELISPOT assays, Mamu-A∗01 monkeys have been favored for evaluation of AIDS vaccine candidates (1, 2, 29). Infection with a pathogenic isolate of simian-human immunodeficiency virus (SHIV 89.6P), causes severe CD4 lymphopenia and rapid disease progression in rhesus monkeys (16, 27). Because of the highly pathogenic nature of this virus and its consistent replication kinetics in rhesus monkeys, this challenge model is widely adopted for evaluation of potential AIDS vaccines (1, 2, 29). To further understand the pathogenesis of SHIV 89.6P infection, particularly the impact of expression of the Mamu-A∗01 allele on disease progression, we monitored a cohort of intravenously infected rhesus monkeys with and without expression of the Mamu-A∗01 allele. Here we report that expression of the Mamu-A∗01 allele was associated with delayed disease progression to AIDS in SHIV 89.6P infection. Early virus-specific CTL responses, especially Mamu-A∗01-restricted CTL responses, likely contributed to the significant reduction of initial viral replication observed in lymphoid tissue. Reduced viral replication in lymphoid tissue allowed lymphoid tissue structure to remain relatively intact, thereby resulting in attenuation of disease progression.

Evidence that vaccines capable of inducing a cell-mediated immune (CMI) response (1, 5, 11, 17, 18, 20) may be effective in mitigating infection by human immunodeficiency virus (HIV) is accumulating. Several approaches to the design of such vaccines have been proposed, and some have been implemented. Plasmid DNAs expressing relatively conserved (i.e., nonenvelope) proteins have been investigated (4), and a variety of viral vectors, including canarypox virus (6), modified vaccinia virus Ankara (22), and adenovirus (15), have been used to deliver HIV proteins intracellularly for induction of CMI responses. More recently, mixed-modality vaccine regimens have been used in an effort to overcome or circumvent preexisting immunity to viral vectors. Typically, these regimens include priming with a plasmid DNA vaccine followed by boosting with a protein-based vaccine (14) or with a viral vector (9) expressing the same HIV gene products as are encoded by the DNA vaccine. Characterization and monitoring of the resulting CMI responses by classical methods are both cumbersome and labor-intensive because of the major histocompatibility complex (MHC) diversity of the human population. Cytolytic T-lymphocyte (CTL) responses have been induced by a variety of vaccines, but CTL assays are difficult to perform and are not very quantitative. Lymphoproliferation assays have also been used, but again, these assays are not very quantitative, and they do not adequately measure CD8 T-cell responses. Previously, Larsson et al. (13) described a recombinant vaccinia virus enzyme-linked immunospot (ELISPOT) assay for detection of HIV polymerase responses in HIV-positive subjects. This assay has the advantage of delivering target protein antigen into the cells' cytosol, where the expressed protein can be processed into peptides capable of restimulating T-cell immune responses. A disadvantage is that a live, albeit attenuated, virus must be used for the assay, and construction of the recombinant vaccinia virus vector is not trivial. An alternative is the use of peptides known to bind to MHC class I or class II molecules. Tetramer assays have proven useful in monitoring responses to dominant epitopes (25), but this approach appears to overestimate the number of responding T cells since it measures binding but not function (8). The tetramer assay is also MHC restricted and is limited to detecting responses to known epitopes. When studying an outbred population, assays reliant on measuring responses to a few dominant epitopes will underestimate the response to vaccines or natural infection. To circumvent these issues, we have developed a strategy for using the gamma interferon (IFN-γ) ELISPOT assay to monitor T-cell responses to HIV vaccines. We determined that pools containing as many as 50 peptides spanning the HIV Gag protein could elicit a response equivalent to that induced by infecting peripheral blood mononuclear cells (PBMCs) with a vaccinia virus vector expressing Gag. We further determined the fine specificity of the response to Gag by measuring the ELISPOT response to each individual peptide in the pool. This assay was used to show that immunization of rhesus monkeys with an HIV gag DNA vaccine induced long-lasting IFN-γ memory responses involving CD4 and CD8 T cells.

Recent studies of human immunodeficiency virus type 1 (HIV-1) infection in humans and of simian immunodeficiency virus (SIV) in rhesus monkeys have shown that resolution of the acute viral infection and control of the subsequent persistent infection are mediated by the antiviral cellular immune response. We comparatively assessed several vaccine vector delivery systems-three formulations of a plasmid DNA vector, the modified vaccinia Ankara (MVA) virus, and a replication incompetent adenovirus type 5 (Ad5) vector-expressing the SIV gag protein for their ability to elicit such immune responses in monkeys. The vaccines were tested either as a single modality or in combined modality regimens. Here we show that the most effective responses were elicited by a replication-incompetent Ad5 vector, used either alone or as a booster inoculation after priming with a DNA vector. After challenge with a pathogenic HIV-SIV hybrid virus (SHIV), the animals immunized with Ad5 vector exhibited the most pronounced attenuation of the virus infection. The replication-defective adenovirus is a promising vaccine vector for development of an HIV-1 vaccine.

