Antineoplastic Cytotoxicity and Immune Adjuvancy of a Recombinant Oncolytic Poliovirus

Our group has pioneered the development of a live-attenuated poliovirus, called PVSRIPO, for the purpose of targeting cancer. Despite clinical progress, the cancer selective cytotoxicity and immunotherapeutic potential of PVSRIPO has not yet been mechanistically dissected. Defining such mechanisms may inform its clinical application.

Herein I describe the discovery of a mechanism by which the MAP-Kinase Interacting Kinases (MNKs) regulate PVSRIPO cytotoxicity in cancer. In doing so, I delineate a novel, intricate network connecting the MNK and mTOR signaling pathway that regulates activity of a splicing kinase called the Ser-Arg Rich Protein Kinase (SRPK), and define SRPK as an impediment to IRES mediated translation. Moreover, I demonstrate that MNK regulates mTORC1 associations that determine its substrate proximity and thus, activity. In a collaborative effort, we found that PVSRIPO oncolysis produces antigen specific, cytolytic anti-tumor immunity in an in vitro human system and that much of the observed adjuvancy is due to the direct infection of dendritic cells (DCs) by the virus itself; implicating PVSRIPO as a potent adjuvant. In summary, oncogenic signaling in part through MNK leads to cancer specific cytotoxicity by PVSRIPO that engages an inflammatory environment conducive to DC activation and antigen specific T cell antigen immunity.

The Macrophage-Specific Promoter mfap4 Allows Live, Long-Term Analysis of Macrophage Behavior during Mycobacterial Infection in Zebrafish.

Transgenic labeling of innate immune cell lineages within the larval zebrafish allows for real-time, in vivo analyses of microbial pathogenesis within a vertebrate host. To date, labeling of zebrafish macrophages has been relatively limited, with the most specific expression coming from the mpeg1 promoter. However, mpeg1 transcription at both endogenous and transgenic loci becomes attenuated in the presence of intracellular pathogens, including Salmonella typhimurium and Mycobacterium marinum. Here, we describe mfap4 as a macrophage-specific promoter capable of producing transgenic lines in which transgene expression within larval macrophages remains stable throughout several days of infection. Additionally, we have developed a novel macrophage-specific Cre transgenic line under the control of mfap4, enabling macrophage-specific expression using existing floxed transgenic lines. These tools enrich the repertoire of transgenic lines and promoters available for studying zebrafish macrophage dynamics during infection and inflammation and add flexibility to the design of future macrophage-specific transgenic lines.

Magnitude and breadth of a nonprotective neutralizing antibody response in an efficacy trial of a candidate HIV-1 gp120 vaccine.

BACKGROUND: A candidate vaccine consisting of human immunodeficiency virus type 1 (HIV-1) subunit gp120 protein was found previously to be nonprotective in an efficacy trial (Vax004) despite strong antibody responses against the vaccine antigens. Here we assessed the magnitude and breadth of neutralizing antibody responses in Vax004. METHODS: Neutralizing antibodies were measured against highly sensitive (tier 1) and moderately sensitive (tier 2) strains of HIV-1 subtype B in 2 independent assays. Vaccine recipients were stratified by sex, race, and high versus low behavioral risk of HIV-1 acquisition. RESULTS: Most vaccine recipients mounted potent neutralizing antibody responses against HIV-1(MN) and other tier 1 viruses. Occasional weak neutralizing activity was detected against tier 2 viruses. The response against tier 1 and tier 2 viruses was significantly stronger in women than in men. Race and behavioral risk of HIV-1 acquisition had no significant effect on the response. Prior vaccination had little effect on the neutralizing antibody response that arose after infection. CONCLUSIONS: Weak overall neutralizing antibody responses against tier 2 viruses is consistent with a lack of protection in this trial. The magnitude and breadth of neutralization reported here should be useful for identifying improved vaccines.

Safety and immunogenicity of a replication-defective adenovirus type 5 HIV vaccine in Ad5-seronegative persons: a randomized clinical trial (HVTN 054).

