Glycosphingolipids are involved in the first steps of virus-cell interaction, where they mediate specific recognition of the host cell membrane. We have employed total-internal-reflection fluorescence microscopy to explore the interaction kinetics between individual unlabeled noroviruslike particles, which are attached to a glycosphingolipid-containing lipid bilayer, and fluorescent vesicles containing different types and concentrations of glycosphingolipids. Under association equilibrium, the vesicle-binding rate is found to be kinetically limited, yielding information on the corresponding activation energy. The dissociation kinetics are logarithmic over a wide range of time. The latter is explained by the vesicle-size-related distribution of the dissociation activation energy. The biological, pharmaceutical, and diagnostic relevance of the study is briefly discussed.

n/a

Background

Hepatitis C virus (HCV) represents one of the persistent viral infections afflicting humankind, and a significant proportion of chronic HCV disease progresses over time through liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). One potential mechanism underlying the chronic disease is believed to be viral escape from immune surveillance via upregulation of inhibitory molecules on immune cells by HCV. We investigated the diverse expression of various inhibitory molecules in PBMCs of healthy non-HCV controls and chronically HCV infected patients.

Methods

The expression of inhibitory molecules on PBMCs was investigated in chronic HCV infected patients relative to healthy non-HCV controls using standard immunological and molecular methods. The serum levels of indoleamine 2, 3 deoxygenase (IDO) and cyclooxygenase-2 (COX-2) were also investigated.

Results

The gene expression profile of chronically HCV infected patients was significantly different from control individuals. Our results showed upregulation of TIM-3 (p≤0.01), PD-1 (p≤0.01), FOXP-3 (p≤0.01), BLIMP-1 (p≤0.01), CD160 (p≤0.01), CTLA-4 (p≤0.01), TRAIL (p≤0.01), BTLA (p≤0.01) and LAG-3 (p≤0.01) with fold change of 1.3, 0.4, 14.6, 0.87, 6.6, 0.4, 14.7, 10.9 and 2.5 respectively in chronically HCV infected patients. The plasma IDO and COX-2 levels were significantly higher (p=0.001) in chronically HCV infected subjects relative to healthy control.

Conclusion

The upregulation of inhibitory molecules on PBMCs in chronically HCV infected patients suggest the contribution of these molecules to immune cells impairment in HCV infection. Viral persistence and eventual progression following potential evasion of the host immune armory via viral impregnation of inhibitory immune biosignatures in HCV disease pathogenesis warrants further elucidation.

HIV-1 only infects humans and chimpanzees. SIV or SHIV are, therefore, used as models for HIV in rhesus, cynomologus and pigtail macaques. Since conducting experiments in primate models does not fully mimic infection or vaccination against HIV-1 and is expensive, there is a great need for small-animal models in which it is possible to study HIV-1 infection, immunity and vaccine efficacy. This review summarizes the available murine models for studying HIV-1 infection with an emphasis on our experience of the HIV-1-infected-cell challenge as a model for evaluating candidate HIV-1 vaccines. In the cell-based challenge model, several important factors that, hopefully, can be related to vaccine efficacy in humans were discovered: the efficiency of combining plasmid DNA representing several of the viral genes originating from multiple clades of HIV-1, the importance of adjuvants activating innate and induced immunity and the enhanced HIV eradication by drug-conjugated antibody. © 2008 Future Drugs Ltd.

We increase our understanding of augmenting a cellular immune response, by using an HIV-1 protease-derived epitope (PR75−84), and variants thereof, coupled to the C-terminal, of the B subunit of cholera toxin (CTB). Fusion proteins were used for immunizations of HLA-A0201 transgenic C57BL/6 mice. We observed different capacities to elicit a cellular immune response by peptides with additions of five to ten amino acids to the PR epitope. There was a positive correlation between the magnitude of the elicited cellular immune response and the capacity of the fusion protein to bind GM-1. This binding capacity is affected by its ability to form natural pentamers of CTB. Our results suggest that functional CTB pentamers containing a foreign amino acid-modified epitope is a novel way to overcome the limited cellular immunogenicity of minimal peptide antigens. This way of using a functional assay as readout for improved cellular immunogenicity might become highly valuable for difficult immunogens such as short peptides (epitopes).

Delivery of DNA encoding foreign antigens into mammalian cells can induce adaptive immune responses. There are currently many DNA-based vaccines in clinical trials against infectious diseases and cancer but there is a lack of adjuvants for improvement of responses to DNA-based vaccines. Here, we show augmented systemic and mucosa-associated B cell responses after immunization with a cocktail of seven different plasmids (3 env, 2 gag, 1 rev, 1 RT) combined with mitogen activated apoptotic syngeneic lymphocytes in mice. In addition we show that apoptotic cells can function as adjuvant for induction of cellular immune responses in a magnitude comparable to the cytokine adjuvant GM-CSF in mice. These data suggest that activated apoptotic lymphocytes can act independent as adjuvants to improve antigen-specific DNA vaccines.

This study was designed to determine the impact of maternal HIV-1 specific immunity on HIV-DNA immunization of 2 weeks old pups during the breast-feeding period.

Adult female mice received intranasal or intradermal HIV DNA (gp160Env, p37Gag, Nef, Tat and Rev) prime and recombinant protein booster immunizations that induced mucosal and systemic HIV-1 specific B and T cell responses. Intranasal administration of the immunogens induced higher serum IgG titers to HIV antigens than the intradermal immunization. Furthermore, predominantly higher HIV-1 specific fecal IgA titers were obtained in nasally immunized mice. The capacity to respond to one single prime with DNA and one boost with recombinant protein was then compared in pups born to mothers displaying HIV-1-specific immune responses and in pups born to non-vaccinated mothers. Immune responses to the greatest number of antigens were detected in pups born to mothers having the highest HIV-1 specific immune responses. These data suggest that HIV-1 DNA-plasmid immunization during breast-feeding and recombinant protein boosting shortly thereafter enhances the breadth of the humoral HIV-1 specific immune responses.

One of the major challenges for the development of an HIV vaccine is to induce potent virus-specific immune responses at the mucosal surfaces where transmission of virus occurs. Intranasal delivery of classical vaccines has been shown to induce good mucosal antibody responses, but so far for genetic vaccines the success has been limited. This study shows that young individuals are sensitive to nasal immunization with a genetic vaccine delivered in a formulation of a lipid adjuvant, the Eurocine N3. Intranasal delivery of a multiclade/multigene HIV-1 genetic vaccine gave rise to vaginal and rectal IgA responses as well as systemic humoral and cellular responses. As electroporation might become the preferred means of delivering genetic vaccines for systemic HIV immunity, nasal delivery by droplet formulation in a lipid adjuvant might become a means of priming or boosting the mucosal immunity. © 2008 Elsevier Ltd. All rights reserved.

