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We previously demonstrated that NK cells from HIV-infected individuals have elevated expression of activation markers, spontaneously degranulate ex vivo, and decrease expression of a signal-transducing protein for NK-activating receptors, FcRγ. Importantly, these changes were maintained in virologically suppressed (VS) individuals receiving combination antiretroviral therapy (cART). In this study, we show that loss of FcRγ is caused by the expansion of a novel subset of FcRγ(-)CD56(dim) NK cells with an altered activation receptor repertoire and biological properties. In a cross-sectional study, FcRγ(-) NK cells as a proportion of total CD56(dim) NK cells increased in cART-naive viremic HIV-infected individuals (median [interquartile range] = 25.9 [12.6-56.1] compared with 3.80 [1.15-11.5] for HIV(-) controls, p < 0.0001) and in VS HIV-infected individuals (22.7 [13.1-56.2] compared with 3.80 [1.15-11.5], p = 0.0004), with no difference between cART-naive and VS patients (p = 0.93). FcRγ(-) NK cells expressed no NKp30 or NKp46. They showed greater Ab-dependent cellular cytotoxicity activity against rituximab-opsonized Raji cells and in a whole-blood assay measuring NK responses to overlapping HIV peptides, despite having reduced CD16 expression compared with conventional NK cells. Their prevalence correlated with CMV Ab titers in HIV(-) subjects but not in HIV(+) individuals, and with the inflammatory marker CXCL10 in both groups. The expansion of a subset of NK cells that lacks NKp30 and NKp46 to ∼90% of CD56(dim) NK cells in some VS HIV(+) individuals may influence NK-mediated immunosurveillance in patients receiving cART., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Background: Combination antiretroviral therapy (cART) effectively controls human immunodeficiency virus (HIV) infection but does not eliminate HIV, and lifelong treatment is therefore required. HIV-specific cytotoxic T lymphocyte (CTL) responses decline following cART initiation. Alterations in other HIV-specific immune responses that may assist in eliminating latent HIV infection, specifically antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADP), are unclear., Methods: A cohort of 49 cART-naive HIV-infected subjects from Thailand (mean baseline CD4 count, 188 cells/µL; mean viral load, 5.4 log10 copies/mL) was followed for 96 weeks after initiating cART. ADCC and ADP assays were performed using serum samples obtained at baseline and after 96 weeks of cART., Results: A 35% reduction in HIV type 1 envelope (Env)-specific ADCC-mediated killing of target cells (P<.001) was observed after 96 weeks of cART. This was corroborated by a significant reduction in the ability of Env-specific ADCC antibodies to activate natural killer cells (P<.001). Significantly reduced ADP was also observed after 96 weeks of cART (P=.018)., Conclusions: This longitudinal study showed that cART resulted in significant reductions of HIV-specific effector antibody responses, including ADCC and ADP. Therapeutic vaccines or other immunomodulatory approaches may be required to improve antibody-mediated control of HIV during cART., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Intravenous immunoglobulin (IVIG) is a purified pool of human antibodies from thousands of donors that is used to prevent or treat primary immune deficiency, several infectious diseases, and autoimmune diseases. The antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) against heterologous influenza strains may be present in IVIG preparations., Methods: We tested 8 IVIG preparations prior to the 2009 H1N1 swine-origin influenza pandemic and 10 IVIG preparations made after 2010 for their ability to mediate influenza-specific ADCC., Results: ADCC mediating antibodies to A(H1N1)pdm09 hemagglutinin (HA) and neuraminidase (NA) were detected in IVIG preparations prior to the 2009-H1N1 pandemic. The HA-specific ADCC targeted both the HA1 and HA2 regions of A(H1N1)pdm09 HA and was capable of recognizing a broad range of HA proteins including those from recent avian influenza strains A(H5N1) and A(H7N9). The low but detectable ADCC recognition of A(H7N9) was likely due to rare individuals in the population contributing cross-reactive antibodies to IVIG., Conclusions: IVIG preparations contain broadly cross-reactive ADCC mediating antibodies. IVIG may provide at least some level of protection for individuals at high risk of severe influenza disease, especially during influenza pandemics prior to the development of effective vaccines., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Antibody-dependent phagocytosis (ADP) is a potentially important immune mechanism to clear HIV. How HIV-specific ADP responses mature during HIV infection or in response to vaccinations administered, including the partially successful RV144 HIV vaccine, is not known. We established a modified ADP assay to measure internalisation of HIV antibody (Ab)-opsonised targets using a specific hybridisation internalisation probe. Labelled beads were coated with both biotinylated HIV gp140 envelope protein and a fluorescent internalisation probe, opsonised with Abs and incubated with a monocytic cell line. The fluorescence derived from the fluorescent internalisation probe on surface-bound beads, but not from internalised beads, was quenched by the addition of a complementary quencher probe. HIV Env-specific ADP was measured in 31 subjects during primary infection and early chronic HIV infection. Although ADP responses were present early during HIV infection, a significant increase in ADP responses in all 31 subjects studied was detected (P<0.001). However, when we tested 30 HIV-negative human subjects immunised with the Canarypox/gp120 vaccine regimen (subjects from the RV144 trial) we did not detect HIV-specific ADP activity. In conclusion, a modified assay was developed to measure HIV-specific ADP. Enhanced ADP responses early in the course of HIV infection were observed but no ADP activity was detected following the vaccinations administered in the RV144 trial. Improved vaccine regimens may be needed to capitalise on ADP-mediated immunity against HIV.