Virus-specific CD4+ T cell responses have been shown to play a critical role in controlling HIV-1 replication. Candidate HIV-1 vaccines should therefore elicit potent CD4+ as well as CD8+ T cell responses. In this report we investigate the ability of plasmid GM-CSF to augment CD4+ T cell responses elicited by an HIV-1 gp120 DNA vaccine in mice. Coadministration of a plasmid expressing GM-CSF with the gp120 DNA vaccine led to only a marginal increase in gp120-specific splenocyte CD4+ T cell responses. However, immunization with a bicistronic plasmid that coexpressed gp120 and GM-CSF under control of a single promoter led to a dramatic augmentation of vaccine-elicited CD4+ T cell responses, as measured by both cellular proliferation and ELISPOT assays. This augmentation of CD4+ T cell responses was selective, since vaccine-elicited Ab and CD8+ T cell responses were not significantly changed by the addition of GM-CSF. A 100-fold lower dose of the gp120/GM-CSF bicistronic DNA vaccine was required to elicit detectable gp120-specific splenocyte proliferative responses compared with the monocistronic gp120 DNA vaccine. Consistent with these findings, i.m. injection of the gp120/GM-CSF bicistronic DNA vaccine evoked a more extensive cellular infiltrate at the site of inoculation than the monocistronic gp120 DNA vaccine. These results demonstrate that bicistronic DNA vaccines containing GM-CSF elicit remarkably potent CD4+ T cell responses and suggest that optimal Th cell priming requires the precise temporal and spatial codelivery of Ag and GM-CSF.

Recently it has been shown that immunization with plasmid DNA encoding genes for viral or bacterial antigens can elicit both humoral and cellular immune responses in rodents and nonhuman primates. In this study, mice and nonhuman primates were vaccinated by intramuscular injection with plasmids that express either a secreted form of HIV-1 gp120 or rev proteins. Mice receiving the tPA-gp120 DNA developed antigen-specific antibody responses against recombinant gp120 protein and the V2 peptide neutralization epitope as determined by ELISA. Vaccinated mice also exhibited gp120-specific T cell responses, such as in vitro proliferation of splenocytes and MHC Class I-restricted cytotoxic T lymphocyte (CTL) activities, following antigen restimulation. In addition, supernatants from these lymphocyte cultures showed high levels of gamma-interferon production compared with IL-4, suggesting that primarily type 1-like helper T (Th1) lymphocyte responses were induced by both vaccines. Th1-like responses were also obtained for mice vaccinated with rev DNA. Immune responses induced by gp120 or rev vaccines were dose-dependent, boostable, and long-lived (or = 6 months). Nonhuman primates vaccinated with tPA-gp120 DNA also showed antigen-specific T lymphocyte proliferative and humoral responses, including moderate levels of neutralizing sera against homologous HIV. These results suggest that plasmid DNA may provide a powerful means for eliciting humoral and cellular immune responses against HIV.

2'-Nor-2'-deoxyguanosine (2'NDG), previously reported by us to effectively treat acute herpes simplex infections in mice, was used therapeutically to significantly enhance healing of established herpetic corneal lesions and prevent stromal disease in rabbits. Treatment using 0.06% 2'NDG drops (5 times daily) starting 3 days after infection resulted in more rapid healing of corneal epithelial lesions, rapid resolution of conjunctival inflammation, and prevention of stromal clouding compared to placebo-treated animals. In comparative dose-response titrations, the relative potency of 2'NDG to acyclovir was 6.4, which was significant. In addition, soluble ophthalmic inserts were developed for delivery of 2'NDG. Once a day treatment using ophthalmic inserts which released 100 micrograms 2'NDG significantly enhanced corneal and conjunctival healing and prevented stromal disease; 2'NDG eye drops (100 micrograms) delivered once a day were also effective in inhibiting the progression of corneal lesions. These results indicate that 2'NDG may be therapeutically effective in treatment of herpes keratitis, and further suggest that for use as eye drops or in an ophthalmic insert, 2'NDG may be effective even if applied once per day.