Rotaviruses (RVs) are a major cause of severe diarrhoea in young children. Nicaragua introduced routine immunization with the pentavalent RV vaccine (RV5) in 2006, which greatly reduced the incidence of diarrhoea. A remaining concern has been the possible emergence of new RV strains to which the vaccination has less effect. In this study, 837 children with diarrhoea in hospital settings were investigated for RV between May 2011 and July 2013. RVs were subsequently typed by multiplex PCR and/or sequencing. Fecal anti-RV IgA titres for a subset of RV-infected (n = 137) and noninfected children (n = 52) were determined with an in-house enzyme-linked immunosorbent assay. The RV detection rate was 8% in 2011, followed by a sharp increase to 29% in 2012 and 19% in 2013. This was associated with emergence and predominance of genotype G12 RV, from 0% in 2011 to 66% in 2012 and 82% in 2013, infecting children from 1 month to 10 years of age. Two sequenced G12 strains showed a Wa-like genome with genotype G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, similar to the globally emerging G12 strains. Fecal anti-RV IgA analysis showed that most G12-infected and noninfected children had been in contact with either vaccine or wild RV strains, but such antibodies did not prevent symptomatic G12 infection. A marked increase of RV was evident in the hospital setting associated with a nationwide emergence and predominance of RV G12 genotype in a population with high RV5 vaccine coverage.

We describe the genetic diversity of sapovirus (SaV) in children in Nicaragua and investigate the role of host genetic factors and susceptibility to SaV infections. Our results indicate that neither ABO blood group, Lewis phenotype, nor secretor status affects susceptibility to SaV infection in Nicaragua.

Host genetic resistance to Norovirus (NoV) has been observed in challenge and outbreak studies in populations from Europe, Asia, and USA. In this study, we have investigated if histo-blood group antigens can predict susceptibility to diarrhea caused by NoV in Nicaragua, Central America, and if this can be reflected in antibody-prevalence and titer to NoV among individuals with different histo-blood group antigen phenotypes. Investigation of 28 individuals infected with NoV and 131 population controls revealed 6% of non-secretors in the population and nil non-secretors among patients infected with NoV, suggesting that non-secretors may be protected against NoV disease in Nicaragua. Surprisingly, 25% of the population was Lewis negative (Le(a-b-)). NoV infections with genogroup I (GI) and GII occurred irrespective of Lewis genotype, but none of the Lewis a positive (Le(a + b-)) were infected. The globally dominating GII.4 virus infected individuals of all blood groups except AB (n = 5), while the GI viruses (n = 4) infected only blood type O individuals. Furthermore, O blood types were susceptible to infections with GI.4, GII.4, GII.7, GII.17, and GII.18-Nica viruses, suggesting that secretors with blood type O are susceptible (OR = 1.52) and non-secretors resistant. The overall antibody-prevalence to NoV GII.3 VLP was 62% with the highest prevalence among blood type B carriers (70%) followed by A (68%) and O (62%). All four investigated individuals carrying blood type AB were antibody-negative. Among secretors, 63% were antibody-positive compared to 33% among non-secretors (P = 0.151). This study extends previous knowledge about the histo-blood group antigens role in NoV disease in a population with different genetic background than North American and European.

Rotavirus (RV) and norovirus (NoV) are major causes of pediatric diarrhea and are altogether associated with approximately 800,000 deaths in young children every year. In Nicaragua, national RV vaccination program using the pentavalent RV5 vaccine from Merck was implemented in October 2006. To determine whether RV vaccination decreased the overall number of RV infections, we investigated the occurrence of RV and NoV in wastewater in the city of Leon from July 2007 to July 2008 and compared these data with pre-vaccination data. The major finding was the low prevalence of RV compared to NoV in all sampling points (11% vs 44%, pandlt;0.05), and that RV concentration was lower as compared to NoV. RV was observed mainly during the rainy season (July-September), and the majority of all RV detected (6/9) belonged to subgroup (SG) I. The partial VP7-gene obtained from one RV positive sample was similar (99% nt identity) to a G6 VP7-gene of bovine origin and similar to the corresponding gene of the vaccine strain (98%). Furthermore RV G-types 2 and 4 were found in the incoming wastewater. NoV strains were detected throughout the year, of which a majority (20/21) were of genotype GII.4. We conclude that the introduction of RV vaccination reduced the transmission of RV in the community in Nicaragua. However, the burden of diarrhea in the country remains high, and the high prevalence of NoVs in hospital and municipal wastewater is noteworthy. This study highlights the need for further assessment of NoV following RV vaccine introduction.

Information about norovirus (NoV) infections in Central America is limited. Through a passive community and hospital pediatric diarrhea surveillance program, a total of 542 stool samples were collected between March 2005 and February 2006 in León, Nicaragua. NoV was detected in 12% (65/542) of the children; of these, 11% (45/409) were in the community and 15% (20/133) were in the hospital, with most strains (88%) belonging to genogroup II. NoV infections were age and gender associated, with children of <2 years of age (P < 0.05) and girls (P < 0.05) being most affected. Breast-feeding did not reduce the number of NoV infections. An important proportion (57%) of NoV-infected children were coinfected with diarrheagenic Escherichia coli. A significant proportion (18/31) of NoV-positive children with dehydration required intravenous rehydration. Nucleotide sequence analysis (38/65) of the N-terminal and shell region in the capsid gene revealed that at least six genotypes (GI.4, GII.2, GII.4, GII.7, GII.17, and a potentially novel cluster termed "GII.18-Nica") circulated during the study period, with GII.4 virus being predominant (26/38). The majority (20/26) of those GII.4 strains shared high nucleotide homology (99%) with the globally emerging Hunter strain. The mean viral load was approximately 15-fold higher in children infected with GII.4 virus than in those infected with other G.II viruses, with the highest viral load observed for the group of children infected with GII.4 and requiring intravenous rehydration. This study, the first of its type from a Central American country, suggests that NoV is an important etiological agent of acute diarrhea among children of <2 years of age in Nicaragua.