Unlabelled: The mechanisms by which hepatitis B virus (HBV) establishes and maintains chronic hepatitis B infection (CHB) are poorly defined. Innate immune responses play an important role in reducing HBV replication and pathogenesis. HBV has developed numerous mechanisms to escape these responses, including the production of the secreted hepatitis B e antigen (HBeAg), which has been shown to regulate antiviral toll-like receptor (TLR) and interleukin-1 (IL-1) signaling. IL-18 is a related cytokine that inhibits HBV replication in hepatoma cell lines and in the liver through the induction of gamma interferon (IFN-γ) by NK cells and T cells. We hypothesized that HBV or HBV proteins inhibit IFN-γ expression by NK cells as an accessory immunomodulatory function. We show that HBeAg protein inhibits the NF-κB pathway and thereby downregulates NK cell IFN-γ expression. Additionally, IFN-γ expression was significantly inhibited by exposure to serum from individuals with HBeAg-positive but not HBeAg-negative chronic HBV infection. Further, we show that the HBeAg protein suppresses IL-18-mediated NF-κB signaling in NK and hepatoma cells via modulation of the NF-κB pathway. Together, these findings show that the HBeAg inhibits IL-18 signaling and IFN-γ expression, which may play an important role in the establishment and/or maintenance of persistent HBV infection., Importance: It is becoming increasingly apparent that NK cells play a role in the establishment and/or maintenance of chronic hepatitis B infection. The secreted HBeAg is an important regulator of innate and adaptive immune responses. We now show that the HBeAg downregulates NK cell-mediated IFN-γ production and IL-18 signaling, which may contribute to the establishment of infection and/or viral persistence. Our findings build on previous studies showing that the HBeAg also suppresses the TLR and IL-1 signaling pathways, suggesting that this viral protein is a key regulator of antiviral innate immune responses., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Objective: The objective of this study is to determine the breadth of HIV-1 Env-specific antibody-dependent cellular cytotoxicity (ADCC) in HIV controllers and HIV progressors with a view to design globally relevant HIV vaccines., Design: The breadth of ADCC towards four major HIV-1 Env subtypes was measured in vitro for 11 HIV controllers and 11 HIV progressors., Methods: Plasma from 11 HIV controllers (including long-term slow progressors, viremic controllers, elite controller and posttreatment controller) and 11 HIV progressors, mostly infected with HIV-1 subtype B, was analysed for ADCC responses. ADCC assays were performed against 10 HIV-1 gp120 and 8 gp140 proteins from four major HIV-1 subtypes (A, B, C and E) and 3 glycosylation-mutant gp140 proteins., Results: ADCC-mediated natural killer cell activation was significantly broader (P = 0.02) and of higher magnitude (P < 0.001) in HIV controllers than in HIV progressors. HIV controllers also showed significantly higher magnitude of ADCC-mediated killing of Env-coated target cells than HIV progressors to both HIV-1 subtype B and the heterologous subtype E gp140 (P = 0.001). We found good ADCC reactivity to subtype B and E Envs, less cross-reactivity to subtype A and minimal cross-reactivity to subtype C Envs. Glycosylation-dependent ADCC epitopes comprise a significant proportion of the total Env-specific ADCC response, as evident from the reduction in ADCC to nonglycosylated form of HIV-1 gp140 (P = 0.004)., Conclusion: HIV controllers have robust ADCC responses that recognize a broad range of HIV-1 Env. Glycosylation of Env was found to be important for recognition of ADCC epitopes. Identifying conserved ADCC epitopes will assist in designing globally relevant ADCC-based HIV vaccines.

