Evaluation of Functional NK Cell Responses in Vaccinated and SIV-Infected Rhesus Macaques

NK cells are crucial components of the innate immune system due to their capacity to exert rapid cytotoxic and immunomodulatory function in the absence of prior sensitization. NK cells can become activated by exposure to target cells and/or by cytokines produced by antigen-presenting cells. In this study, we examined the effects of a simian immunodeficiency virus (SIV) vaccine regimen and subsequent SIV infection on the cytotoxic and immunomodulatory functions of circulatory NK cells. While vaccination did not significantly impact the capacity of NK cells to kill MHC-devoid 721.221 target cells, SIV-infection led to a significant decrease in target cell killing. NK cells from un-infected macaques were responsive to a low dose (5 ng/ml) of IL-15 pre-activation, leading to significant increases in their cytotoxic potential, however, NK cells from SIV-infected macaques required a higher dose (50 ng/ml) of IL-15 pre-activation in order to significantly increase their cytotoxic potential. In contrast, no differences were observed in the capacity of NK cells from vaccinated and SIV-infected macaques to respond to IL-12 and IL-18. Similarly, NK cells both before and after infection exhibited equivalent responses to Fc-mediated activation. Collectively our results show that early SIV-infection impairs the natural cytotoxic capacity of circulatory NK cells without affecting Fc-mediated or cytokine-producing function

Impact of antibody quality and anamnestic response on viremia control post-challenge in a combined Tat/Env vaccine regimen in rhesus macaques

AbstractPreviously, priming rhesus macaques with Adenovirus type 5 host range mutant-recombinants encoding Tat and Env and boosting with Tat and Env protein in MPL-SE controlled chronic viremia by 4 logs following homologous intravenous SHIV89.6P challenge. Here we evaluated Tat, Env, and Tat/Env regimens for immunogenicity and protective efficacy using clade C Env, alum adjuvant, and a heterologous intrarectal SHIV1157ipd3N4 challenge. Despite induction of strong cellular and humoral immunity, Tat/Env group T and B-cell memory responses were not significantly enhanced over Tat- or Env-only groups. Lack of viremia control post-challenge was attributed to lower avidity Env antibodies and no anamnestic ADCC response or SHIV1157ipd3N4 neutralizing antibody development post-challenge. Poor biologic activity of the Tat immunogen may have impaired Tat immunity. In the absence of sterilizing immunity, strong anamnestic responses to heterologous virus can help control viremia. Both antibody breadth and optimal adjuvanticity are needed to elicit high-quality antibody for protective efficacy

Innate adaptive immune cell dynamics in tonsillar tissues during chronic SIV infection

HIV-infected patients are at higher risk of developing oral mucosal infection and Epstein–Barr virus (EBV)-associated B cell malignancies. However, the potential role of oral immunity in the pathogenesis of oral lesions is unknown. Tonsils are oral-pharyngeal mucosal-associated lymphoid tissues that play an important role in oral mucosal immunity. In this study, we investigated the changes of innate and adaptive immune cells in macaque tonsils during chronic SIV infection. We found significantly higher frequencies of classical monocytes, CD3+CD56+ (NKT-like) cells, CD3+CD4+CD8+ (DP), and CD161+ CD4 T cells in tonsils from chronic infected compared to naïve animals. On the contrary, intermediate monocytes and CD3+CD4-CD8- (DN) cells were lower in chronic SIV-infected macaques. We further confirmed a recently described small B-cell subset, NKB cells, were higher during chronic infection. Furthermore, both adaptive and innate cells showed significantly higher TNF-α and cytotoxic marker CD107a, while IL-22 production was significantly reduced in innate and adaptive immune cells in chronic SIV-infected animals. A dramatic reduction of IFN-γ production by innate immune cells might indicate enhanced susceptibility to EBV infection and potential transformation of B cells in the tonsils. In summary, our observation shows that the SIV-associated immune responses are distinct in the tonsils compared to other mucosal tissues. Our data extends our understanding of the oral innate immune system during SIV infection and could aid future studies in evaluating the role of tonsillar immune cells during HIV-associated oral mucosal infections