Background: It has been previously reported that histo-blood group antigens (HBGAs) and particularly secretor status provides protection against symptomatic norovirus infection, but it remains unclear to what extent this includes asymptomatic infections in children. Methods: To explore whether HBGAs or certain viral genotypes are associated with asymptomatic norovirus infections in a pediatric population in Nicaragua, we investigated 163 children andlt;= 5 years of age, without a recent history of diarrhea (andlt;= 10 days). Results: Asymptomatic norovirus infections were observed in 11.7% (19/163), with children andlt;= 6 months of age being most frequently infected (16%). Of the 19 norovirus-positive children, 4 (21%) and 10 (53%) were infected with genogroups GI and GII, respectively, and 4 children (21%) were infected with viruses of both genogroups. Most children had andgt;= 10(6) viral genomes per gram of feces. Nucleotide sequence analysis (15/19) revealed uncommon genotypes, such as, GII. 7 (n = 5) and GII. 2 (n = 3). An interesting observation was the low frequency of norovirus GII. 4 strains among the asymptomatic children. AB blood type, Lewis a (Lea(a+b-)) phenotype and nonsecretor genotype (se(428)se(428)) were not found among the asymptomatic children, but they occurred in population controls. Conclusions: Frequency of asymptomatic norovirus infections was similar to that observed in symptomatic children from Nicaragua. Norovirus GII. 2 and GII. 7 were frequently detected but the globally dominating GII. 4 was infrequent. Host genetic factors previously observed to be associated with protection against symptomatic norovirus infection were not found in this study.

Rotavirus (RV) vaccination programs have been established in several countries using the human-attenuated G1P[8] monovalent vaccine Rotarix (TM) (GlaxoSmithKline) and/or the human-bovine reassortant G1, G2, G3, G4, P[8] pentavalent vaccine Rotaleq (TM) (Merck). The efficacy of both vaccines is high (similar to 90%) in developed countries, but can be remarkably lower in developing countries. For example, a vaccine efficacy against severe diarrhea of only 58% was observed in a 2007-2009 Nicaraguan study using RotaTeq. To gain insight into the significant level of vaccine failure in this country, we sequenced the genomes of RVs recovered from vaccinated Nicaraguan children with gastroenteritis. The results revealed that all had genotype specificities typical for human RVs (11G1P[8], 1G3P[8]) and that the sequences and antigenic epitopes of the outer capsid proteins (VP4 and VP7) of these viruses were similar to those reported for RVs isolated elsewhere in the world. As expected, nine of the G1 P[8] viruses and the single G3P[8] virus had genome constellations typical of human G1 P[8] and G3P[8] RVs: G1/3-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. However, two of the G1P[8] viruses had atypical constellations, G1-P[8]-I1-R1-C1-M1-A1-N2-T1-E1-H1, due to the presence of a genotype-2 NSP2 (N2) gene. The sequence of the N2 NSP2 gene was identical to the bovine N2 NSP2 gene of RotaTeq, indicating that the two atypical viruses originated via reassortment of human G1P[8] RVs with RotaTeq viruses. Together, our data suggest that the high level of vaccine failure in Nicaraguan is probably not due to antigenic drift of commonly circulating virus strains nor the emergence of new antigenetically distinct virus strains. Furthermore, our data suggest that the widespread use of the RotaTeq vaccine has led to the introduction of vaccine genes into circulating human RVs.

Female rhesus macaques were immunized with HIV virus-like particles (HIV-VLPs) or HIV DNA administered as sequential combinations of mucosal (intranasal) and systemic (intramuscular) routes, according to homologous or heterologous prime-boost schedules. The results show that in rhesus macaques only the sequential intranasal and intramuscular administration of HIV-VLPs, and not the intranasal alone, is able to elicit humoral immune response at the systemic as well as the vaginal level.

The viruses described in this thesis are the norovirus and sapoviruses, which belong to the family of human caliciviruses and are known to cause gastroenteritis in humans. Gastroenteritis has emerged as a global health problem and is based on the large number of infected considered as one of the most common diseases today. According to estimates of the World Health Organization (WHO), gastroenteritis causes over five times more pediatric deaths compared to pediatric deaths caused by HIV/AIDS worldwide. Norovirus, the cause of the famous “winter vomiting disease”, is alone responsible for more than 200 000 deaths each year in children less than 5 years of age.

The mechanism for emergence and evolution of new human calicivirus strains, as well as protective immunity in the human population is poorly understood. The main focus for this thesis was to elucidate the possible correlation between human calicivirus evolution, host genetics and disease susceptibility. One of the main findings presented in this thesis is the documentation of in vivo capsid gene evolution and quasispecies dynamics during chronic NoV GI.3 infection (Paper 1). In paper II, we reported that the G428A nonsense mutation in the FUT2 gene provides strong but not absolute protection against symptomatic GII.4 NoV infection. In my last two papers (Paper III and IV), we were the first to investigate host genetic susceptibility factors during authentic SaV infection.

To summarize, the results presented in this thesis show that the success of human calicivirus infection probably is determined by a delicate interplay between virus evolution and susceptibility of the host, both genetically and immunologically.

In November 2004, 116 individuals in an elderly nursing home in El Grao de Castellón, Spain were symptomatically infected with genogroup II.4 (GII.4) norovirus. The global attack rate was 54.2%. Genotyping of 34 symptomatic individuals regarding the FUT2 gene revealed that one patient was, surprisingly, a non-secretor, hence indicating secretor-independent infection. Lewis genotyping revealed that Lewis-positive and negative individuals were susceptible to symptomatic norovirus infection indicating that Lewis status did not predict susceptibility. Saliva based ELISA assays were used to determine binding of the outbreak virus to saliva samples. Saliva from a secretor-negative individual bound the authentic outbreak GII.4 Valencia/2004/Es virus, but did not in contrast to secretor-positive saliva bind VLP of other strains including the GII.4 Dijon strain. Amino acid comparison of antigenic A and B sites located on the external loops of the P2 domain revealed distinct differences between the Valencia/2004/Es and Dijon strains. All three aa in each antigenic site as well as 10/11 recently identified evolutionary hot spots, were unique in the Valencia/2004/Es strain compared to the Dijon strain. To the best of our knowledge, this is the first example of symptomatic GII.4 norovirus infection of a Le(a+b-) individual homozygous for the G428A nonsense mutation in FUT2. Taken together, our study provides new insights into the host genetic susceptibility to norovirus infections and evolution of the globally dominating GII.4 viruses.