Background: During the 2009 pandemic of influenza A virus subtype H1N1 (A[H1N1]pdm09) infection, older individuals were partially protected from severe disease. It is not known whether preexisting antibodies with effector functions such as antibody-dependent cellular cytotoxicity (ADCC) contributed to the immunity observed., Methods: We tested serum specimens obtained from 182 individuals aged 1-72 years that were collected either immediately before or after the A(H1N1)pdm09 pandemic for ADCC antibodies to the A(H1N1)pdm09 hemagglutinin (HA) protein., Results: A(H1N1)pdm09 HA-specific ADCC antibodies were detected in almost all individuals aged >45 years (28/31 subjects) before the 2009 A(H1N1) pandemic. Conversely, only approximately half of the individuals aged 1-14 years (11/31) and 15-45 years (17/31) had cross-reactive ADCC antibodies before the 2009 A(H1N1) pandemic. The A(H1N1)pdm09-specific ADCC antibodies were able to efficiently mediate the killing of influenza virus-infected respiratory epithelial cells. Further, subjects >45 years of age had higher ADCC titers to a range of seasonal H1N1 HA proteins, including from the 1918 virus, compared with younger individuals., Conclusions: ADCC antibodies may have contributed to the protection exhibited in older individuals during the 2009 A(H1N1) pandemic. This work has significant implications for improved vaccination strategies for future influenza pandemics.

Strategies to eliminate infectious HIV that persists despite present treatments and with the potential to cure HIV infection are of great interest. One patient seems to have been cured of HIV infection after receiving a bone marrow transplant with cells resistant to the virus, although this strategy is not viable for large numbers of infected people. Several clinical trials are underway in which drugs are being used to activate cells that harbour latent HIV. In a recent study, investigators showed that activation of latent HIV infection in patients on antiretroviral therapy could be achieved with a single dose of vorinostat, a licensed anticancer drug that inhibits histone deacetylase. Although far from a cure, such studies provide some guidance towards the logical next steps for research. Clinical studies that use a longer duration of drug dosing, alternative agents, combination approaches, gene therapy, and immune-modulation approaches are all underway., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The partial success of the RV144 trial re-energized the field of HIV vaccine research, which had stalled after vaccines based on neutralizing antibody and cytotoxic T cells had failed to induce protection. A large post-vaccine research effort has focused attention on the role of non-neutralizing antibodies in the protection afforded by the RV144 vaccine. These binding antibodies can initiate immune responses such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) and combine elements of the adaptive and innate immune system in the form of antibodies and effector cells (including NK cells, monocytes and granulocytes). A complex interplay exists between the variable portion of the binding antibody and its HIV antigen target on one hand and the constant region of the antibody and the Fcγ-receptor of the effector cell on the other hand. Technical advances have revolutionized the abilities of scientist to detect the targets of non-neutralizing antibodies, including both envelope and non-envelope epitopes, and their role in forcing escape. Our understanding of the antibody characteristics (including IgG subclasses and Fc glycan profile) is providing valuable insights into their optimal structure and function. We expand on critical research on ADCC effector cells, particularly education of NK cells. We introduce the concept of HIV antibodydependent trogocytosis by monocytes as a potentially important aspect of HIV immunity. In summary, this review highlights recent advances in HIV-specific antibody immunity mediated through NK cells and monocytes.

