Simian-Human Immunodeficiency Virus SHIV.CH505 Infection of Rhesus Macaques Results in Persistent Viral Replication and Induces Intestinal Immunopathology.

Simian-human immunodeficiency viruses (SHIVs) have been utilized to test vaccine efficacy and characterize mechanisms of viral transmission and pathogenesis. However, the majority of SHIVs currently available have significant limitations in that they were developed using sequences from chronically HIV-infected individuals or uncommon HIV subtypes or were optimized for the macaque model by serially passaging the engineered virus in vitro or in vivo Recently, a newly developed SHIV, SHIV.C.CH505.375H.dCT (SHIV.CH505), which incorporates vpu-env (gp140) sequences from a transmitted/founder HIV-1 subtype C strain, was shown to retain attributes of primary HIV-1 strains. However, a comprehensive analysis of the immunopathology that results from infection with this virus, especially in critical tissue compartments like the intestinal mucosa, has not been completed. In this study, we evaluated the viral dynamics and immunopathology of SHIV.CH505 in rhesus macaques. In line with previous findings, we found that SHIV.CH505 is capable of infecting and replicating efficiently in rhesus macaques, resulting in peripheral viral kinetics similar to that seen in pathogenic SIV and HIV infection. Furthermore, we observed significant and persistent depletions of CCR5+ and CCR6+ CD4+ T cells in mucosal tissues, decreases in CD4+ T cells producing Th17 cell-associated cytokines, CD8+ T cell dysfunction, and alterations of B cell and innate immune cell function, indicating that SHIV.CH505 elicits intestinal immunopathology typical of SIV/HIV infection. These findings suggest that SHIV.CH505 recapitulates the early viral replication dynamics and immunopathogenesis of HIV-1 infection of humans and thus can serve as a new model for HIV-1 pathogenesis, treatment, and prevention research.IMPORTANCE The development of chimeric SHIVs has been instrumental in advancing our understanding of HIV-host interactions and allowing for in vivo testing of novel treatments. However, many of the currently available SHIVs have distinct drawbacks and are unable to fully reflect the features characteristic of primary SIV and HIV strains. Here, we utilize rhesus macaques to define the immunopathogenesis of the recently developed SHIV.CH505, which was designed without many of the limitations of previous SHIVs. We observed that infection with SHIV.CH505 leads to peripheral viral kinetics and mucosal immunopathogenesis comparable with those caused by pathogenic SIV and HIV. Overall, these data provide evidence of the value of SHIV.CH505 as an effective model of SIV/HIV infection and an important tool that can be used in future studies, including preclinical testing of new therapies or prevention strategies.

KSHV induces immunoglobulin rearrangements in mature B lymphocytes.

Kaposi sarcoma herpesvirus (KSHV/HHV-8) is a B cell tropic human pathogen, which is present in vivo in monotypic immunoglobulin λ (Igλ) light chain but polyclonal B cells. In the current study, we use cell sorting to infect specific B cell lineages from human tonsil specimens in order to examine the immunophenotypic alterations associated with KSHV infection. We describe IL-6 dependent maturation of naïve B lymphocytes in response to KSHV infection and determine that the Igλ monotypic bias of KSHV infection in vivo is due to viral induction of BCR revision. Infection of immunoglobulin κ (Igκ) naïve B cells induces expression of Igλ and isotypic inclusion, with eventual loss of Igκ. We show that this phenotypic shift occurs via re-induction of Rag-mediated V(D)J recombination. These data explain the selective presence of KSHV in Igλ B cells in vivo and provide the first evidence that a human pathogen can manipulate the molecular mechanisms responsible for immunoglobulin diversity.

Cytomegalovirus CC chemokine promotes immune cell migration.

Cytomegaloviruses manipulate the host chemokine/receptor axis by altering cellular chemokine expression and by encoding multiple chemokines and chemokine receptors. Similar to human cytomegalovirus (HCMV), rat cytomegalovirus (RCMV) encodes multiple CC chemokine-analogous proteins, including r129 (HCMV UL128 homologue) and r131 (HCMV UL130 and MCMV m129/130 homologues). Although these proteins play a role in CMV entry, their function as chemotactic cytokines remains unknown. In the current study, we examined the role of the RCMV chemokine r129 in promoting cellular migration and in accelerating transplant vascular sclerosis (TVS) in our rat heart transplant model. We determined that r129 protein is released into culture supernatants of infected cells and is expressed with late viral gene kinetics during RCMV infection and highly expressed in heart and salivary glands during in vivo rat infections. Using the recombinant r129 protein, we demonstrated that r129 induces migration of lymphocytes isolated from rat peripheral blood, spleen, and bone marrow and from a rat macrophage cell line. Using antibody-mediated cell sorting of rat splenocytes, we demonstrated that r129 induces migration of naïve/central memory CD4(+) T cells. Through ligand-binding assays, we determined that r129 binds rat CC chemokine receptors CCR3, CCR4, CCR5, and CCR7. In addition, mutational analyses identified functional domains of r129 resulting in recombinant proteins that fail to induce migration (r129-ΔNT and -C31A) or alter the chemotactic ability of the chemokine (r129-F43A). Two of the mutant proteins (r129-C31A and -ΔNT) also act as dominant negatives by inhibiting migration induced by wild-type r129. Furthermore, infection of rat heart transplant recipients with RCMV containing the r129-ΔNT mutation prevented CMV-induced acceleration of TVS. Together our findings indicate that RCMV r129 is highly chemotactic, which has important implications during RCMV infection and reactivation and acceleration of TVS.