Background. Sapovirus (SaV) infections are increasing globally but there is no information available regarding factors determining susceptibility to SaV infections in the Caucasian population.

Methods. Saliva samples were collected from 64 individuals with sapovirus gastroenteritis in Denmark between October 2008 and November 2010. These were genotyped for the FUT2 G428A nonsense mutation (secretor status) and phenotyped for ABO and Lewis histo-blood groups.

Results. We found that neither secretor status nor Lewis phenotype, were associated with susceptibility to symptomatic infection with SaV. However, individuals of histo-blood groups B and AB had significantly lower risk to be infected (OR 0.18, p≤0.01 and OR 0.10, p<0.05, respectively). For 39 of the 64 SaV positive samples viral strains were genotyped and 41%, (16/39) belonged to genotype GI.2, 10% was GI.1 (4/39), 2.5% was GI.5 (1/39), 8% was GII.1 (3/39), 5% was GII.4 (2/39), 18% was GIV (7/39) and 15.5% was GV (6/39).

Conclusion. This is the first report investigating the role of host genetic factors in SaV susceptibility in the Caucasian population. We found a reduced risk of infection in individuals with blood group B (and AB), but no association to the FUT2 G428A nonsense mutation determining secretor status nor to the Lewis status.

We constructed novel HIV-1 fusion inhibitors that may overcome the current limitations of enfuvirtide, the first such therapeutic in this class. The three prototypes generated by the Dock-and-Lock (DNL) technology to comprise four copies of enfuvirtide tethered site-specifically to the Fc end of different humanized monoclonal antibodies potently neutralize primary isolates (both R5-tropic and X4-tropic), as well as T-cell-adapted strains of HIV-1 in vitro. All three prototypes show EC₅₀ values in the subnanomolar range, which are 10- to 100-fold lower than enfuvirtide and attainable whether or not the constitutive antibody targets HIV-1. The potential of such conjugates to purge latently infected cells was also demonstrated in a cell-to-cell viral inhibition assay by measuring their efficacy to inhibit the spread of HIV-1_LAI from infected human peripheral blood mononuclear cells to Jurkat T cells over a period of 30 days following viral activation with 100 nM SAHA (suberoylanilide hydroxamic acid). The IgG-like half-life was not significantly different from that of the parental antibody, as shown by the mean serum concentration of one prototype in mice at 72 h. These encouraging results provide a rationale to develop further novel anti-HIV agents by coupling additional antibodies of interest with alternative HIV-inhibitors via recombinantly-produced, self-assembling, modules.

Priming of T cells in lymphoid tissues of HIV-infected individuals occurs in the presence of HIV-1. DC in this milieu activate T cells and disseminate HIV-1 to newly activated T cells, the outcome of which may have serious implications in the development of optimal antiviral responses. We investigated the effects of HIV-1 on DC-naïve T-cell interactions using an allogeneic in vitro system. Our data demonstrate a dramatic decrease in the primary expansion of naïve T cells when cultured with HIV-1-exposed DC. CD4(+) and CD8(+) T cells showed enhanced expression of PD-1 and TRAIL, whereas CTLA-4 expression was observed on CD4(+) T cells. It is worth noting that T cells primed in the presence of HIV-1 suppressed priming of other naïve T cells in a contact-dependent manner. We identified PD-1, CTLA-4, and TRAIL pathways as responsible for this suppresion, as blocking these negative molecules restored T-cell proliferation to a higher degree. In conclusion, the presence of HIV-1 during DC priming produced cells with inhibitory effects on T-cell activation and proliferation, i.e. suppressor T cells, a mechanism that could contribute to the enhancement of HIV-1 pathogenesis.

The human immunodeficiency virus (HIV)-1 is the causative agent of acquired immune deficiency syndrome (AIDS) worldwide. Till date there are no vaccines or cure for this infection as the virus has adapted myriad ways to remain persistent in the host where it causes severe damage to the immune system. Both humoral and cellular immune responses are mounted against HIV-1 during the initial phase of infection but fail to control viral replication as these responses are severely depleted during disease progression. Of great importance in HIV-1 research today is the in depth understanding of the types of immune responses elicited, the mechanisms behind their decline and how these responses can be  maintained overtime.

The focus of this thesis was to examine the possibility of priming HIV-1 specific T cell responses in vitro from whole viral particles and in detail, scrutinize the type of T cell responses and epitope specificities generated. Next was to investigate in vitro the factors responsible for impaired immune responses in HIV-1 infected individuals. We were also interested in understanding the underlying mechanisms through which HIV-1 initiate suppression of T cell functionality.

Results showed that using HIV-1 pulsed monocyte derived dendritic cells (DCs), we were able to prime HIV-1 specific CD4+ and CD8+ T cells from naïve T cells in vitro. The epitopes primed in vitro were located within the HIV-1 envelope, gag, and pol proteins and were confirmed ex vivo to exist in acute and chronically infected individuals. We established that many of the novel CD4+ T cell epitopes primed in vitro also existed in vivo in HIV-1 infected individuals during acute infection. These responses declined/disappeared early on, which is in line with HIV-1 preferential infection of HIV-1 specific CD4+ T cells.

Besides declining HIV-1 specific T cell responses, many HIV-1 infected individuals also have impaired T cell functionality. We established that one reason behind the decline and impairment in immune responses was the increased expression of inhibitory molecules PD-1, CTLA-4, and TRAIL on HIV-1 primed T cells. These T cells had the capacity to suppress new responses in a cell-cell contact dependent manner. The ability of the HIV-1 primed T cells to proliferate was severely impaired and this condition was reversed after a combined blockade of PD-1, CTLA-4 and TRAIL. Furthermore, more inhibitory molecules TIM-3, LAG-3, CD160, BLIMP-1, and FOXP3 were also found increased at both gene and protein levels on HIV-1 primed T cells. Additionally, we showed decreased levels of functional cytokines IL-2, IFN-γ and TNF-α, and the cytolytic proteins perforin and granzyme in DC T cell priming cocultures containing HIV-1. This could be as a result of the decreased T cell activation or impaired production by T cells. The mechanisms responsible for the elevated levels of inhibitory molecules emanated mainly from the P38MAPK/STAT3 pathways. Blockade of these pathways in both allogeneic and autologous DC-T cell assays significantly suppressed expression of inhibitory molecules and subsequently rescued T cell proliferation.