Background: Natural control of HIV infection is associated with CD8 T-cell responses to Gag-encoded antigens of the HIV core and carriage of 'protective' human leukocyte antigen (HLA)-B alleles, but some HIV controllers do not possess these attributes. As slower HIV disease progression is associated with high levels of antibodies to HIV Gag proteins, we have examined antibodies to HIV proteins in controllers with and without 'protective' HLA-B alleles., Methods: Plasma from 32 HIV controllers and 21 noncontrollers was examined for immunoglobulin G1 (IgG1) and IgG2 antibodies to HIV proteins in virus lysates by western blot assay and to recombinant (r) p55 and gp140 by ELISA. Natural killer (NK) cell-activating antibodies and FcγRIIa-binding immune complexes were also assessed., Results: Plasma levels of IgG1 antibodies to HIV Gag (p18, p24, rp55) and Pol-encoded (p32, p51, p66) proteins were higher in HIV controllers. In contrast, IgG1 antibodies to Env proteins were less discriminatory, with only antigp120 levels being higher in controllers. High-level IgG2 antibodies to any Gag protein were most common in HIV controllers not carrying a 'protective' HLA-B allele, particularly HLA-B*57 (P = 0.016). HIV controllers without 'protective' HLA-B alleles also had higher plasma levels of IgG1 antip32 (P = 0.04). NK cell-activating antibodies to gp140 Env protein were higher in elite controllers but did not differentiate HIV controllers with or without 'protective' HLA-B alleles. IgG1 was increased in FcγRIIa-binding immune complexes from noncontrollers., Conclusion: We hypothesize that isotype-switched (IgG2+) antibodies to HIV Gag proteins and possibly IgG1 antip32 may provide alternative or additional immune control mechanisms to HLA-restricted CD8 T-cell responses in HIV controllers.

A better understanding of immunity to influenza virus is needed to generate cross-protective vaccines. Engagement of Ab-dependent cellular cytotoxicity (ADCC) Abs by NK cells leads to killing of virus-infected cells and secretion of antiviral cytokines and chemokines. ADCC Abs may target more conserved influenza virus Ags compared with neutralizing Abs. There has been minimal interest in influenza-specific ADCC in recent decades. In this study, we developed novel assays to assess the specificity and function of influenza-specific ADCC Abs. We found that healthy influenza-seropositive young adults without detectable neutralizing Abs to the hemagglutinin of the 1968 H3N2 influenza strain (A/Aichi/2/1968) almost always had ADCC Abs that triggered NK cell activation and in vitro elimination of influenza-infected human blood and respiratory epithelial cells. Furthermore, we detected ADCC in the absence of neutralization to both the recent H1N1 pandemic strain (A/California/04/2009) as well as the avian H5N1 influenza hemagglutinin (A/Anhui/01/2005). We conclude that there is a remarkable degree of cross-reactivity of influenza-specific ADCC Abs in seropositive humans. Targeting cross-reactive influenza-specific ADCC epitopes by vaccination could lead to improved influenza vaccines.

