A long-term stable cold-chain-friendly HIV mRNA vaccine encoding multi-epitope viral protease cleavage site immunogens inducing immunogen-specific protective T cell immunity.

AbstractThe lack of success in clinical trials for HIV vaccines highlights the need to explore novel strategies for vaccine development. Research on highly exposed seronegative (HESN) HIV-resistant Kenyan female sex workers revealed naturally protective immunity is correlated with a focused immune response mediated by virus-specific CD8 T cells. Further studies indicated that the immune response is unconventionally focused on highly conserved sequences around HIV viral protease cleavage sites (VPCS). Thus, taking an unconventional approach to HIV vaccine development, we designed lipid nanoparticles loaded with mRNA that encodes multi-epitopes of VPCS (MEVPCS-mRNA LNP), a strategic design to boost antigen presentation by dendritic cells, promoting effective cellular immunity. Furthermore, we developed a novel cold-chain compatible mRNA LNP formulation, ensuring long-term stability and compatibility with cold-chain storage/transport, widening accessibility of mRNA LNP vaccine in low-income countries. The in-vivo mouse study demonstrated that the vaccinated group generated VPCS-specific CD8 memory T cells, both systemically and at mucosal sites of viral entry. The MEVPCS-mRNA LNP vaccine-induced CD8 T cell immunity closely resembled that of the HESN group and displayed a polyfunctional profile. Notably, it induced minimal to no activation of CD4 T cells. This proof-of-concept study underscores the potential of the MEVPCS-mRNA LNP vaccine in eliciting CD8 T cell memory specific to the highly conserved multiple VPCS, consequently having a broad coverage in human populations and limiting viral escape mutation. The MEVPCS-mRNA LNP vaccine holds promise as a candidate for an effective prophylactic HIV vaccine.

Simian immunodeficiency virus-infected rhesus macaques with AIDS co-develop cardiovascular pathology and encephalitis.

Despite effective antiretroviral therapy, HIV co-morbidities remain where central nervous system (CNS) neurocognitive disorders and cardiovascular disease (CVD)-pathology that are linked with myeloid activation are most prevalent. Comorbidities such as neurocogntive dysfunction and cardiovascular disease (CVD) remain prevalent among people living with HIV. We sought to investigate if cardiac pathology (inflammation, fibrosis, cardiomyocyte damage) and CNS pathology (encephalitis) develop together during simian immunodeficiency virus (SIV) infection and if their co-development is linked with monocyte/macrophage activation. We used a cohort of SIV-infected rhesus macaques with rapid AIDS and demonstrated that SIV encephalitis (SIVE) and CVD pathology occur together more frequently than SIVE or CVD pathology alone. Their co-development correlated more strongly with activated myeloid cells, increased numbers of CD14+CD16+ monocytes, plasma CD163 and interleukin-18 (IL-18) than did SIVE or CVD pathology alone, or no pathology. Animals with both SIVE and CVD pathology had greater numbers of cardiac macrophages and increased collagen and monocyte/macrophage accumulation, which were better correlates of CVD-pathology than SIV-RNA. Animals with SIVE alone had higher levels of activated macrophage biomarkers and cardiac macrophage accumulation than SIVnoE animals. These observations were confirmed in HIV infected individuals with HIV encephalitis (HIVE) that had greater numbers of cardiac macrophages and fibrosis than HIV-infected controls without HIVE. These results underscore the notion that CNS and CVD pathologies frequently occur together in HIV and SIV infection, and demonstrate an unmet need for adjunctive therapies targeting macrophages.

Spatially Resolved and Highly Multiplexed Protein and RNA In Situ Detection by Combining CODEX With RNAscope In Situ Hybridization.

Highly multiplexed protein and RNA in situ detection on a single tissue section concurrently is highly desirable for both basic and applied biomedical research. CO-detection by inDEXing (CODEX) is a new and powerful platform to visualize up to 60 protein biomarkers in situ, and RNAscope in situ hybridization (RNAscope) is a novel RNA detection system with high sensitivity and unprecedent specificity at a single-cell level. Nevertheless, to our knowledge, the combination of CODEX and RNAscope remained unreported until this study. Here, we report a simple and reproducible combination of CODEX and RNAscope. We also determined the cross-reactivities of CODEX anti-human antibodies to rhesus macaques, a widely used animal model of human disease.