In conclusion, HIV-1 pulsed DCs have the capacity to prime HIV-1 specific responses in vitro that do exist in HIV-1 infected individuals and we found evidence that many of these responses were eliminated rapidly in HIV-1 infected individuals. HIV-1 triggers through P38MAPK/STAT3 pathway the synthesis of inhibitory molecules, namely CTLA-4, PD-1, TRAIL, TIM-3, LAG-3, CD160, and suppression associated transcription factors FOXP3, BLIMP-1 and DTX1. This is followed by decreased T cell proliferation and functionality which are much needed to control viral replication.

HIV-1 infection enhances the expression of negative costimulatory molecules on T cellsleading to T cell impairment. The signaling pathway underlying the regulation ofinhibitory molecules and the subsequent onset of T cell impairment remains to beinvestigated. Herein, we showed that the T cells activated by HIV-pulsed dendritic cells(DCs) upregulated CTLA-4, TRAIL, LAG-3, TIM-3, and CD160 and suppressionassociated transcription factors BLIMP-1, DTX1, and FOXP3, leading to T cellsuppression. The induction of suppressor T cells was regulated by the signal transducerand activator of transcription 3 (STAT3) molecules as blockade of this pathwaysignificantly down regulates the expression of inhibitory molecules. The cytokines IL-6and IL-10 were not responsible for STAT3 activation as their neutralization could neitherrecover T cell proliferation nor decrease the expression of negative costimulatorymolecules. Contrarily, we demonstrated that the intracytoplasmic cross-talk of P38Mitogen-Activated Protein Kinase (MAPK) with STAT3 was responsible as blockade ofthe P38MAPK significantly impaired negative costimulatory molecular expression andthe subsequent recovery of T cell proliferation. Notably, the blockade of viral access toDC cytosol, via CD4 binding and fusion, significantly reduced the negative effects DCsimposed on the primed T cells. In conclusion, viral access to cytosol modulated theDCs- T cell priming to induce T cells with upreguled expression of negativecostimulatory molecules in a P38MAPK/STAT3 pathway dependent fashion

Human immunodeficiency virus type 1 (HIV-1) infection enhances the expression of inhibitory molecules on T cells, leading to T-cell impairment. The signaling pathways underlying the regulation of inhibitory molecules and subsequent onset of T-cell impairment remain elusive. We showed that both autologous and allogeneic T cells exposed to HIV-pulsed dendritic cells (DCs) upregulated cytotoxic T-lymphocyte antigen (CTLA-4), tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), lymphocyte-activation gene-3 (LAG3). T-cell immunoglobulin mucin-3 (TIM-3), CD160 and certain suppression-associated transcription factors, such as B-lymphocyte induced maturation protein-1 (BLIMP-1), deltex homolog 1 protein (DTX1) and forkhead box P3 (FOXP3), leading to T-cell suppression. This induction was regulated by p38 mitogen-activated protein kinase/signal transducer and activator of transcription-3 (P38MAPK/STAT3) pathways, because their blockade significantly abrogated expression of all the inhibitory molecules studied and a subsequent recovery in T-cell proliferation. Neither interleukin-6 (IL-6) nor IL-10 nor growth factors known to activate STAT3 signaling events were responsible for STAT3 activation. Involvement of the P38MAPK/STAT3 pathways was evident because these proteins had a higher level of phosphorylation in the HIV-1-primed cells. Furthermore, blockade of viral CD4 binding and fusion significantly reduced the negative effects DCs imposed on primed T cells. In conclusion, HIV-1 interaction with DCs modulated their functionality, causing them to trigger the activation of the P38MAPK/STAT3 pathway in T cells, which was responsible for the upregulation of inhibitory molecules. Online address: http://www.molmed.org doi: 10.2119/molmed.2012.00103

Nanoparticles (NPs) are attractive carriers for vaccines. We have previously shown that a short peptide (Hp91) activates dendritic cells (DCs), which are critical for initiation of immune responses. In an effort to develop Hp91 as a vaccine adjuvant with NP carriers, we evaluated its activity when encapsulated in or conjugated to the surface of poly(D, L-lactic-co-glycolic) acid (PLGA) NPs. We found that Hp91, when encapsulated in or conjugated to the surface of PLGA-NPs, not only activates both human and mouse DCs, but is in fact more potent than free Hp91. Hp91 packaged within NPs was about fivefold more potent than the free peptide, and Hp91 conjugated to the surface of NPs was similar to 20-fold more potent than free Hp91. Because of their capacity to activate DCs, such NP-Hp91 systems are promising as delivery vehicles for subunit vaccines against infectious disease or cancer.

The main pathway for the induction of type I interferons (IFN) by viruses is through the recognition of viral RNA by cytosolic receptors and the subsequent activation of interferon regulatory factor 3 (IRF-3), which drives IFN-a/ß transcription. In addition to their role in inducing an antiviral state, type I IFN also play a role in modulating adaptive immune responses, in part via their effects on dendritic cells (DCs). Many viruses have evolved mechanisms to interfere with type I IFN induction, and one recently reported strategy for achieving this is by targeting IRF-3 for degradation, as shown for rotavirus nonstructural protein 1 (NSP1). It was therefore of interest to investigate whether rotavirus-exposed DCs would produce type I IFN and/or mature in response to the virus. Our results demonstrate that IRF-3 was rapidly degraded in rotavirus-infected mouse embryonic fibroblasts (MEFs) and type I IFN was not detected in these cultures. In contrast, rotavirus induced type I IFN production in myeloid DCs (mDCs), resulting in their activation. Type I IFN induction in response to rotavirus was reduced in mDCs from IRF-3-/- mice, indicating that IRF-3 was important for mediating the response. Exposure of mDCs to UV-treated rotavirus induced significantly higher type I IFN levels, suggesting that rotavirus-encoded functions also antagonized the response in DCs. However, in contrast to MEFs, this action was not sufficient to completely abrogate type I IFN induction, consistent with a role for DCs as sentinels for virus infection. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Purpose of review less thanbrgreater than less thanbrgreater thanThe early stage of HIV-1 infection is when the virus is most vulnerable, and should therefore offer the best opportunity for therapeutic interventions. This review addresses the recent progress in the understanding of innate immune responses against HIV-1 with focus on the potential targets for prevention of viral acquisition, replication and dissemination. less thanbrgreater than less thanbrgreater thanRecent findings less thanbrgreater than less thanbrgreater thanResearch indicates that the host-derived factor trappin-2/elafin is protective against HIV, whereas semen-derived enhancer of viral infection and defensins 5 and 6 enhance viral transmission. Further, studies suggest that stimulation of TLR4 and inhibition of TLR7-9 pathways may be HIV suppressive. The regulation and function of viral restriction factors tetherin and APOBEC3G have been investigated and a molecule mimicking the premature uncoating achieved by TRIM5 alpha, PF74, has been identified. Chloroquine has been shown to inhibit plasmacytoid dendritic cell activation and suppress negative modulators of T-cell responses. Blockade of HMBG1 has been found to restore natural-killer-cell-mediated killing of infected dendritic cells, normally suppressed by HIV-1. Interestingly, when used as adjuvants, EAT-2 and heat shock protein gp96 reportedly enhance innate immune responses. less thanbrgreater than less thanbrgreater thanSummary less thanbrgreater than less thanbrgreater thanSeveral targets for innate immunity-mediated therapeutics have been identified. Nonetheless, more research is required to unveil their underlying mechanisms and interactions before testing these molecules in clinical trials.