Antibodies (Abs) that mediate antibody-dependent cellular cytotoxicity (ADCC) activity against HIV-1 are of major interest. A widely used method to measure ADCC Abs is the rapid and fluorometric antibody-dependent cellular cytotoxicity (RFADCC) assay. Antibody-dependent killing of a labelled target cell line by PBMC is assessed by loss of intracellular CFSE but retention of membrane dye PKH26 (CFSE-PKH26+). Cells of this phenotype are assumed to be derived from CFSE+PKH26+ target cells killed by NK cells. We assessed the effector cells that mediate ADCC in this assay. Backgating analysis and phenotyping of CFSE-PKH26+ revealed that the RFADCC assay's readout mainly represents CD3-CD14+ monocytes taking up the PKH26 dye. This was confirmed for 53 HIV+plasma-purified IgG samples when co-cultured with PBMC from three separate healthy donors. Emergence of the CFSE-PKH26+ monocyte population was observed upon co-culture of targets with purified monocytes but not with purified NK cells. Image flow cytometry and microscopy showed a monocyte-specific interaction with target cells without typical morphological changes associated with phagocytosis, suggesting a monocyte-mediated ADCC process. We conclude that the RFADCC assay primarily reflects Ab-mediated monocyte function. Further studies on the immunological importance of HIV-specific monocyte-mediated ADCC are warranted., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Antibodies with antibody-dependent cellular cytotoxicity (ADCC) activity play an important role in protection against HIV-1 infection, but generating sufficient amounts of antibodies to study their protective efficacy is difficult. HIV-specific IgG can be easily and inexpensively produced in large quantities using bovine colostrum. We previously vaccinated cows with HIV-1 envelope gp140 and elicited high titers of anti-gp140-binding IgG in colostrum. In the present study, we determined whether bovine antibodies would also demonstrate specific cytotoxic activity. We found that bovine IgG bind to Fcγ-receptors (FcγRs) on human neutrophils, monocytes, and NK cells in a dose-dependent manner. Antibody-dependent killing was observed in the presence of anti-HIV-1 colostrum IgG but not nonimmune colostrum IgG. Killing was dependent on Fc and FcγR interaction since ADDC activity was not seen with F(ab')(2) fragments. ADCC activity was primarily mediated by CD14(+) monocytes with FcγRIIa (CD32a) as the major receptor responsible for monocyte-mediated ADCC in response to bovine IgG. In conclusion, we demonstrate that bovine anti-HIV colostrum IgG have robust HIV-1-specific ADCC activity and therefore offer a useful source of antibodies able to provide a rapid and potent response against HIV-1 infection. This could assist the development of novel Ab-mediated approaches for prevention of HIV-1 transmission., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

FcRγ is an ITAM-containing adaptor required for CD16 signaling and function in NK cells. We have previously shown that NK cells from HIV patients receiving combination antiretroviral therapy (cART) have decreased FcRγ expression, but the factors causing this are unknown. We conducted a cross-sectional study of cART-naive viremic patients (ART(-)), virologically suppressed patients receiving cART (ART(+)), and HIV-uninfected controls. CD8(+) T cells were activated, as assessed by CD38(+)HLA-DR(+) expression, in ART(-) patients (p < 0.0001), which was significantly reduced in ART(+) patients (p = 0.0005). In contrast, CD38(+)HLA-DR(+) NK cells were elevated in ART(-) patients (p = 0.0001) but did not decrease in ART(+) patients (p = 0.88). NK cells from both ART(-) and ART(+) patients showed high levels of spontaneous degranulation in ex vivo whole blood assays as well as decreased CD16 expression (p = 0.0001 and p = 0.0025, respectively), FcRγ mRNA (p < 0.0001 for both groups), FcRγ protein expression (p = 0.0016 and p < 0.0001, respectively), and CD16-dependent Syk phosphorylation (p = 0.0001 and p = 0.003, respectively). HIV-infected subjects showed alterations in NK activation, degranulation, CD16 expression and signaling, and elevated plasma markers of inflammation and macrophage activation, that is, neopterin and sCD14, which remained elevated in ART(+) patients. Alterations in NK cell measures did not correlate with viral load or CD4 counts. These data show that in HIV patients who achieve viral suppression following cART, NK cell activation persists. This suggests that NK cells respond to factors different from those driving T cell activation, but which are associated with inflammation in HIV patients.

