No abstract

Transmission studies have suggested that an optimal human immunodeficiency virus type 1 (HIV-1) vaccine should induce both neutralzing antibodies and cytolytic T cells to eliminate free virus and infected cells. A phase I trial in healthy HIV-1-seronegative persons was conducted with a combination HIV-1 vaccine regimen (strain RIB) consiting of priming with a recombinant vaccinia (vac/env) virus expressing HIV-1 envelope and boosting with a gpl6O glycoprotein derived from a recombinant baculovirus (rgpl6O). T-cell and antibody responses detected after immunization with either vac/env alone or rgpl6O alone were generally oflow magnitude and transient, and no subject developed neutraling antibodies. In contrast, recipients of the combination regimen demonstrated in vitro T-cell proliferative responses to homologous HIV-1 antigens that were 3-to 10-fold higher than responses with either vaccine alone, and these responses were sustained for >18 months in 75% of recipients. Moreover, both CD8+ and CD4+ cytolytic T cells were detected. Antibody responses (titer, 1:800 to 1:102,400) to homologous HIV envelope developed in all recipients of the combination regimen, and neutralizing antibodies were detected in 7 of 13. Thus, immunization with a live virus vaccine followed by boosting with a soluble protein offers promise for inducing the broad immunity needed in an HIV vaccine.

Organ infiltration by donor T cells is critical to the development of acute graft-versus-host disease (aGVHD) in recipients after allogeneic hematopoietic stem cell transplant (allo-HCT). However, deconvoluting the transcriptional programs of newly recruited donor T cells from those of tissue-resident T cells in aGVHD target organs remains a challenge. Here, we combined the serial intravascular staining technique with single-cell RNA sequencing to dissect the tightly connected processes by which donor T cells initially infiltrate tissues and then establish a pathogenic tissue residency program in a rhesus macaque allo-HCT model that develops aGVHD. Our results enabled creation of a spatiotemporal map of the transcriptional programs controlling donor CD8+ T cell infiltration into the primary aGVHD target organ, the gastrointestinal (GI) tract. We identified the large and small intestines as the only two sites demonstrating allo-specific, rather than lymphodepletion-driven, T cell infiltration. GI-infiltrating donor CD8+ T cells demonstrated a highly activated, cytotoxic phenotype while simultaneously developing a canonical tissue-resident memory T cell (TRM) transcriptional signature driven by interleukin-15 (IL-15)/IL-21 signaling. We found expression of a cluster of genes directly associated with tissue invasiveness, including those encoding adhesion molecules (ITGB2), specific chemokines (CCL3 and CCL4L1) and chemokine receptors (CD74), as well as multiple cytoskeletal proteins. This tissue invasion transcriptional signature was validated by its ability to discriminate the CD8+ T cell transcriptome of patients with GI aGVHD from those of GVHD-free patients. These results provide insights into the mechanisms controlling tissue occupancy of target organs by pathogenic donor CD8+ TRM cells during aGVHD in primate transplant recipients.

Allogeneic transplantation (allo-HCT) has led to the cure of HIV in one individual, raising the question of whether transplantation can eradicate the HIV reservoir. To test this, we here present a model of allo-HCT in SHIV-infected, cART-suppressed nonhuman primates. We infect rhesus macaques with SHIV-1157ipd3N4, suppress them with cART, then transplant them using MHC-haploidentical allogeneic donors during continuous cART. Transplant results in ~100% myeloid donor chimerism, and up to 100% T-cell chimerism. Between 9 and 47 days post-transplant, terminal analysis shows that while cell-associated SHIV DNA levels are reduced in the blood and in lymphoid organs post-transplant, the SHIV reservoir persists in multiple organs, including the brain. Sorting of donor-vs.-recipient cells reveals that this reservoir resides in recipient cells. Moreover, tetramer analysis indicates a lack of virus-specific donor immunity post-transplant during continuous cART. These results suggest that early post-transplant, allo-HCT is insufficient for recipient reservoir eradication despite high-level donor chimerism and GVHD.

Accurate species delimitation and description are necessary to guide effective conservation of imperiled species, and this synergy is maximized when multiple data sources are used to delimit species. We illustrate this point by examining Drymarchon couperi (Eastern Indigo Snake), a large, federally-protected species in North America that was recently divided into two species based on gene sequence data from three loci and heuristic morphological assessment. Here, we re-evaluate the two-species hypothesis for D. couperi by evaluating both population genetic and gene sequence data. Our analyses of 14 microsatellite markers revealed 6–8 genetic population clusters with significant admixture, particularly across the contact zone between the two hypothesized species. Phylogenetic analyses of gene sequence data with maximum-likelihood methods suggested discordance between mitochondrial and nuclear markers and provided phylogenetic support for one species rather than two. For these reasons, we place Drymarchon kolpobasileus into synonymy with D. couperi. We suggest inconsistent patterns between mitochondrial and nuclear DNA are driven by high dispersal of males relative to females. We advocate for species delimitation exercises that evaluate admixture and gene flow in addition to phylogenetic analyses, particularly when the latter reveal monophyletic lineages. This is particularly important for taxa, such as squamates, that exhibit strong sex-biased dispersal. Problems associated with over-delimitation of species richness can become particularly acute for threatened and endangered species, because of high costs to conservation when taxonomy demands protection of more individual species than are supported by accumulating data.

