KIR3DL05 and KIR3DS02 Recognition of a Nonclassical MHC Class I Molecule in the Rhesus Macaque Implicated in Pregnancy Success.

Knowledge of the MHC class I ligands of rhesus macaque killer-cell Ig-like receptors (KIRs) is fundamental to understanding the role of natural killer (NK) cells in this species as a nonhuman primate model for infectious diseases, transplantation and reproductive biology. We previously identified Mamu-AG as a ligand for KIR3DL05. Mamu-AG is a nonclassical MHC class I molecule that is expressed at the maternal-fetal interface of the placenta in rhesus macaques similar to HLA-G in humans. Although Mamu-AG and HLA-G share similar molecular features, including limited polymorphism and a short cytoplasmic tail, Mamu-AG is considerably more polymorphic. To determine which allotypes of Mamu-AG serve as ligands for KIR3DL05, we tested reporter cell lines expressing five different alleles of KIR3DL05 (KIR3DL05*001, KIR3DL05*004, KIR3DL05*005, KIR3DL05*008 and KIR3DL05*X) for responses to target cells expressing eight different alleles of Mamu-AG. All five allotypes of KIR3DL05 responded to Mamu-AG2*01:01, two exhibited dominant responses to Mamu-AG1*05:01, and three had low but detectable responses to Mamu-AG3*03:01, -AG3*03:02, -AG3*03:03 and -AG3*03:04. Since KIR3DL05*X is the product of recombination between KIR3DL05 and KIR3DS02, we also tested an allotype of KIR3DS02 (KIR3DS02*004) and found that this activating KIR also recognizes Mamu-AG2*01:01. Additional analysis of Mamu-AG variants with single amino acid substitutions identified residues in the α1-domain essential for recognition by KIR3DL05. These results reveal variation in KIR3DL05 and KIR3DS02 responses to Mamu-AG and define Mamu-AG polymorphisms that differentially affect KIR recognition.

BRCA1 and RNAi factors promote repair mediated by small RNAs and PALB2-RAD52.

Strong connections exist between R-loops (three-stranded structures harbouring an RNA:DNA hybrid and a displaced single-strand DNA), genome instability and human disease1-5. Indeed, R-loops are favoured in relevant genomic regions as regulators of certain physiological processes through which homeostasis is typically maintained. For example, transcription termination pause sites regulated by R-loops can induce the synthesis of antisense transcripts that enable the formation of local, RNA interference (RNAi)-driven heterochromation6. Pause sites are also protected against endogenous single-stranded DNA breaks by BRCA17. Hypotheses about how DNA repair is enacted at pause sites include a role for RNA, which is emerging as a normal, albeit unexplained, regulator of genome integrity8. Here we report that a species of single-stranded, DNA-damage-associated small RNA (sdRNA) is generated by a BRCA1-RNAi protein complex. sdRNAs promote DNA repair driven by the PALB2-RAD52 complex at transcriptional termination pause sites that form R-loops and are rich in single-stranded DNA breaks. sdRNA repair operates in both quiescent (G0) and proliferating cells. Thus, sdRNA repair can occur in intact tissue and/or stem cells, and may contribute to tumour suppression mediated by BRCA1.

Diversification of Bw4 Specificity and Recognition of a Nonclassical MHC Class I Molecule Implicated in Maternal-Fetal Tolerance by Killer Cell Ig-like Receptors of the Rhesus Macaque.

The rhesus macaque is an important animal model for AIDS and other infectious diseases; however, studies to address NK cell function in this species have been limited by the lack of defined ligands for killer cell Ig-like receptors (KIRs). To identify ligands for rhesus macaque KIRs, we adopted a novel approach based on a pair of stable cell lines. NFAT-responsive luciferase reporter cell lines expressing the extracellular domains of macaque KIRs fused to the transmembrane and cytoplasmic domains of CD28 and CD3ζ were incubated with target cells expressing individual MHC class I molecules, and ligand recognition was detected by the MHC class I-dependent upregulation of luciferase. Using this approach, we found that Mamu-KIR3DL01, -KIR3DL06, -KIR3DL08, and -KIR3DSw08 all recognize Mamu-Bw4 molecules but with differing allotype specificity. In contrast, Mamu-KIR3DL05 recognizes Mamu-A and Mamu-A-related molecules, including Mamu-A1*002 and -A3*13, Mamu-B*036, the product of a recombinant Mamu-B allele with α1 and α2 domain sequences derived from a MHC-A gene, and Mamu-AG*01, a nonclassical molecule expressed on placental trophoblasts that originated from an ancestral duplication of a MHC-A gene. These results reveal an expansion of the lineage II KIRs in macaques that recognize Bw4 ligands and identify a nonclassical molecule implicated in placental development and pregnancy as a ligand for Mamu-KIR3DL05. In addition to offering new insights into KIR-MHC class I coevolution, these findings provide an important foundation for investigating the role of NK cells in the rhesus macaque as an animal model for infectious diseases and reproductive biology.