Bovine colostrum (first milk) contains very high concentrations of IgG, and on average 1 kg (500 g/liter) of IgG can be harvested from each immunized cow immediately after calving. We used a modified vaccination strategy together with established production systems from the dairy food industry for the large-scale manufacture of broadly neutralizing HIV-1 IgG. This approach provides a low-cost mucosal HIV preventive agent potentially suitable for a topical microbicide. Four cows were vaccinated pre- and/or postconception with recombinant HIV-1 gp140 envelope (Env) oligomers of clade B or A, B, and C. Colostrum and purified colostrum IgG were assessed for cross-clade binding and neutralization against a panel of 27 Env-pseudotyped reporter viruses. Vaccination elicited high anti-gp140 IgG titers in serum and colostrum with reciprocal endpoint titers of up to 1 × 10(5). While nonimmune colostrum showed some intrinsic neutralizing activity, colostrum from 2 cows receiving a longer-duration vaccination regimen demonstrated broad HIV-1-neutralizing activity. Colostrum-purified polyclonal IgG retained gp140 reactivity and neutralization activity and blocked the binding of the b12 monoclonal antibody to gp140, showing specificity for the CD4 binding site. Colostrum-derived anti-HIV antibodies offer a cost-effective option for preparing the substantial quantities of broadly neutralizing antibodies that would be needed in a low-cost topical combination HIV-1 microbicide.

Zoonotic orthopoxvirus (OPV) can induce severe disease in man and the virus has potential for use in bioterrorism. New vaccines and therapeutics against OPV infections must be tested in animal models. The aim of this study was to characterize the clinical course and pathology of a new OPV isolate, calpox virus, which is infectious in marmosets. Infection experiments were performed with 28 common marmosets (Callithrix jacchus) exposed to different challenge doses of calpox virus by the intravenous, oropharyngeal and intranasal (IN) routes. The median marmoset IN infectious dose corresponded to 8.3 × 10(2)plaque forming units of calpox virus. Infected animals developed reproducible clinical signs and died within 4-15 days post infection. Characteristic pox-like lesions developed in affected organs, particularly in the skin, mucous membranes, lymph nodes, liver and spleen. Calpox virus disease progression and pathological findings in the common marmoset appear to be consistent with lethal OPV infections in man and in other non-human primate (NHP) models. IN inoculation with low virus doses mimics the natural route of the human variola virus infection. Thus, the marmoset model of calpox virus infection can be considered to be relevant to investigation of the mechanisms of OPV pathogenesis and pathology and for the evaluation of new vaccines and antiviral therapies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Most patients with human immunodeficiency virus (HIV) who remain CD4(+) T-cell deficient on antiretroviral therapy (ART) exhibit marked immune activation. As CD4(+) T-cell activation may be mediated by microbial translocation or interferon-alpha (IFN-α), we examined these factors in HIV patients with good or poor CD4(+) T-cell recovery on long-term ART. Messenger RNA levels for 3 interferon-stimulated genes were increased in CD4(+) T cells of patients with poor CD4(+) T-cell recovery, whereas levels in patients with good recovery did not differ from those in healthy controls. Poor CD4(+) T-cell recovery was also associated with CD4(+) T-cell expression of markers of activation, senescence, and apoptosis, and with increased serum levels of the lipopolysaccharide receptor and soluble CD14, but these were not significantly correlated with expression of the interferon-stimulated genes. Therefore, CD4(+) T-cell recovery may be adversely affected by the effects of IFN-α, which may be amenable to therapeutic intervention.

We developed a real-time PCR to quantify 16S rRNA gene levels in plasma from HIV-infected patients as a marker of microbial translocation. The assay uses shrimp nuclease (SNuc) to eliminate DNA contamination, giving high sensitivity and low variability. The 16S rRNA gene levels measured in plasma from HIV patients correlated significantly with lipopolysaccharide levels.