Sustained heat stress elevated corneal and body surface temperatures and altered circulating leukocytes and metabolic indicators in wether lambs supplemented with ractopamine or zilpaterol.

Understanding how ß adrenergic agonists influence the physiology of heat stress could lead to mitigation options. We sought to investigate body surface temperatures in feedlot wethers supplemented with ractopamine or zilpaterol and exposed to heat stress for 18 d. Corneal and skin temperatures were assessed via infrared thermography at 1- and 2-m distances. Rectal temperatures and circulating leukocytes, metabolites, and electrolytes were also measured. Heat stress increased (P < 0.05) rectal temperatures in unsupplemented and zilpaterol-supplemented lambs but not in ractopamine-supplemented lambs. Heat stress also increased (P < 0.05) surface temperatures of the cornea, nose, ear, and back, regardless of supplement. Observations were comparable between thermography performed at 1 and 2 m, and higher emissivity settings generally produced less variation. Heat stress tended to increase (P = 0.08) blood monocytes in unsupplemented but not ractopamine- or zilpaterol-supplemented lambs. Granulocytes were increased (P < 0.05) by heat stress in ractopamine-supplemented lambs but decreased (P < 0.05) in zilpaterol-supplemented lambs. Blood glucose, triglycerides, and cholesterol did not differ among groups, and blood lactate was reduced (P < 0.05) by heat stress in zilpaterol-supplemented lambs only. Blood Na+ was reduced (P < 0.05) and Ca2+ increased (P < 0.05) by heat stress, regardless of supplement. These findings indicate that ß1- and ß2-adrenergic agonists differentially relieve some but not all heat stress-induced changes in stress indicators. Moreover, corneal and skin surface temperatures measured by infrared thermography reasonably identified body temperature changes at a distance of 2 m.

Whole blood transcriptome analysis in feedlot cattle after 35 days of supplementation with a ß1-adrenergic agonist.

Ractopamine HCl (RHC) is supplemented to feedlot cattle to improve feed efficiency and increase carcass weight. Supplementation of RHC clearly benefits livestock production, but it is of note that the adrenergic system through which it acts is typically associated with stress. The purpose of this study was to identify changes in the transcriptome of whole blood in RHC-supplemented feedlot cattle. We hypothesized that transcripts related to inflammatory processes would be upregulated after 35 days of dietary RHC supplementation. To test this hypothesis, RNA from whole blood collected from 16 cattle before and after supplementation with 300 mg/day of RHC was sequenced using 3' tag-seq. Eight transcripts were differentially expressed (Adjp < 0.10) between pre- and post-supplementation blood samples. Although several of these transcripts including IFI35, TYROBP, and TP53INP1 are associated with inflammation, a systemic dysregulation of inflammatory pathways was not evident. These data provide insight into the response of cattle to RHC supplementation that will direct future studies examining how the transcriptome of whole blood and other tissues responds during acute exposure to RHC and how this supplement mechanistically improves growth performance.

Evidence for a de novo, dominant germ-line mutation causative of osteogenesis imperfecta in two Red Angus calves.

A genetic disorder, osteogenesis imperfecta (OI) is broadly characterized by connective tissue abnormalities and bone fragility most commonly attributed to alterations in Type I collagen. Two Red Angus calves by the same sire presented with severe bone and dental fragility, blue sclera, and evidence of in utero fractures consistent with OI congenita. Comparative analyses with human cases suggested the OI in these calves most closely resembled that classified as OI Type II. Due to the phenotypic classification and shared paternity, a dominant, germ-line variant was hypothesized as causative although recessive genotypes were also considered due to a close relationship between the sire and dam of one calf. Whole-genome sequencing revealed the presence of a missense mutation in the alpha 1 chain of collagen Type I (COL1A1), for which both calves were heterozygous. The variant resulted in the substitution of a glycine residue with serine in the triple helical domain of the protein; in this region, glycine normally occupies every third position as is critical for correct formation of the Type I collagen molecule. Allele-specific amplification by droplet digital PCR further quantified the variant at a frequency of nearly 4.4% in the semen of the sire while it was absent in his blood, supporting the hypothesis of a de novo causative variant for which the germ line of the sire was mosaic. The identification of novel variants associated with unwanted phenotypes in livestock is critical as the high prolificacy of breeding stock has the potential to rapidly disseminate undesirable variation.