Abstract In an earlier study, our group vaccinated rhesus macaques with vesicular stomatitis virus (VSV) vectors expressing Gag, Pol, and Env proteins from a hybrid simian/human immunodeficiency ...virus (SHIV). This was followed by a single boost with modified vaccinia virus Ankara (MVA) vectors expressing the same proteins. Following challenge with SHIV89.6P, vaccinated animals cleared challenge virus RNA from the blood by day 150 and maintained normal CD4 T cell counts for 8 months. Here we report on the long-term (>5-year post-challenge) status of these animals and the immunological correlates of long-term protection. Using real-time PCR, we found that viral DNA in peripheral blood mononuclear cells (PBMCs) of the vaccinees declined continuously and fell to below detection (<5 copies/105 cells) by approximately 3 years post-challenge. SHIV DNA was also below the limit of detection in the lymph nodes of two of the four animals at 5 years post-challenge. We detected long-term persistence of multi-functional Gag-specific CD8+ T cells in both PBMCs and lymph nodes of the two protected animals with the Mamu A*01+ MHC I allele. All animals also maintained SHIV89.6P neutralizing antibody titers for 5 years. Our results show that this vaccine approach generates solid, long-term control of SHIV infection, and suggest that it is mediated by both cytotoxic T lymphocytes and neutralizing antibody.
Staphylococcus aureus was shown to transport iron complexed to a variety of hydroxamate type siderophores, including ferrichrome, aerobactin, and desferrioxamine. An S. aureus mutant defective in the ...ability to transport ferric hydroxamate complexes was isolated from a Tn917-LTV1 transposon insertion library after selection on iron-limited media containing aerobactin and streptonigrin. Chromosomal DNA flanking the Tn917-LTV1 insertion was identified by sequencing of chromosomal DNA isolated from the mutant. This information localized the transposon insertion to a gene whose predicted product shares significant similarity with FhuG of Bacillus subtilis. DNA sequence information was then used to clone a larger fragment of DNA surrounding the fhuG gene, and this resulted in the identification of an operon of three genes, fhuCBG, all of which show significant similarities to ferric hydroxamate uptake (fhu) genes in B. subtilis. FhuB and FhuG are highly hydrophobic, suggesting that they are embedded within the cytoplasmic membrane, while FhuC shares significant homology with ATP-binding proteins. Given this, the S. aureus FhuCBG proteins were predicted to be part of a binding protein-dependent transport system for ferric hydroxamates. Exogenous iron levels were shown to regulate ferric hydroxamate uptake in S. aureus. This regulation is attributable to Fur in S. aureus because a strain containing an insertionally inactivated fur gene showed maximal levels of ferric hydroxamate uptake even when the cells were grown under iron-replete conditions. By using the Fur titration assay, it was shown that the Fur box sequences upstream of fhuCBG are recognized by the Escherichia coli Fur protein.
Most successful vaccines elicit neutralizing antibodies and this property is a high priority when developing an HIV vaccine. Indeed, passively administered neutralizing antibodies have been shown to ...protect against HIV challenge in some of the best available animal models. For example, antibodies given intravenously can protect macaques against intravenous or mucosal SHIV (an HIV/SIV chimaera) challenge and topically applied antibodies can protect macaques against vaginal SHIV challenge. However, the mechanism(s) by which neutralizing antibodies afford protection against HIV is not understood and, in particular, the role of antibody Fc-mediated effector functions is unclear. Here we report that there is a dramatic decrease in the ability of a broadly neutralizing antibody to protect macaques against SHIV challenge when Fc receptor and complement-binding activities are engineered out of the antibody. No loss of antibody protective activity is associated with the elimination of complement binding alone. Our in vivo results are consistent with in vitro assays indicating that interaction of Fc-receptor-bearing effector cells with antibody-complexed infected cells is important in reducing virus yield from infected cells. Overall, the data suggest the potential importance of activity against both infected cells and free virus for effective protection against HIV.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Rhesus macaques intrabronchially inoculated with simian varicella virus (SVV), the counterpart of human varicella-zoster virus (VZV), developed primary infection with viremia and rash, which resolved ...upon clearance of viremia, followed by the establishment of latency. To assess the role of CD4 T cell immunity in reactivation, monkeys were treated with a single 50-mg/kg dose of a humanized monoclonal anti-CD4 antibody; within 1 week, circulating CD4 T cells were reduced from 40 to 60% to 5 to 30% of the total T cell population and remained low for 2 months. Very low viremia was seen only in some of the treated monkeys. Zoster rash developed after 7 days in the monkey with the most extensive CD4 T cell depletion (5%) and in all other monkeys at 10 to 49 days posttreatment, with recurrent zoster in one treated monkey. SVV DNA was detected in the lung from two of five monkeys, in bronchial lymph nodes from one of the five monkeys, and in ganglia from at least two dermatomes in three of five monkeys. Immunofluorescence analysis of skin rash, lungs, lymph nodes, and ganglia revealed SVV ORF63 protein at the following sites: sweat glands in skin; type II cells in lung alveoli, macrophages, and dendritic cells in lymph nodes; and the neuronal cytoplasm of ganglia. Detection of SVV antigen in multiple tissues upon CD4 T cell depletion and virus reactivation suggests a critical role for CD4 T cell immunity in controlling varicella virus latency.