Shewanella oneidensis Hfq promotes exponential phase growth, stationary phase culture density, and cell survival.

BACKGROUND: Hfq is an RNA chaperone protein that has been broadly implicated in sRNA function in bacteria. Here we describe the construction and characterization of a null allele of the gene that encodes the RNA chaperone Hfq in Shewanella oneidensis strain MR-1, a dissimilatory metal reducing bacterium. RESULTS: Loss of hfq in S. oneidensis results in a variety of mutant phenotypes, all of which are fully complemented by addition of a plasmid-borne copy of the wild type hfq gene. Aerobic cultures of the hfq∆ mutant grow more slowly through exponential phase than wild type cultures, and hfq∆ cultures reach a terminal cell density in stationary phase that is ~2/3 of that observed in wild type cultures. We have observed a similar growth phenotype when the hfq∆ mutant is cultured under anaerobic conditions with fumarate as the terminal electron acceptor, and we have found that the hfq∆ mutant is defective in Cr(VI) reduction. Finally, the hfq∆ mutant exhibits a striking loss of colony forming units in extended stationary phase and is highly sensitive to oxidative stress induced by H2O2 or methyl viologen (paraquat). CONCLUSIONS: The hfq mutant in S. oneidensis exhibits pleiotropic phenotypes, including a defect in metal reduction. Our results also suggest that hfq mutant phenotypes in S. oneidensis may be at least partially due to increased sensitivity to oxidative stress.

Effects of persistent modulation of intestinal microbiota on SIV/HIV vaccination in rhesus macaques.

An effective vaccine to prevent HIV transmission has not yet been achieved. Modulation of the microbiome via probiotic therapy has been suggested to result in enhanced mucosal immunity. Here, we evaluated whether probiotic therapy could improve the immunogenicity and protective efficacy of SIV/HIV vaccination. Rhesus macaques were co-immunized with an SIV/HIV DNA vaccine via particle-mediated epidermal delivery and an HIV protein vaccine administered intramuscularly with Adjuplex™ adjuvant, while receiving daily oral Visbiome® probiotics. Probiotic therapy alone led to reduced frequencies of colonic CCR5+ and CCR6+ CD4+ T cells. Probiotics with SIV/HIV vaccination led to similar reductions in colonic CCR5+ CD4+ T cell frequencies. SIV/HIV-specific T cell and antibody responses were readily detected in the periphery of vaccinated animals but were not enhanced with probiotic treatment. Combination probiotics and vaccination did not impact rectal SIV/HIV target populations or reduce the rate of heterologous SHIV acquisition during the intrarectal challenge. Finally, post-infection viral kinetics were similar between all groups. Thus, although probiotics were well-tolerated when administered with SIV/HIV vaccination, vaccine-specific responses were not significantly enhanced. Additional work will be necessary to develop more effective strategies of microbiome modulation in order to enhance mucosal vaccine immunogenicity and improve protective immune responses.

Aberrant proliferation in CXCR7+ endothelial cells via degradation of the retinoblastoma protein.

Angiogenesis is a critical factor in the growth and dissemination of solid tumors. Indeed, tumor vasculature is abnormal and contributes to the development and spread of malignancies by creating a hostile microenvironment. The alternative SDF-1/CXCL12 receptor, CXCR7, is frequently and specifically expressed in tumor-associated vessels. In this study, we examine the role of endothelium-expressed CXCR7 in tumor vascular dysfunction by specifically examining the contribution of CXCR7 to endothelial cell (EC) proliferation. We demonstrate that CXCR7 expression is sufficient to drive post-confluent growth in EC cultures. Further, we provide a novel mechanism for CXCR7-mediated proliferation via proteasomal degradation of the tumor suppressor protein Rb. These findings identify a heretofore unappreciated role for CXCR7 in vascular dysfunction and confirm this receptor as a plausible target for anti-tumor therapy.