Prior exposure to an attenuated Listeria vaccine does not reduce immunogenicity: pre-clinical assessment of the efficacy of a Listeria vaccine in the induction of immune responses against HIV

Abstract Background We have evaluated an attenuated Listeria monocytogenes (Lm) candidate vaccine vector in nonhuman primates using a delivery regimen relying solely on oral vaccination. We sought to determine the impact of prior Lm vector exposure on the development of new immune responses against HIV antigens. Findings Two groups of rhesus macaques one Lm naive, the other having documented prior Lm vector exposures, were evaluated in response to oral inoculations of the same vector expressing recombinant HIV-1 Gag protein. The efficacy of the Lm vector was determined by ELISA to assess the generation of anti-Listerial antibodies; cellular responses were measured by HIV-Gag specific ELISpot assay. Our results show that prior Lm exposures did not diminish the generation of de novo cellular responses against HIV, as compared to Listeria-naïve monkeys. Moreover, empty vector exposures did not elicit potent antibody responses, consistent with the intracellular nature of Lm. Conclusions The present study demonstrates in a pre-clinical vaccine model, that prior oral immunization with an empty Lm vector does not diminish immunogenicity to Lm-expressed HIV genes. This work underscores the need for the continued development of attenuated Lm as an orally deliverable vaccine

Cosmogenic ^(10)Be and ^(36)Cl geochronology of offset alluvial fans along the northern Death Valley fault zone: Implications for transient strain in the eastern California shear zone

The northern Death Valley fault zone (NDVFZ) has long been recognized as a major right-lateral strike-slip fault in the eastern California shear zone (ECSZ). However, its geologic slip rate has been difficult to determine. Using high-resolution digital topographic imagery and terrestrial cosmogenic nuclide dating, we present the first geochronologically determined slip rate for the NDVFZ. Our study focuses on the Red Wall Canyon alluvial fan, which exposes clean dextral offsets of seven channels. Analysis of airborne laser swath mapping data indicates ∼297 ± 9 m of right-lateral displacement on the fault system since the late Pleistocene. In situ terrestrial cosmogenic ^(10)Be and ^(36)Cl geochronology was used to date the Red Wall Canyon fan and a second, correlative fan also cut by the fault. Beryllium 10 dates from large cobbles and boulders provide a maximum age of 70 +22/−20 ka for the offset landforms. The minimum age of the alluvial fan deposits based on ^(36)Cl depth profiles is 63 ± 8 ka. Combining the offset measurement with the cosmogenic ^(10)Be date yields a geologic fault slip rate of 4.2 +1.9/−1.1 mm yr^(−1), whereas the ^(36)Cl data indicate 4.7 +0.9/−0.6 mm yr^(−1) of slip. Summing these slip rates with known rates on the Owens Valley, Hunter Mountain, and Stateline faults at similar latitudes suggests a total geologic slip rate across the northern ECSZ of ∼8.5 to 10 mm yr^(−1). This rate is commensurate with the overall geodetic rate and implies that the apparent discrepancy between geologic and geodetic data observed in the Mojave section of the ECSZ does not extend north of the Garlock fault. Although the overall geodetic rates are similar, the best estimates based on geology predict higher strain rates in the eastern part of the ECSZ than to the west, whereas the observed geodetic strain is relatively constant

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Safety and Immunogenicity in Neonatal Mice of a Hyperattenuated Listeria Vaccine Directed against Human Immunodeficiency Virus

CD8(+) T cells are a major component of the adaptive response of a host to infections by viruses and other intracellular pathogenic agents. However, because of the intrinsic immaturity of the immune system of neonatal animals, neonates are highly sensitive to a variety of pathogens and may be unable to respond in a protective manner. Here we explore whether a hyperattenuated strain of Listeria monocytogenes that can be used as a live vaccine vector in adults is safe and able to induce an effective response in neonates. We answer both questions affirmatively

Vaginal Protection and Immunity after Oral Immunization of Mice with a Novel Vaccine Strain of Listeria monocytogenes Expressing Human Immunodeficiency Virus Type 1 gag