Reactivation of latent VZV in humans can result in serious neurological complications. VZV-specific cell-mediated immunity is critical for the maintenance of latency. Similar to VZV in humans, SVV causes varicella in monkeys, establishes latency in ganglia, and reactivates to produce shingles. Here, we show that depletion of CD4 T cells in rhesus macaques results in SVV reactivation, with virus antigens found in zoster rash and SVV DNA and antigens found in lungs, lymph nodes, and ganglia. These results suggest the critical role of CD4 T cell immunity in controlling varicella virus latency.
Clostridium difficile-associated disease (CDAD) constitutes a large majority of nosocomial diarrhea cases in industrialized nations and is mediated by the effects of two secreted toxins, toxin A ...(TcdA) and toxin B (TcdB). Patients who develop strong antitoxin antibody responses can clear C. difficile infection and remain disease free. Key toxin-neutralizing epitopes have been found within the carboxy-terminal receptor binding domains (RBDs) of TcdA and TcdB, which has generated interest in developing the RBD as a viable vaccine target. While numerous platforms have been studied, very little data describes the potential of DNA vaccination against CDAD. Therefore, we created highly optimized plasmids encoding the RBDs from TcdA and TcdB in which any putative N-linked glycosylation sites were altered. Mice and nonhuman primates were immunized intramuscularly, followed by in vivo electroporation, and in these animal models, vaccination induced significant levels of both anti-RBD antibodies (blood and stool) and RBD-specific antibody-secreting cells. Further characterization revealed that sera from immunized mice and nonhuman primates could detect RBD protein from transfected cells, as well as neutralize purified toxins in an in vitro cytotoxicity assay. Mice that were immunized with plasmids or given nonhuman-primate sera were protected from a lethal challenge with purified TcdA and/or TcdB. Moreover, immunized mice were significantly protected when challenged with C. difficile spores from homologous (VPI 10463) and heterologous, epidemic (UK1) strains. These data demonstrate the robust immunogenicity and efficacy of a TcdA/B RBD-based DNA vaccine in preclinical models of acute toxin-associated and intragastric, spore-induced colonic disease.
Simian varicella virus (SVV), the primate counterpart of varicella-zoster virus, causes varicella (chickenpox), establishes latency in ganglia, and reactivates to produce zoster. We previously ...demonstrated that a recombinant SVV expressing enhanced green fluorescent protein (rSVV.eGFP) is slightly attenuated both in culture and in infected monkeys. Here, we generated two additional recombinant SVVs to visualize infected cells
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One harbors eGFP fused to the N terminus of open reading frame 9 (ORF9) (rSVV.eGFP-2a-ORF9), and another harbors eGFP fused to the C terminus of ORF66 (rSVV.eGFP-ORF66). Both recombinant viruses efficiently expressed eGFP in cultured cells. Both recombinant SVV infections in culture were comparable to that of wild-type SVV (SVV.wt). Unlike SVV.wt, eGFP-tagged SVV did not replicate in rhesus cells in culture. Intratracheal (i.t.) or i.t. plus intravenous (i.v.) inoculation of rhesus macaques with these new eGFP-tagged viruses resulted in low viremia without varicella rash, although SVV DNA was abundant in bronchoalveolar lavage (BAL) fluid at 10 days postinoculation (dpi). SVV DNA was also found in trigeminal ganglia of one monkey inoculated with rSVV.eGFP-ORF66. Intriguingly, a humoral response to both SVV and eGFP was observed. In addition, monkeys inoculated with the eGFP-expressing viruses were protected from superinfection with SVV.wt, suggesting that the monkeys had mounted an efficient immune response. Together, our results show that eGFP expression could be responsible for their reduced pathogenesis.
