Deborah Smith

Low circulating CD4 cell numbers and CD4 cell dysfunction are distinguishing features of HIV-mediated disease. The current study delineates the in vivo effects of HIV on distinct functional subsets of CD4 cells in homosexually active men who have been infected with HIV for different lengths of time, and examines the capacity of lymphocytes from these men to proliferate in vitro in response to soluble antigen. Although peripherial blood mononuclear cells from most acquired immune deficiency syndrome (AIDS) patients did not proliferate in response to either tetanus toxoid or Candida albicans, cells from most HIV seropositive men without AIDS, many of whom had been infected for more than 18 mo, responded normally to both. Non-responsiveness in HIV-infected men without AIDS was a late event and was associated with longer duration of infection, lower CD4 cell numbers, and subsequent development of AIDS. A defect in this response was observed in only one of 19 HIV seropositive men whose CD4 levels were greater than 300/mm3, but in eight of 10 with levels less than 300/mm3. The defect could not be attributed to a selective depletion of defined CD4 subpopulations that respond to soluble antigen. Dual-color immunofluorescent flow cytometry indicated that 4B4+, 2H4-, and HB-11- CD4 cells were not lost at a faster rate than other CD4 subsets.

Vaccination of Mauritian cynomolgus macaques with the attenuated nef-truncated C8 variant of SIVmac251/32H (SIVmacC8) induces early, potent protection against pathogenic, heterologous challenge before the maturation of cognate immunity. To identify processes that contribute to early protection in this model the pathogenesis, anatomical distribution and viral vaccine kinetics were determined in relation to localised innate responses triggered by vaccination. The early biodistribution of SIVmacC8 was defined by rapid, widespread dissemination amongst multiple lymphoid tissues, detectable after 3 days. Cell-associated viral RNA dynamics identified mesenteric lymph nodes (MLN) and spleen, as well as the gut mucosae, as early major contributors of systemic virus burden. Rapid, localised infection was populated by discrete foci of persisting virus-infected cells. Localised productive infection triggered a broad innate response, with type-1 interferon sensitive IRF-7, STAT-1, TRIM5α and ApoBEC3G genes all upregulated during the acute phase but induction did not prevent viral persistence. Profound changes in vaccine-induced cell-surface markers of immune activation were detected on macrophages, B-cells and dendritic cells (DC-SIGN, S-100, CD40, CD11c, CD123 and CD86). Notably, high DC-SIGN and S100 staining for follicular and interdigitating DCs respectively, in MLN and spleen were detected by 3 days, persisting 20 weeks post-vaccination. Although not formally evaluated, the early biodistribution of SIVmacC8 simultaneously targets multiple lymphoid tissues to induce strong innate immune responses coincident at the same sites critical for early protection from wild-type viruses. HIV vaccines which stimulate appropriate innate, as well as adaptive responses, akin to those generated by live attenuated SIV vaccines, may prove the most efficacious.

BACKGROUND: Vaccination with live attenuated SIV can protect against detectable infection with wild-type virus. We have investigated whether target cell depletion contributes to the protection observed. Following vaccination with live attenuated SIV the frequency of intestinal CD4+CCR5+ T cells, an early target of wild-type SIV infection and destruction, was determined at days 3, 7, 10, 21 and 125 post inoculation. RESULTS: In naive controls, modest frequencies of intestinal CD4+CCR5+ T cells were predominantly found within the LPL TTrM-1 and IEL TTrM-2 subsets. At day 3, LPL and IEL CD4+CCR5+ TEM cells were dramatically increased whilst less differentiated subsets were greatly reduced, consistent with activation-induced maturation. CCR5 expression remained high at day 7, although there was a shift in subset balance from CD4+CCR5+ TEM to less differentiated TTrM-2 cells. This increase in intestinal CD4+CCR5+ T cells preceded the peak of SIV RNA plasma loads measured at day 10. Greater than 65.9% depletion of intestinal CD4+CCR5+ T cells followed at day 10, but overall CD4+ T cell homeostasis was maintained by increased CD4+CCR5- T cells. At days 21 and 125, high numbers of intestinal CD4+CCR5- naive TN cells were detected concurrent with greatly increased CD4+CCR5+ LPL TTrM-2 and IEL TEM cells at day 125, yet SIV RNA plasma loads remained low. CONCLUSIONS: This increase in intestinal CD4+CCR5+ T cells, following vaccination with live attenuated SIV, does not correlate with target cell depletion as a mechanism of protection. Instead, increased intestinal CD4+CCR5+ T cells may correlate with or contribute to the protection conferred by vaccination with live attenuated SIV.

<h3>Introduction</h3> Vascular adhesion protein (VAP)-1 is an adhesion molecule and potent amine-oxidase. Activation on hepatic sinusoidal endothelial cells (HSEC) leads to H₂O₂release, NFκB activation and expression of gut-homing receptor MAdCAM-1, which promotes homing of gut-tropic lymphocytes to the liver. Given the proposed role of this pathway in hepatic disorders complicating inflammatory bowel disease (IBD); we quantified serum (sVAP-1) titre and intrahepatic/colonic enzyme activity in primary sclerosing cholangitis (PSC)/IBD, as well as investigated consequences of activation with variant amine substrates. <h3>Method</h3> sVAP-1 was quantified by ELISA in PSC (n = 105); PBC (90); AIH (99); IBD-alone (50) and healthy controls (21). Correlation with clinical outcome was assessed using Cox proportional hazards assumption/KM-estimates. VAP-1 activity was determined (Amplex red assay) in protein lysates extracted from (a) explanted liver (PSC=9; PBC=10, AIH=5, normal donor, n = 10) and (b) colonic resections (n = 7). Putative VAP-1 substrates were selected based on inclusion in the human metabolome database, and induced kinetic rates measured (Michaelis-Menton analysis). Induction of MAdCAM-1 on HSEC was evaluated quantitatively (cell-ELISA) and functionally (flow-adhesion assays). <h3>Results</h3> PSC patients had higher sVAP-1 concentration (median 517 ng/mL) than those with AIH (475), PBC (472), IBD-alone (413) and healthy controls (425) (p &lt; 0.001). sVAP-1 tires &gt;530 ng/ml in PSC were associated with significantly worse transplant-free survival (unadj. HR:2.94, p = 0.008), and retained independent predictive value when controlling for other significant risk factors (cirrhosis, ascending cholangitis and liver biochemistry; adj. HR:3.85, p = 0.003). Intrahepatic VAP-1 enzyme activity was significantly greater in PSC (227 pmol H₂O₂/min/mg protein) compared with PBC (124), AIH (128) and donor liver (109) (p &lt; 0.001), yet comparable to activity in colonic tissue (220; p=n.s.). The substrate associated with highest VAP-1 enzymatic efficiency was cysteamine (k_cat/K_m:5.4 × 10⁷); an amine secreted by Escherichia. which induces colitis in mice. Cysteamine+TNFa provision resulted in greater MAdCAM-1 expression by HSEC ELISA than that with other substrates or TNFa alone (p &lt; 0.01), with increased a4b7-dependent adhesion under flow (p &lt; 0.01). <h3>Conclusion</h3> Elevated levels of circulating sVAP-1 exist in PSC and predictive of poor outcome. Intrahepatic VAP-1 enzyme activity is also increased in PSC and akin to that seen in the colon. The ability of VAP-1 to catabolise amine substrates secreted by gut commensals/enteric pathogens, provides a theoretical link between altered colonic microbiota and mucosal immunity in the pathogenesis of PSC. <h3>Disclosure of interest</h3> None Declared.