BACKGROUND: Individuals without prior immunity to a vaccine vector may be more sensitive to reactions following injection, but may also show optimal immune responses to vaccine antigens. To assess safety and maximal tolerated dose of an adenoviral vaccine vector in volunteers without prior immunity, we evaluated a recombinant replication-defective adenovirus type 5 (rAd5) vaccine expressing HIV-1 Gag, Pol, and multiclade Env proteins, VRC-HIVADV014-00-VP, in a randomized, double-blind, dose-escalation, multicenter trial (HVTN study 054) in HIV-1-seronegative participants without detectable neutralizing antibodies (nAb) to the vector. As secondary outcomes, we also assessed T-cell and antibody responses. METHODOLOGY/PRINCIPAL FINDINGS: Volunteers received one dose of vaccine at either 10(10) or 10(11) adenovector particle units, or placebo. T-cell responses were measured against pools of global potential T-cell epitope peptides. HIV-1 binding and neutralizing antibodies were assessed. Systemic reactogenicity was greater at the higher dose, but the vaccine was well tolerated at both doses. Although no HIV infections occurred, commercial diagnostic assays were positive in 87% of vaccinees one year after vaccination. More than 85% of vaccinees developed HIV-1-specific T-cell responses detected by IFN-γ ELISpot and ICS assays at day 28. T-cell responses were: CD8-biased; evenly distributed across the three HIV-1 antigens; not substantially increased at the higher dose; and detected at similar frequencies one year following injection. The vaccine induced binding antibodies against at least one HIV-1 Env antigen in all recipients. CONCLUSIONS/SIGNIFICANCE: This vaccine appeared safe and was highly immunogenic following a single dose in human volunteers without prior nAb against the vector. TRIAL REGISTRATION: ClinicalTrials.gov NCT00119873.

Dental microwear in live, wild-trapped Alouatta palliata from Costa Rica.

One problem with dental microwear analyses of museum material is that investigators can never be sure of the diets of the animals in question. An obvious solution to this problem is to work with live animals. Recent work with laboratory primates has shown that high resolution dental impressions can be obtained from live animals. The purpose of this study was to use similar methods to begin to document rates and patterns of dental microwear for primates in the wild. Thirty-three Alouatta palliata were captured during the wet season at Hacienda La Pacifica near Canas, Costa Rica. Dental impressions were taken and epoxy casts of the teeth were prepared using the methods of Teaford and Oyen (1989a). Scanning electron micrographs were taken of the left mandibular second molars at magnifications of 200x and 500x. Lower magnification images were used to calculate rates of wear, and higher magnification images were used to measure the size and shape of microwear features. Results indicate that, while basic patterns of dental microwear are similar in museum samples and samples of live, wild-trapped animals of the same species, ecological differences between collection locales may lead to significant intraspecific differences in dental microwear. More importantly, rates of microwear provide the first direct evidence of differences in molar use between monkeys and humans.

Magnetic resonance imaging of graded skeletal muscle injury in live rats.

INTRODUCTION: Increasing number of stretch-shortening contractions (SSCs) results in increased muscle injury. METHODS: Fischer Hybrid rats were acutely exposed to an increasing number of SSCs in vivo using a custom-designed dynamometer. Magnetic resonance imaging (MRI) imaging was conducted 72 hours after exposure when rats were infused with Prohance and imaged using a 7T rodent MRI system (GE Epic 12.0). Images were acquired in the transverse plane with typically 60 total slices acquired covering the entire length of the hind legs. Rats were euthanized after MRI, the lower limbs removed, and tibialis anterior muscles were prepared for histology and quantified stereology. RESULTS: Stereological analyses showed myofiber degeneration, and cellular infiltrates significantly increased following 70 and 150 SSC exposure compared to controls. MRI images revealed that the percent affected area significantly increased with exposure in all SSC groups in a graded fashion. Signal intensity also significantly increased with increasing SSC repetitions. DISCUSSION: These results suggest that contrast-enhanced MRI has the sensitivity to differentiate specific degrees of skeletal muscle strain injury, and imaging data are specifically representative of cellular histopathology quantified via stereological analyses.

Live Imaging of Host-Parasite Interactions in a Zebrafish Infection Model Reveals Cryptococcal Determinants of Virulence and Central Nervous System Invasion.