Analysis and expression of the rat complement proteins factor H and factor I

In der vorliegenden Arbeit wurde die zuvor nicht bekannte komplette cDNA-Sequenz des Ratten FH identifiziert. Zusätzlich wurden zwei verkürzte rekombinante Varianten des Ratten FH hergestellt. Eine bestand aus den ersten 7 SCR des FH und wurde in HEK293- und Mono-Mac-6-Säugerzellen exprimiert, die andere bestand aus den ersten 4 SCR des FH und wurde mit Hilfe des Baculovirussystems in SF21-Insektenzellen exprimiert. Beide rekombinanten Faktoren FH(SCR1-7) und FH(SCR1-4) zeigten eine Komplement-inhibitorische Wirkung im CH50-Hämolysetest und konnten damit als funktionell aktiv charakterisiert werden. Mit Hilfe des in dieser Arbeit hergestellten monoklonalen anti-FH Antikörpers (mAK 4-7D) konnte eine immunaffinitätschromatographische Aufreinigung des FH aus Rattenserum etabliert werden, die den Gesamt-FH(SCR1-20) im zweistelligen mg-Bereich für strukturelle und funktionelle Untersuchungen zur Verfügung stellte. In Verbindung mit dem ebenfalls neu generierten anti-FH IgG (polyklonaler Antikörper des Kaninchens) wurde für die Detektion und Quantifizierung des FH ein Sandwich-ELISA etabliert. Durch die bereits im Rahmen der eigenen Diplomarbeit („Induktion und Regulation des Komplementproteins Faktor I“) erfolgte Generierung eines monoklonalen anti-Ratten FI Antikörpers und die innerhalb dieser Arbeit zusätzlich erfolgte Herstellung eines polyklonalen anti-Ratten FI IgG (ebenfalls aus dem Kaninchen) wurden weitere Werkzeuge für die Detektion des FI im Immunblotverfahren geschaffen. Durch die Immobilisierung des anti-Ratten FI mAK15F11-2 an einer Matrix war es zusätzlich möglich, den Ratten FI in funktionell aktiver Form aus Serum zu isolieren. Die beiden mittels Immunaffinitätschromatographie aus Serum isolierten Faktoren H und I erwiesen sich wie auch die beiden rekombinanten FH-Varianten (SCR1-4 und SCR1-7) im CH50-Hämolysetest als aktive Proteine, so dass sie für weitere in vitro aber auch für geplante in vivo Experimente zur Verfügung stehen. Zusammenfassend sei festgestellt, dass mit dieser Arbeit eine breite Basis an Werkzeugen für die Detektion, Quantifizierung und strukturelle Untersuchung der Komplement-inhibitorischen Faktoren H und I der Ratte geschaffen wurde. Darüberhinaus stehen beide Faktoren in zur Homogenität aufgereinigter Form, der Faktor H zusätzlich in zwei rekombinanten Varianten (SCR1-4 und SCR1-7) funktionell aktiv für einen therapeutischen Einsatz in verschiedenen Krankheitsmodellen der Ratte zur Verfügung

Loss of marginal zone B-cells in SHIVSF162P4 challenged rhesus macaques despite control of viremia to low or undetectable levels in chronic infection