HIV-1 reportedly augments the expression of certain negative costimulatory and inhibitory molecules on T cells, leading to immune impairment. The signaling mechanisms underlying the induction of suppressor molecules and subsequent onset of T-cell impairment in HIV infection remain ambiguous. Our experiments with both autologous and allogeneic T cells exposed to HIV-pulsed dendritic cells showed increased expression of LAG-3, TIM-3, CD160 CTLA-4, TRAIL, and certain suppression-associated transcription factors, namely Blimp-1, DTX1 and FoxP3, whose recruitments were closely regulated by P38MAPK/STAT3 signal transduction pathways. Blockade of P38MAPK/STAT3 significantly decreased the expression of the inhibitory molecules studied and significantly restored T-cell proliferation. The P38MAPK/STAT3 proteins had a higher degree of phosphorylation in the HIV-1-primed cells. We also found that IL-6 and IL-10, and certain other growth factors commonly known to activate STAT3 signaling events were not responsible for STAT3 activation. Blockade of viral CD4 binding and fusion with DCs significantly reduced the negative effects DCs imposed on primed T cells. We concluded that HIV-1 negatively modulate DC functions, causing the activation of the P38MAPK/STAT3 pathway in T cells, leading to recruitment of inhibitory molecules and subsequent onset of T-cell impairment.

The present study reports the diversity of rotavirus strains circulating in León, Nicaragua during three years. There was a shift of G and P genotypes with increment of one specific genotype during the second most important peak of diarrhea occurring in the beginning of every year. © 2006 Lippincott Williams & Wilkins, Inc.

Oseltamivir is the main antiviral for treatment and prevention of pandemic influenza. The increase in oseltamivir resistance reported recently has therefore sparked a debate on how to use oseltamivir in non pandemic influenza and the risks associated with wide spread use during a pandemic. Several questions have been asked about the fate of oseltamivir in the sewage treatment plants and in the environment. We have assessed the fate of oseltamivir and discuss the implications of environmental residues of oseltamivir regarding the occurrence of resistance. A series of batch experiments that simulated normal sewage treatment with oseltamivir present was conducted and the UV-spectra of oseltamivir were recorded. Findings. Our experiments show that the active moiety of oseltamivir is not removed in normal sewage water treatments and is not degraded substantially by UV light radiation, and that the active substance is released in waste water leaving the plant. Our conclusion is that a ubiquitous use of oseltamivir may result in selection pressures in the environment that favor development of drug-resistance. © 2007 Fick et al.

Dendritic cell (DC)-based adoptive tumor immunotherapy approaches have shown promising results, but the incidence of tumor regression is low and there is an evident call for identifying culture conditions that produce DCs with a more potent Th1 potential. Routinely, DCs are differentiated in CO(2) incubators under atmospheric oxygen conditions (21% O(2)), which differ from physiological oxygen levels of only 3-5% in tissue, where most DCs reside. We investigated whether differentiation and maturation of DCs under physiological oxygen levels could produce more potent T-cell stimulatory DCs for use in adoptive immunotherapy. We found that immature DCs differentiated under physiological oxygen levels showed a small but significant reduction in their endocytic capacity. The different oxygen levels did not influence their stimuli-induced upregulation of cluster of differentiation 54 (CD54), CD40, CD83, CD86, C-C chemokine receptor type 7 (CCR7), C-X-C chemokine receptor type 4 (CXCR4) and human leukocyte antigen (HLA)-DR or the secretion of interleukin (IL)-6, tumor necrosis factor (TNF)-alpha and IL-10 in response to lipopolysaccharide (LPS) or a cytokine cocktail. However. DCs differentiated under physiological oxygen level secreted higher levels of IL-12(p70) after exposure to LPS or CD40 ligand. Immature DCs differentiated at physiological oxygen levels caused increased T-cell proliferation, but no differences were observed for mature DCs with regard to T-cell activation. In conclusion, we show that although DCs generated under atmospheric or physiological oxygen conditions are mostly similar in function and phenotype, DCs differentiated under physiological oxygen secrete larger amounts of IL-12(p70). This result could have implications for the use of ex vivo-generated DCs for clinical studies, since DCs differentiated at physiological oxygen could induce increased Th1 responses in vivo.

otavirus (RV) is the major cause of severe gastroenteritis in young children. A virus-encoded enterotoxin, NSP4 is proposed to play a major role in causing RV diarrhoea but how RV can induce emesis, a hallmark of the illness, remains unresolved. In this study we have addressed the hypothesis that RV-induced secretion of serotonin (5-hydroxytryptamine, 5-HT) by enterochromaffin (EC) cells plays a key role in the emetic reflex during RV infection resulting in activation of vagal afferent nerves connected to nucleus of the solitary tract (NTS) and area postrema in the brain stem, structures associated with nausea and vomiting. Our experiments revealed that RV can infect and replicate in human EC tumor cells ex vivo and in vitro and are localized to both EC cells and infected enterocytes in the close vicinity of EC cells in the jejunum of infected mice. Purified NSP4, but not purified virus particles, evoked release of 5-HT within 60 minutes and increased the intracellular Ca(2+) concentration in a human midgut carcinoid EC cell line (GOT1) and ex vivo in human primary carcinoid EC cells concomitant with the release of 5-HT. Furthermore, NSP4 stimulated a modest production of inositol 1,4,5-triphosphate (IP(3)), but not of cAMP. RV infection in mice induced Fos expression in the NTS, as seen in animals which vomit after administration of chemotherapeutic drugs. The demonstration that RV can stimulate EC cells leads us to propose that RV disease includes participation of 5-HT, EC cells, the enteric nervous system and activation of vagal afferent nerves to brain structures associated with nausea and vomiting. This hypothesis is supported by treating vomiting in children with acute gastroenteritis with 5-HT(3) receptor antagonists.