9-[(2-Hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]guanine P-Oxide (2′-nor-cGMP), the cyclic phosphate of 2′-nor-deoxyguanosine (2′-NDG) was synthesized by phosphorylation of 2′-NDG and evaluated for antiherpetic activity in cell cultures and in animal protection studies. 2′-nor-cGMP was effective in cell culture against both thymidine kinase deficient and wild-type herpes simplex virus type 1 strains and also against herpes simplex virus type 2. The anti-herpes activity of 2′-nor-cGMP against thymidine kinase deficient HSV-1 was confirmed by animal protection studies. Also, in comparative cell culture protection studies, the ED50 (μM) of 2′-nor-cGMP was approximately 10-fold lower than that of 2′-NDG against three strains of varicella zoster virus. In addition, 2′-nor-cGMP was effective orally in preventing HSV-1 orofacial infection and HSV-2 genital infection of mice. Topical therapeutic applications of 2′-nor-cGMP prevented orofacial HSV-1 lesion development in mice and development of HSV-2 genital lesions in guinea pigs. Subcutaneous application of 2′-nor-cGMP to intracerebral HSV-1 challenged weanling mice significantly prolonged survival. These studies indicate that 2′-nor-cGMP is not dependent on viral thymidine kinase for its antiviral activity and is highly effective in preventing experimental HSV infections.

We sought to determine how effectively a CD8+ T cell inducing vaccine controls SHIV-89.6P infection in rhesus macaques at a range of challenge times post-vaccination. To this end, twenty eight Mamu-A⁎01+ rhesus macaques were given replication incompetent human serotype 5 adenovirus vector expressing SIVmac239 gag DNA and boosted 24 weeks later. Groups of 4 monkeys were then challenged with SHIV-89.6P at 1, 3, 6, 12, and 24 weeks after the boost. We compared the kinetics of viral load, CD4+ and virus-specific CD8+ T cells in these macaques. Measurements of CD8+ T cells taken before challenge show an exponential decay between 1 and 12 weeks following vaccination (p

Evidence is presented that indicates a deep crevice located on the surface of human rhinovirus type 14 is involved in virion attachment to cellular receptors. By using mutagenesis of an infectious cDNA clone, 11 mutants were created by single amino acid substitutions or insertions at positions 103, 155, 220, 223, and 273 of the structural protein VP1. Seven of the recovered mutants had a small plaque phenotype and exhibited binding affinities significantly lower than wild-type virus. One mutant, in which glycine replaced proline at amino acid position 155, showed a greatly enhanced binding affinity. Single-cycle growth kinetics suggested that 5 of the mutants had delayed growth cycles due to intracellular deficiencies apart from receptor binding.

2'-Nor-2'-deoxyguanosine (2'NDG), a new antiviral agent, conferred protection when given orally or topically to hairless mice after the mice were subjected to orofacial infection with herpes simplex virus type 1. The average severity of orofacial lesions was significantly reduced in mice receiving oral gavage treatments twice daily for 7 days beginning 3 h postinfection. The minimum effective dose of 2'NDG was 0.2 mg/kg per day. A minimum of eight treatments over 4 days resulted in a significant reduction in lesion severity. Topical treatment begun 3 h postinfection and continued four times daily for 3 days resulted in a minimum effective dose of 0.06%. Oral treatment with 2'NDG begun as late as 72 h postinfection or topical treatment begun as late as 48 h postinfection resulted in significantly reduced lesion severity compared with lesion severity among placebo-treated animals. In addition, significant prevention of ganglionic infection occurred when 2'NDG was administered either orally or topically within 24 h after infection.

Poly I:C/poly-L-lysine (poly ICL) was effective in preventing or delaying the development of osteogenic sarcoma (OGS) in mice. C57BL/6J mice were inoculated subcutaneously with 5 X 10(4) OGS cells and treatment was evaluated by palpable tumor development and subsequent day of death. A significant antitumor effect resulted from injection of 150 microgram of poly ICL into the tumor site starting immediately after tumor implant and followed by four subsequent treatments. Seventy percent of treated animals remained tumor free at 50 days, a time at which 70% of placebo treated animals had died as a result of tumor development. A similar treatment regimen of mice inoculated with 2 X 10(5) OGS cells resulted in a significant delay of time to tumor and subsequent day of death. Treatments with poly ICL were ineffective if they were initiated after development of palpable tumor or if they were administered at a nontumorous site on the animal. These findings indicate that the optimal therapy resulted from repeated intratumor treatment prior to development of extensive tumor burden.