UNLABELLED: The human fungal pathogen Cryptococcus neoformans is capable of infecting a broad range of hosts, from invertebrates like amoebas and nematodes to standard vertebrate models such as mice and rabbits. Here we have taken advantage of a zebrafish model to investigate host-pathogen interactions of Cryptococcus with the zebrafish innate immune system, which shares a highly conserved framework with that of mammals. Through live-imaging observations and genetic knockdown, we establish that macrophages are the primary immune cells responsible for responding to and containing acute cryptococcal infections. By interrogating survival and cryptococcal burden following infection with a panel of Cryptococcus mutants, we find that virulence factors initially identified as important in causing disease in mice are also necessary for pathogenesis in zebrafish larvae. Live imaging of the cranial blood vessels of infected larvae reveals that C. neoformans is able to penetrate the zebrafish brain following intravenous infection. By studying a C. neoformans FNX1 gene mutant, we find that blood-brain barrier invasion is dependent on a known cryptococcal invasion-promoting pathway previously identified in a murine model of central nervous system invasion. The zebrafish-C. neoformans platform provides a visually and genetically accessible vertebrate model system for cryptococcal pathogenesis with many of the advantages of small invertebrates. This model is well suited for higher-throughput screening of mutants, mechanistic dissection of cryptococcal pathogenesis in live animals, and use in the evaluation of therapeutic agents. IMPORTANCE: Cryptococcus neoformans is an important opportunistic pathogen that is estimated to be responsible for more than 600,000 deaths worldwide annually. Existing mammalian models of cryptococcal pathogenesis are costly, and the analysis of important pathogenic processes such as meningitis is laborious and remains a challenge to visualize. Conversely, although invertebrate models of cryptococcal infection allow high-throughput assays, they fail to replicate the anatomical complexity found in vertebrates and, specifically, cryptococcal stages of disease. Here we have utilized larval zebrafish as a platform that overcomes many of these limitations. We demonstrate that the pathogenesis of C. neoformans infection in zebrafish involves factors identical to those in mammalian and invertebrate infections. We then utilize the live-imaging capacity of zebrafish larvae to follow the progression of cryptococcal infection in real time and establish a relevant model of the critical central nervous system infection phase of disease in a nonmammalian model.

Potential To Streamline Heterologous DNA Prime and NYVAC/Protein Boost HIV Vaccine Regimens in Rhesus Macaques by Employing Improved Antigens.

UNLABELLED: In a follow-up to the modest efficacy observed in the RV144 trial, researchers in the HIV vaccine field seek to substantiate and extend the results by evaluating other poxvirus vectors and combinations with DNA and protein vaccines. Earlier clinical trials (EuroVacc trials 01 to 03) evaluated the immunogenicity of HIV-1 clade C GagPolNef and gp120 antigens delivered via the poxviral vector NYVAC. These showed that a vaccination regimen including DNA-C priming prior to a NYVAC-C boost considerably enhanced vaccine-elicited immune responses compared to those with NYVAC-C alone. Moreover, responses were improved by using three as opposed to two DNA-C primes. In the present study, we assessed in nonhuman primates whether such vaccination regimens can be streamlined further by using fewer and accelerated immunizations and employing a novel generation of improved DNA-C and NYVAC-C vaccine candidates designed for higher expression levels and more balanced immune responses. Three different DNA-C prime/NYVAC-C+ protein boost vaccination regimens were tested in rhesus macaques. All regimens elicited vigorous and well-balanced CD8(+)and CD4(+)T cell responses that were broad and polyfunctional. Very high IgG binding titers, substantial antibody-dependent cellular cytotoxicity (ADCC), and modest antibody-dependent cell-mediated virus inhibition (ADCVI), but very low neutralization activity, were measured after the final immunizations. Overall, immune responses elicited in all three groups were very similar and of greater magnitude, breadth, and quality than those of earlier EuroVacc vaccines. In conclusion, these findings indicate that vaccination schemes can be simplified by using improved antigens and regimens. This may offer a more practical and affordable means to elicit potentially protective immune responses upon vaccination, especially in resource-constrained settings. IMPORTANCE: Within the EuroVacc clinical trials, we previously assessed the immunogenicity of HIV clade C antigens delivered in a DNA prime/NYVAC boost regimen. The trials showed that the DNA prime crucially improved the responses, and three DNA primes with a NYVAC boost appeared to be optimal. Nevertheless, T cell responses were primarily directed toward Env, and humoral responses were modest. The aim of this study was to assess improved antigens for the capacity to elicit more potent and balanced responses in rhesus macaques, even with various simpler immunization regimens. Our results showed that the novel antigens in fact elicited larger numbers of T cells with a polyfunctional profile and a good Env-GagPolNef balance, as well as high-titer and Fc-functional antibody responses. Finally, comparison of the different schedules indicates that a simpler regimen of only two DNA primes and one NYVAC boost in combination with protein may be very efficient, thus showing that the novel antigens allow for easier immunization protocols.