Objectives: To examine the relationship between plasma markers of microbial translocation and antibodies to lipopolysaccharide (LPS) and circulating memory B cells in patients with HIV infection., Design: Cross-sectional study in antiretroviral therapy (ART)-naive (n = 23) and ART-treated (n = 27) HIV patients., Methods: Antibodies to LPS and immunoglobulins, assayed in stored serum, and matched memory B-cell counts were correlated with levels of LPS and bacterial 16S ribosome DNA (16S rDNA), assayed in stored plasma., Results: In ART-naive patients, plasma LPS levels correlated inversely with serum levels of IgG and IgA antibodies to LPS (P = 0.03 and 0.006, respectively), serum levels of IgA anti-LPS correlated with total IgA (P < 0.0001) and levels of IgG anti-LPS correlated with IgM(+) memory B-cell counts (P = 0.025). In ART-treated patients, plasma LPS levels were not related to levels of LPS antibodies, but were related to CD4(+) T-cell and switched memory B-cell counts. There were no correlations with plasma levels of 16S rDNA., Conclusion: Plasma LPS levels were associated with antibody and possibly B-cell responses to LPS in ART-naive HIV patients, whereas they were associated with the degree of immune reconstitution in ART-treated patients.

Effective immunity to HIV is poorly understood. In particular, a role for antibody-dependent cellular cytotoxicity (ADCC) in controlling HIV is controversial. We hypothesized that significant pressure from HIV-specific ADCC would result in immune-escape variants. A series of ADCC epitopes in HIV-infected subjects to specific consensus strain HIV peptides were mapped using a flow cytometric assay for natural killer cell activation. We then compared the ADCC responses to the same peptide epitope derived from the concurrent HIV sequence(s) expressed in circulating virus. In 9 of 13 epitopes studied, ADCC antibodies were unable to recognize the concurrent HIV sequence. Our studies suggest ADCC responses apply significant immune pressure on the virus. This result has implications for the induction of ADCC responses by HIV vaccines.

Combination antiretroviral therapy (cART) has significantly reduced morbidity and mortality of HIV-infected patients, yet their life expectancy remains reduced compared with the general population. Most HIV-infected patients receiving cART have some persistent immune dysfunction characterized by chronic immune activation and premature aging of the immune system. Here we review biomarkers of T-cell activation (CD69, -25 and -38, HLA-DR, and soluble CD26 and -30); generalized immune activation (C-reactive protein, IL-6 and D-dimer); microbial translocation (lipopolysaccharide, 16S rDNA, lipopolysaccharide-binding protein and soluble CD14); and immune dysfunction of specific cellular subsets (T cells, natural killer cells and monocytes) in HIV-infected patients on cART and their relationship to adverse clinical outcomes including impaired CD4 T-cell recovery, as well as non-AIDS clinical events, such as cardiovascular disease.

Increasing infections with Monkeypox and Cowpox viruses pose a continuous and growing threat to human health. The standard method for detecting poxvirus neutralizing antibodies is the plaque-reduction neutralization test that is specific but also time-consuming and laborious. Therefore, a rapid and reliable method was developed to determine neutralizing antibody titers within twelve hours. The new assay measures viral mRNA transcription as a marker for actively replicating virus after incomplete neutralization using real-time PCR., (© 2011 Kramski et al; licensee BioMed Central Ltd.)

Small non-coding micro-RNAs (miRNA) are important post-transcriptional regulators of mammalian gene expression that can be used to direct the knockdown of expression from targeted genes. We examined whether DNA vaccine vectors co-expressing miRNA with HIV-1 envelope (Env) antigens could influence the magnitude or quality of the immune responses to Env in mice. Human miR-155 and flanking regions from the non-protein encoding gene mirhg155 were introduced into an artificial intron within an expression vector for HIV-1 Env gp140. Using the miR-155-expressing intron as a scaffold, we developed novel vectors for miRNA-mediated targeting of the cellular antiviral proteins PKR and PERK, which significantly down-modulated target gene expression and led to increased Env expression in vitro. Finally, vaccinating BALB/c mice with a DNA vaccine vector delivering miRNA targeting PERK, but not PKR, was able to augment the generation of Env-specific T-cell immunity. This study provides proof-of-concept evidence that miRNA effectors incorporated into vaccine constructs can positively influence vaccine immunogenicity. Further testing of vaccine-encoded miRNA will determine if such strategies can enhance protective efficacy from vaccines against HIV-1 for eventual human use.