While the clinical importance of human rotavirus (RV) disease is well recognized and potent vaccines have been developed, our understanding of how human RV causes diarrhoea, vomiting and death remains unresolved. The fact that oral rehydration corrects electrolyte and water loss, indicates that enterocytes in the small intestine have a functional sodium-glucose co-transporter. Moreover, RV infection delays gastric emptying and loperamide appears to attenuate RV diarrhoea, thereby suggesting activation of the enteric nervous system. Serotonin (5-HT) receptor antagonists attenuate vomiting in young children with gastroenteritis while zinc and enkephalinase inhibitors attenuate RV-induced diarrhoea. In this review we discuss clinical symptoms, pathology, histology and treatment practices for human RV infections and compile the data into a simplified disease model.

Recent results by us and others have shown that the accessory protein Vpu determines plasma membrane versus endosomal accumulation of the HIV-1 core protein Gag and progeny virions in the HeLa model of HIV-1 infection, since Vpu suppresses endocytosis of cell surface-associated Gag. In this report, we used pulse-chase studies and subcellular fractionations to investigate endocytosis of newly synthesized Gag in HeLa HI cells. The uptake of Gag in Delta Vpu-virus background was not blocked by inhibitors of clathrin-mediated endocytosis and macropinocytosis. The cholesterol-sequestering drug filipin inhibited the uptake, but only if the drug was applied before extensive multimerization of Gag had taken place. Thus, the uptake mechanism most likely is only indirectly dependent on cholesterol. Our results also indicated that targeting phenotype of Gag was different in confluent versus subconfluent cell cultures, which could perhaps explain some of the controversies in intracellular targeting of Gag. (c) 2007 Elsevier Inc. All rights reserved.

Mycobacterium tuberculosis (Mtb) manipulates cells of the innate immune system to provide the bacteria with a sustainable intracellular niche. Mtb spread through aerosol carrying them deep into the lungs, where they are internalized by phagocytic cells, such as neutrophils (PMNs), dendritic cells (DCs), and macrophages. PMNs undergo accelerated apoptosis after interaction with the bacterium, and apoptotic cells are sequestered by neighboring phagocytes. Removal of aged apoptotic cells because of natural tissue turnover is described as an immunologically silent process facilitating resolution of inflammation and inhibition of DC maturation. Silencing of immune cells could be favorable for intracellular bacteria. The aim of this study was to clarify the interaction between Mtb-induced apoptotic PMNs and DCs, and evaluate whether this interaction follows the proposed anti-inflammatory pathway. In contrast to aged apoptotic cells, Mtb-induced apoptotic PMNs induced functional DC maturation. We found that the cell fraction from Mtb-induced apoptotic PMNs contained almost all stimulatory capacity, suggesting that cell-cell interaction is crucial for DC activation. Inhibitory studies showed that this cell contact-dependent activation required binding of the PMN Mac-1 (CD11b/CD18) to the DC via DC-SIGN and endocytic activity involving the alpha(v)beta(5) but did not involve the scavenger receptor CD36. Taken together, this study demonstrates that the DCs can distinguish between normal and infected apoptotic PMNs via cellular crosstalk, where the DCs can sense the presence of danger on the Mtb-infected PMNs and modulate their response accordingly.

In Mycobacterium tuberculosis (Mtb)-infected individuals cells of the innate immune system accumulate in the spleen and in granulomas, but how this relates to the protection against Mtb or in the pathogenesis is unknown. Mtb is internalized in the lung by phagocytic cells, such as neutrophils (PMNs), dendritic cells (DCs) and macrophages. PMNs undergo accelerated apoptosis after internalization of the bacterium and are subsequently sequestered by neighbouring phagocytes. Removal of aged apoptotic cells is an immunologically silent process and the aim of this study was to clarify the interaction between Mtb-induced apoptotic PMNs and DCs, and evaluate if this interaction induced functional maturation of the DCs. In fact, Mtb-induced apoptotic PMNs induced DC maturation, whereas exposure to spontaneous apoptotic PMNs had no effect on DCs maturation status. We found that the cell fraction contained almost all stimulatory capacity, suggesting that the cell-cell interaction is crucial for DC activation. Inhibitory studies showed that this cell contact-dependent activation required binding of the PMN Mac-1 (CD11b/CD18) to the DC via DC-SIGN and endocytic activity. Taken together, this study proves that the DCs can distinguish between normal and infected apoptotic PMNs via cellular cross talk, where the DCs can sense the presence of danger on the Mtb-infected PMNs and modulate their response accordingly.

Background: There is a need for safe and potent adjuvants capable of delivering vaccine candidates over the mucosal barrier, with good capacity to stimulate both mucosal and systemic cell-mediated and humoral immunity. An adjuvant aimed for intranasal delivery should preferably deliver the antigen and minimize the transfer into the close proximity of the central nervous system, thus avoiding damage on the olfactory tissues. Advantages with a mucosal delivery route would be to provide mucosal and systemic immunity, requiring lower vaccine doses then when given parentally. The aim of this study was to study if the N3 adjuvant intranasally administered with HIV DNA plasmids would be transferred into the olfactory tissues and cause local inflammation and tissue damage. Results: The N3 adjuvant alone and when combined with HIV-1 DNA gag plasmid and delivered intranasally did not cause detectable damage to the nasal epithelium or the olfactory epithelium or bulb over a period of 3 days after delivery. The intranasal administration of HIV-1 gagp37 DNA induced both a humoral and a cell-mediated immunity against the gag antigen. Significantly higher HIV-1-specific humoral, but not cell-mediated immune responses were seen in DNA/N3-immunized mice in comparison with HIV-1 DNA/saline-immunized animals. Conclusions: A safe and convenient intranasal mode of HIV-1 DNA plasmid and adjuvant delivery was shown not to interfere with the tissues in close proximity to the central nervous system. The N3 adjuvant combined with HIV-1 plasmids enhances the HIV-1-specific immunogenicity and merits to be clinically tested. © 2008 Elsevier Ltd. All rights reserved.