Thirty-four eastern indigo snakes ( Drymarchon couperi ) naturally infected with Cryptosporidium serpentis were randomly divided into two groups. The first group received 360 mg/kg paromomycin twice weekly in a food item for six weeks, while the second group received the food item with no treatment. Cloacal swabs were collected every two months for six months to measure C. serpentis shedding by quantitative polymerase chain reaction testing (qPCR). Snakes that were qPCR negative after six 6 months were immunosuppressed with a single dose of 4 mg/kg dexamethasone sodium-phosphate SC. These snakes were then screened by qPCR for an additional 6 months as described above. Snakes that were qPCR negative after one year of serial sampling were then re-evaluated for C. serpentis via gastric biopsy for histological and qPCR analyses. The paromomycin-treated group were significantly (p=0.008) more likely to test qPCR negative (8/17; 47%, 95% Confidence Interval [CI]: 23.2-70.7) than the control snakes (1/17; 5.8%, 95% CI: 0.01-16.9) prior to immunosuppression. However, there was no significant difference (p=0.5) in C. serpentis status following immunosuppression as only 2/17 (11.7%, 95% CI: 0.01-26.9) paromomycin-treated snakes were qPCR negative six months after immunosuppression compared to 1/17 (5.8%, 95% CI: 95% CI: 0.01-16.9) control snakes. These findings suggest that 360 mg/kg paromomycin twice weekly for six weeks in a food item is ineffective in eliminating C. serpentis in naturally infected D. couperi .

Wildlife repatriation is an important tool to decrease extinction risk for imperiled species, but successful repatriations require significant time, resources and planning. Because repatriations can be long and expensive processes, clear release strategies and monitoring programs are essential to efficiently use resources and evaluate success. However, monitoring can be challenging and surrounded by significant uncertainty, particularly for secretive species with extremely low detection probability. Here, we simulated how alternative repatriation strategies influence repatriation success for the eastern indigo snake Drymarchon couperi, a federally‐Threatened species that is currently being repatriated in Alabama and Florida. Critically, we demonstrate how observed population growth can differ from true population growth when detection probabilities are low and mark‐recapture analyses are not an option. Specifically, we built a stochastic stage‐based population model to predict population growth and extinction risk under different release strategies and use information from ongoing repatriations to predict success and guide future releases. Because D.  couperi is difficult to monitor, we modeled how detection probability influenced perceptions of abundance and population growth by monitoring programs. Simulated repatriation strategies releasing older, head‐started snakes in greater abundance and frequency created wild populations with decreased extinction risk relative to scenarios releasing fewer and younger snakes less frequently. Ongoing repatriations currently have a 0.23 (Alabama) and 0.61 (Florida) probability of quasi‐extinction, but extinction risk decreased to 0.07 and 0.10 at sites upon achieving the targeted number of releases. Abundances observed under realistic detection thresholds for D.  couperi did not always predict true population growth; specifically, we demonstrate that monitoring programs during repatriations of secretive species may indicate that efforts have been unsuccessful when populations are actually growing. Overall, our modeling framework informs release strategies to maximize repatriation success while demonstrating the need to consider how detection processes influence assessment of success during conservation interventions.

Analysis of major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) capable of killing human immunodeficiency virus type 1 (HIV-1)-infected targets is essential for elucidating the basis for HIV-1 disease progression and the potential efficacy of candidate vaccines. The use of primary CD4+ T cells with variable infectivity as targets for such studies has significant limitations, and immortal autologous cells with high levels of CD4 expression that can be consistently infected with HIV-1 would be of much greater utility. Therefore, we transduced Epstein-Barr-virus-transformed B-lymphoblastoid cell lines (LCL) with a retroviral vector, LT4SN, containing the human CD4 gene. Stable LCL in which more than 95% of cells expressed membrane CD4 were obtained. Aliquots were infected with HIV-1, and, after 4 to 7 days, nearly all of the cells contained cytoplasmic gag and produced high levels of p24 antigen. The ability of major histocompatibility complex-restricted CD8+ CTL to lyse such HIV-1-infected CD4-transduced LCL (LCL-CD4H) was evaluated. These autologous targets were lysed by CTL generated from an HIV-1-uninfected vaccinee over a broad range of effector-to-target ratios. Similarly, the LCL-CD4HWV 1were efficiently lysed by fresh circulating CTL from HIV-1-infected individuals, as well as by CTL activated by in vitro stimulation. Both HIV-1 env-and gag-specific CTL effectors lysed LCL-CD4HIV1, consistent with the cellular expression of both HIV-1 genes. The LCL-CD4HWv also functioned as stimulator cells, and thus are capable of amplifying CTL against multiple HIV-1 gene products in HIV-1-infected individuals. The ability to produce HIV-1-susceptible autologous immortalized cell lines that can be employed as target cells should enable a more detailed evaluation of vaccine-induced CTL against both homologous and disparate HIV-1 strains. Furthermore, the use of