Natural transmission of human immunodeficiency virus (HIV) occurs at mucosal surfaces. During acute infection, intestinal and other mucosae are preferential sites of virus replication and rapidly become depleted of CD4(+) T cells. Therefore, mucosal immunity may be critical to control both initial infection and the massive early spread of virus. An attenuated d-alanine-requiring strain of the oral intracellular microorganism Listeria monocytogenes expressing HIV type 1 gag was shown to induce protective cell-mediated immunity in mice against viruses that express HIV gag when immunization occurs in the presence of a transient supply of d-alanine. In this study, we examined the efficacy of new attenuated strains that are able to synthesize d-alanine from a heterologous dal gene tightly regulated by an actA-promoted resolvase recombination system. In the absence of d-alanine, Gag-specific cytotoxic T lymphocytes (CTLs) were induced systemically after intravenous immunization, and one strain, Lmdd-gag/pARS, induced strong dose-dependent Gag-specific CTLs after oral immunization. A significant level of Gag-specific CD8(+) T cells was induced in the mucosal-associated lymphoid tissues (MALTs). Upon intravaginal challenge of these orally immunized mice with recombinant vaccinia virus (rVV) expressing HIV gag, gamma interferon- and tumor necrosis factor alpha-secreting Gag-specific CD8(+) T cells were dramatically increased in the spleen and MALTs. Oral immunization with Lmdd-gag/pARS led to complete protection against vaginal challenge by a homologous clade B gag-expressing rVV. In addition, strong cross-clade protection was seen against clades A and C and partial protection against clade G gag-expressing rVV. These results suggest that Lmdd-gag/pARS may be considered as a novel vaccine candidate for use against HIV/AIDS

Pathogenicity and Immunogenicity of a Vaccine Strain of Listeria monocytogenes That Relies on a Suicide Plasmid To Supply an Essential Gene Product

Listeria monocytogenes is a bacterial pathogen that elicits a strong cellular immune response and thus has potential use as a vaccine vector. An attenuated strain, L. monocytogenes dal dat, produced by deletion of two genes (dal and dat) used for d-alanine synthesis, induces cytotoxic T lymphocytes and protective immunity in mice following infection in the presence of d-alanine. In order to obviate the dependence of L. monocytogenes dal dat on supplemental d-alanine yet retain its attenuation and immunogenicity, we explored mechanisms to allow transient endogenous synthesis of the amino acid. Here, we report on a derivative strain, L. monocytogenes dal dat/pRRR, that expresses a dal gene and synthesizes d-alanine under highly selective conditions. We constructed the suicide plasmid pRRR carrying a dal gene surrounded by two res1 sites and a resolvase gene, tnpR, which acts at the res1 sites. The resolvase gene is regulated by a promoter activated upon exposure to host cell cytosol. L. monocytogenes dal dat/pRRR was thus able to grow in liquid culture and to infect host cells without d-alanine supplementation. However, after infection of these cells, resolvase-mediated excision of the dal gene resulted in strong down-regulation of racemase expression. As a result, this system allowed only transient growth of L. monocytogenes dal dat/pRRR in infected cells and survival in animals for only 2 to 3 days. Nevertheless, mice immunized with L. monocytogenes dal dat/pRRR generated listeriolysin O-specific effector and memory CD8(+) T cells and were protected against lethal challenge by wild-type Listeria. This vector may be an attractive vaccine candidate for the induction of protective cellular immune responses

Conditional Lethality Yields a New Vaccine Strain of Listeria monocytogenes for the Induction of Cell-Mediated Immunity

Listeria monocytogenes is a gram-positive intracellular pathogen that can enter phagocytic and nonphagocytic cells and colonize their cytosols. Taking advantage of this property to generate an intracellular vaccine delivery vector, we previously described a mutant strain of L. monocytogenes, Δdal Δdat, which is unable to synthesize cell wall by virtue of deletions in two genes (dal and dat) required for d-alanine synthesis. This highly attenuated strain induced long-lived protective systemic and mucosal immune responses in mice when administered in the transient presence of d-alanine. We have now increased the usefulness of this organism as a vaccine vector by use of an inducible complementation system that obviates the need for exogenous d-alanine administration. The strain expresses a copy of the Bacillus subtilis racemase gene under the control of a tightly regulated isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter present on a multicopy plasmid. This bacterium demonstrates strict dose-dependent growth in the presence of IPTG. After removal of inducer, bacterial growth ceased within two replication cycles. Following infection of mice in the absence of IPTG or d-alanine, the bacterium survived in vivo for less than 3 days. Nevertheless, a single immunization elicited a state of long-lasting protective immunity against wild-type L. monocytogenes and induced a subset of effector listeriolysin O-specific CD11a(+) CD8(+) T cells in spleen and other tissues that was strongly enhanced after secondary immunization. This improved L. monocytogenes vector system may have potential use as a live vaccine against human immunodeficiency virus, other infectious diseases, and cancer