The intentional re-introduction of Variola virus (VARV), the agent of smallpox, into the human population is of great concern due its bio-terroristic potential. Moreover, zoonotic infections with Cowpox (CPXV) and Monkeypox virus (MPXV) cause severe diseases in humans. Smallpox vaccines presently available can have severe adverse effects that are no longer acceptable. The efficacy and safety of new vaccines and antiviral drugs for use in humans can only be demonstrated in animal models. The existing nonhuman primate models, using VARV and MPXV, need very high viral doses that have to be applied intravenously or intratracheally to induce a lethal infection in macaques. To overcome these drawbacks, the infectivity and pathogenicity of a particular CPXV was evaluated in the common marmoset (Callithrix jacchus).A CPXV named calpox virus was isolated from a lethal orthopox virus (OPV) outbreak in New World monkeys. We demonstrated that marmosets infected with calpox virus, not only via the intravenous but also the intranasal route, reproducibly develop symptoms resembling smallpox in humans. Infected animals died within 1-3 days after onset of symptoms, even when very low infectious viral doses of 5x10(2) pfu were applied intranasally. Infectious virus was demonstrated in blood, saliva and all organs analyzed.We present the first characterization of a new OPV infection model inducing a disease in common marmosets comparable to smallpox in humans. Intranasal virus inoculation mimicking the natural route of smallpox infection led to reproducible infection. In vivo titration resulted in an MID(50) (minimal monkey infectious dose 50%) of 8.3x10(2) pfu of calpox virus which is approximately 10,000-fold lower than MPXV and VARV doses applied in the macaque models. Therefore, the calpox virus/marmoset model is a suitable nonhuman primate model for the validation of vaccines and antiviral drugs. Furthermore, this model can help study mechanisms of OPV pathogenesis.

Serological and molecular evidence showed that a German female student became infected by Puumala hantavirus during mice trapping efforts in Finland. The incubation period before exhibiting clinical signs of nephropathia epidemica was as long as 6 weeks. Phylogenetic analysis of PUUV nucleic acid sequences amplified from the patient demonstrate that she was infected by the PUUV strain circulating at the place of her occupational exposure in Finland.

Background: Because the clinical course of human infections with hantaviruses can vary from subclinical to fatal, rapid and reliable detection of hantaviruses is essential. To date, the diagnosis of hantavirus infection is based mainly on serologic assays, and the detection of hantaviral RNA by the commonly used reverse transcription (RT)-PCR is difficult because of high sequence diversity of hantaviruses and low viral loads in clinical specimens., Methods: We developed 5 real-time RT-PCR assays, 3 of which are specific for the individual European hantaviruses Dobrava, Puumala, or Tula virus. Two additional assays detect the Asian species Hantaan virus together with Seoul virus and the American species Andes virus together with Sin Nombre virus. Pyrosequencing was established to provide characteristic sequence information of the amplified hantavirus for confirmation of the RT-PCR results or for a more detailed virus typing., Results: The real-time RT-PCR assays were specific for the respective hantavirus species and optimized to run on 2 different platforms, the LightCycler and the ABI 7900/7500. Each assay showed a detection limit of 10 copies of a plasmid containing the RT-PCR target region, and pyrosequencing was possible with 10 to 100 copies per reaction. With this assay, viral genome could be detected in 16 of 552 (2.5%) specimens of suspected hantavirus infections of humans and mice., Conclusions: The new assays detect, differentiate, and quantify hantaviruses in clinical specimens from humans and from their natural hosts and may be useful for in vitro studies of hantaviruses.