AbstractMarginal zone (MZ) B cells generate T-independent antibody responses to pathogens before T-dependent antibodies arise in germinal centers. They have been identified in cynomolgus monkeys and monitored during acute SIV infection, yet have not been well-studied in rhesus macaques. Here we characterized rhesus macaque MZ B cells, present in secondary lymphoid tissue but not peripheral blood, as CD19+, CD20+, CD21hi, IgM+, CD22+, CD38+, BTLA+, CD40+, CCR6+ and BCL-2+. Compared to healthy macaques, SHIVSF162P4-infected animals showed decreased total B cells and MZ B cells and increased MZ B cell Ki-67 expression early in chronic infection. These changes persisted in late chronic infection, despite viremia reductions to low or undetectable levels. Expression levels of additional phenotypic markers and RNA PCR array analyses were in concert with continued low-level activation and diminished function of MZ B cells. We conclude that MZ B-cell dysregulation and dysfunction associated with SIV/HIV infection are not readily reversible

Effects of the Deletion of Early Region 4 (<i>E4</i>) Open Reading Frame 1 (<i>orf1</i>), <i>orf1-2</i>, <i>orf1-3</i> and <i>orf1-4</i> on Virus-Host Cell Interaction, Transgene Expression, and Immunogenicity of Replicating Adenovirus HIV Vaccine Vectors

<div><p>The global health burden engendered by human immunodeficiency virus (HIV)-induced acquired immunodeficiency syndrome (AIDS) is a sobering reminder of the pressing need for a preventative vaccine. In non-human primate models replicating adenovirus (Ad)-HIV/SIV recombinant vaccine vectors have been shown to stimulate potent immune responses culminating in protection against challenge exposures. Nonetheless, an increase in the transgene carrying capacity of these Ad vectors, currently limited to approximately 3000 base pairs, would greatly enhance their utility. Using a replicating, <i>E3</i>-deleted Ad type 5 host range mutant (Ad5 hr) encoding full-length single-chain HIV_BaLgp120 linked to the D1 and D2 domains of rhesus macaque CD4 (rhFLSC) we systematically deleted the genes encoding early region 4 open reading frame 1 (<i>E4orf1</i>) through <i>E4orf4</i>. All the Ad-rhFLSC vectors produced similar levels of viral progeny. Cell cycle analysis of infected human and monkey cells revealed no differences in virus-host interaction. The parental and <i>E4</i>-deleted viruses expressed comparable levels of the transgene with kinetics similar to Ad late proteins. Similar levels of cellular immune responses and transgene-specific antibodies were elicited in vaccinated mice. However, differences in recognition of Ad proteins and induced antibody subtypes were observed, suggesting that the <i>E4</i> gene products might modulate antibody responses by as yet unknown mechanisms. In short, we have improved the transgene carrying capacity by one thousand base pairs while preserving the replicability, levels of transgene expression, and immunogenicity critical to these vaccine vectors. This additional space allows for flexibility in vaccine design that could not be obtained with the current vector and as such should facilitate the goal of improving vaccine efficacy. To the best of our knowledge, this is the first report describing the effects of these <i>E4</i> deletions on transgene expression and immunogenicity in a replicating Ad vector.</p></div

Deletion of <i>E4orf1</i> through <i>E4orf4</i> does not affect transgene or Ad5-specific cellular responses.

<p>(<b>A–B</b>) Intracellular cytokine staining of splenocytes with stacked responses for Env- or Ad5-specific CD8 and CD4 CM or EM T cells secreting IFNγ, IL-2, and TNFα. (A) No differences were observed between the parent and the <i>E4</i>-deleted viruses (p>0.05). Values of the five vaccinated groups were significantly different from those of the empty vector group for Env-specific CD8 CM and CD8 EM T cells (p<0.001 for both) and for Env-specific CD4 EM T cells (p<0.002) but not for Env-specific CD4 EM T cells (p>0.05). (B) No differences were observed between the parent and the E4-deleted viruses for the Ad5-specific memory responses (p>0.05). No differences were observed between vaccinated groups and the empty vector group (p>0.20). For ΔE4orf1-3 only cells from a single mouse were evaluated. For group 6 cells from 4 mice were evaluated. All the other experiments were performed using 5 mice. Stacked mean values of individual cytokines are plotted with error bars indicating SEM.</p