SVV infection in nonhuman primates has served as an extremely useful animal model to study varicella-zoster virus (VZV) pathogenesis. eGFP-tagged viruses are a great tool to investigate their pathogenesis. We constructed and tested two new recombinant SVVs with eGFP inserted into two different locations in the SVV genome. Both recombinant SVVs showed robust replication in culture but reduced viremia compared to that with SVV.wt during primary infection in rhesus macaques. Our results indicate that conclusions on eGFP-tagged viruses based on
results should be handled with care, since eGFP expression could result in attenuation of the virus.
Abstract
Development of a vaccine that drives anti-viral mucosal B cell responses is critical for protection against HIV-1 infection. It is currently unknown whether triggering CCR10/CCL28 pathways ...in a DNA-based vaccine results in induction of HIV-1env specific B cell immunity at mucosal sites of infection. We hypothesized that co-immunization with HIV-1env/CCL28 molecular adjuvant would augment B cell responses at gastrointestinal and vaginal sites and require CCR10. CCL28 co-immunized WT mice displayed a significant enhancement of HIV-1env specific antibody titers in serum, feces and vaginal washes, and enhanced HIV-1 specific IgA responses were abrogated in CCR10KO mice. CCL28 co-immunization did not increase the breadth of linear B cell epitopes in WT mice, but augmented the dominant epitopes elicited by antigen immunization alone. The frequency of splenic and intestinal IgA+CD19+B220+CD138+CCR10+ plasmablasts was augmented in the CCL28 co-immunized WT mice over antigen-only immunized WT controls. The physiological relevance of these findings was confirmed in a NHP model of intravaginal SIVsmE660 challenge in which CCL28 co-immunization resulted in significant increases in serum/vaginal IgG/IgA, decrease in peak viral loads, significant suppression of viral titers over 120 days, and recovery of CD4 T cells. These data support a role for CCL28 in targeting protective anti-viral B cells to mucosal sites when delivered as molecular adjuvants for HIV-1env DNA-based vaccines.
OBJECTIVE:Intravaginal exposure to simian immunodeficiency virus (SIV) acutely recruits interferon-alpha (IFN-α) producing plasmacytoid dendritic cells (pDC) and CD4 T-lymphocyte targets to the ...endocervix of nonhuman primates. We tested the impact of repeated cervicovaginal exposures to noninfectious, defective SIV particles over 72 hours on a subsequent cervicovaginal challenge with replication competent SIV.
METHODS:Thirty-four female Indian Rhesus macaques were given a 3-day twice-daily vaginal exposures to either SIVsmB7, a replication-deficient derivative of SIVsmH3 produced by a T lymphoblast CEMx174 cell clone (n = 16), or to CEM supernatant controls (n = 18). On the fourth day, animals were either euthanized to assess cervicovaginal immune cell infiltration or intravaginally challenged with SIVmac251. Challenged animals were tracked for plasma viral load and CD4 counts and euthanized at 42 days after infection.
RESULTS:At the time of challenge, macaques exposed to SIVsmB7, had higher levels of cervical CD123 pDCs (P = 0.032) and CD4 T cells (P = 0.036) than those exposed to CEM control. Vaginal tissues showed a significant increase in CD4 T-cell infiltrates (P = 0.048) and a trend toward increased CD68 cellular infiltrates. After challenge, 12 SIVsmB7-treated macaques showed 2.5-fold greater daily rate of CD4 decline (P = 0.0408), and viral load rise (P = 0.0036) as compared with 12 control animals.
CONCLUSIONS:Repeated nonproductive exposure to viral particles within a short daily time frame did not protect against infection despite pDC recruitment, resulting instead in an accelerated CD4 T-cell loss with an increased rate of viral replication.
Highlights ► Expression of VSV encoded GMCSF during prime did not enhance immune responses. ► GMCSF reduced the level of protection against subsequent SIVsmE660 challenge. ► Serum neutralizing ...antibody toward Env correlated with protection.
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