Evaluating the Vaccine Potential of a Tetravalent Fusion Protein (rBmHAXT) Vaccine Antigen Against Lymphatic Filariasis in a Mouse Model.

Lymphatic filariasis (LF) is a tropical parasitic infection of human transmitted by mosquitoes. Chronic infection results in severe physical disability in the infected patients. Although several potential vaccine antigens were identified by several groups, there are no licensed prophylactic vaccine to date against this infection in the human. Previous attempts from our laboratory to develop a trivalent prophylactic vaccine against LF showed that >90% protection could be achieved in rodent models. However, this trivalent vaccine gave only 35% protection in non-human primates. The major focus of this study was to develop a tetravalent prophylactic vaccine (rBmHAXT) and test the vaccine potential in a mouse model. We evaluated three different adjuvant formulations; alum, glucopyranosyl lipid adjuvant in stable emulsion (GLA/SE) alum (AL019), and mannosylated chitosan (MCA) to determine the optimum adjuvant formulation for rBmHAXT. Results presented in this study show that rBmHAXT + AL019 gave the highest rate of protection (>88%) against challenge infection, compared to rBmHAXT + AL007 (79%), rBmHAXT + MCA (79%) and controls. Analysis of the immune correlates of protection showed that all three adjuvants elicited high titer of antigen-specific IgG1, IgG2a, and IgG2b antibodies. High number of IFN-γ-producing antigen-specific memory cells were generated in the vaccinated animals irrespective of the adjuvants used. Similarly, spleen cells from rBmHAXT-vaccinated animals secreted IL-4, IL-10, and IFN-γ in response to rBmHAXT suggesting the generation of a balanced Th1/Th2 response. There was also an increase in IL-17-secreting cells in rBmHAXT-vaccinated animals. These findings thus suggest that rBmHAXT + AL019 is a better prophylactic formulation for LF.

Improving the efficacy of a prophylactic vaccine formulation against lymphatic filariasis.

Mass drug administration (MDA) is the current strategy for interrupting the transmission of lymphatic filariasis (LF) infection and control of the disease in endemic areas. However, subject non-compliance has resulted in the presence of several "transmission hotspots" in the endemic regions threatening the reemergence of LF. This situation is further complicated by the fact that the drugs used in MDA are not effective against adult LF worms, a major concern for the control strategy. Thus, there is clearly a need for an effective and sustainable approach to control LF. Prophylactic vaccine combined with targeted treatment of infected patients and vector control is suggested as a more sustainable strategy to eliminate LF infection from endemic regions. A multivalent vaccine (rBmHAT) developed in our laboratory conferred about 90% protection in rodents. However, when we tested the rBmHAT vaccine along with alum in rhesus macaques, only about 40% protection was achieved and the immune response obtained was Th2 biased. In an attempt to improve the vaccine, in this study, we tested two vaccine antigens (rBmHAT and rBmHAX) along with two adjuvant formulations [alum + GLA (AL019) and mannosylated chitosan (MCA)] in a mouse model. Our results show that rBmHAT is a better vaccine antigen than rBmHAX. Combination of rBmHAT with AL019 or MCA adjuvants gave 94 and 88% protection, respectively, against challenge infections. Immunized animals developed antigen-specific memory T cells that secreted significant levels of IL-4, IFN-γ, and IL-17 suggesting the generation of a balanced Th1/Th2 responses following immunization. A major advantage of MCA adjuvant is that the vaccine booster doses can be administered orally. These studies thus showed that rBmHAT is a better vaccine antigen and can be given in combination with AL019 or MCA adjuvant to obtain excellent results.