Dendritic cells present microbial antigens to T cells after uptake of apoptotic vesicles from infected cells. We previously reported that immunizations with apoptotic HIV-1/murine leukemia virus (MuLV) infected cells lead to induction of both cellular and humoral immune responses as well as resistance to mucosal challenge with live HIV-1/MuLV infected cells. Here we extended those studies and investigated whether apoptotic cells from HIV-1/MuLV infected cells stimulate the production of HIV-1 neutralizing activity. We compared different routes of administration and were able to induce p24- and Nef-specific cellular proliferation after intraperitoneal (i.p.), intranasal (i.n.), subcutaneous (s.c.) and intramuscular (i.m.) immunizations. Serum IgG and IgA antibodies directed against gp160, p24, or Nef were also produced regardless of immunization route used. However, the induction of mucosa-associated IgAs from faeces or vaginal secretions were detected only after either i.p. or i.n. immunizations. We were able to measure neutralizing activity in sera of mice after i.p. and i.n. immunization. Neutralizing reactivity was also detected after s.c. and i.m. immunizations in the presence of the cytokine adjuvant granulocyte macrophage-colony stimulating factor (GM-CSF). Conclusively we show induction of cellular and humoral immune responses including neutralizing activity after immunization with apoptotic HIV-1/MuLV infected cells in mice. The results from this study support further evaluations using apoptotic cells as antigen delivery system for vaccination against HIV-1 in other animal models.

Tuberculosis (TB) has emerged as the most prominent bacterial disease found in human immunodeficiency virus (HIV)-positive individuals worldwide. Due to high prevalence of asymptomatic Mycobacterium tuberculosis (Mtb) infections, the future HIV vaccine in areas highly endemic for TB will often be administrated to individuals with an ongoing Mtb infection. The impact of concurrent Mtb infection on the immunogenicity of a HIV vaccine candidate, MultiHIV DNA/protein, was investigated in mice. We found that, depending on the vaccination route, mice infected with Mtb before the administration of the HIV vaccine showed impairment in both the magnitude and the quality of antibody and T cell responses to the vaccine components p24Gag and gp160Env. Mice infected with Mtb prior to intranasal HIV vaccination exhibited reduced p24Gag-specific serum IgG and IgA, and suppressed gp160Env-specific serum IgG as compared to respective titers in uninfected HIV-vaccinated controls. Importantly, in Mtb-infected mice that were HIV-vaccinated by the intramuscular route the virus neutralizing activity in serum was significantly decreased, relative to uninfected counterparts. In addition mice concurrently infected with Mtb had fewer p24Gag-specific IFN-γ-expressing T cells and multifunctional T cells in their spleens. These results suggest that Mtb infection might interfere with the outcome of prospective HIV vaccination in humans.

While IgA is proposed to be essential to control rotavirus disease, no information is available how IgA deficient individuals modulate rotavirus disease and immune responses. We report that individuals (n=62) with selective IgA deficiency ( IgA-D) (<0,05g/l) resolve rotavirus disease and show higher total IgG and IgG1 subclass antibody titers to rotavirus than IgA proficient individuals ( n=62) (GMT 18101 vs 4000 (p<0.005); 8463 vs 1691, (p<0.005). We conclude that IgA is not essential for resolving rotavirus infection in humans.

Rotavirus virus-like particles (RV-VLPs) represent a novel strategy for development of a rotavirus subunit vaccine. In this study, RF 8-2/6/7-VLPs with rotavirus VP8 protein (amino acid 1-241 of VP4) fused to the amino terminal end of a truncated VP2, were evaluated for their immunogenic and protective properties. A single intramuscular dose of, either 2 or 20 μg, RF 8-2/6/7-VLPs alone or combined with a potent adjuvant poly[di(carboxylatophenoxy)]phosphazene] (PCPP) induced rotavirus-specific serum IgG and IgA, fecal IgG titers that were enhanced 5-90-fold by adjuvant. Passive protective immunity was achieved in offspring to dams vaccinated with 2 and 20 μg RV-VLPs in presence of adjuvant and 20 μg RV-VLP without adjuvant. © 2008 Elsevier Ltd. All rights reserved.

A key gap in the development and evaluation of HIV-1 vaccines is insufficient knowledge with regard to sampling techniques and assessment of mucosal immune responses required for early prevention and inhibition of viral dissemination. In an attempt to start bridging this gap, the EUROPRISE network of scientists working on HIV-1 vaccine and microbicide research organized a workshop with the aim to review the types of mucosal responses/biomarkers currently measured in mucosal immunology and to define how the mucosal responses/biomarkers are measured and/or the assays and sampling methods used. The Workshop addressed two critical questions: first whether, with current knowledge, it would be possible to define a consensus set of mucosal sampling methods to facilitate cross-species comparisons and ensure standardized implementation in clinical trials; second to determine the remaining challenges (technical and logistical) and their possible solutions for assessing mucosal responses to HIV-1 vaccines.

In presence of low or high levels of rotavirus-specific maternal antibodies, the ability of newborn mice to respond to immunization with rotavirus RF 8*-2/6/7 VLPs, was evaluated. After parenteral vaccination, 100% of offspring born to low-antibody-titer dams developed rotavirus-specific IgG antibodies (n = 7). In contrast, only 25% of offsprings born to high-antibody-titer dams responded to parenteral immunization (n = 12). When comparing parenteral versus oral immunization in offspring to low-antibody-titer dams only 45% responded after oral immunization (n = 6). In conclusion, the response to parenteral immunization was not hampered by the presence of low levels of maternal antibodies induced by a natural infection while oral immunization was impaired. However, high levels of maternal antibodies impaired the response to parenteral immunization. © 2008 Elsevier Ltd. All rights reserved.

Natural killer (NK) cells are important effectors in resistance to viral infections. The role of NK cells in the acute response to human immunodeficiency virus 1 (HIV-1) infected cells was investigated in a mouse model based on a HIV-1/murine leukemia virus (MuLV) pseudovirus. Splenocytes infected with HIV-1/MuLV were injected intraperitoneally and local immunologic responses and persistence of infected cells were investigated. In vivo depletion with an anti-NK1.1 antibody showed that NK cells are important in resistance to virus infected cells. Moreover, NK cell frequency in the peritoneal cavity increased in response to infected cells and these NK cells had a more mature phenotype, as determined by CD27 and Mac-1 expression. Interestingly, after injection of HIV-1/MuLV infected cells, but not MuLV infected cells, peritoneal NK cells had an increased cytotoxic activity.

Natural killer (NK) cells may be protective in HIV infection and are inhibited by killer cell immunoglobulin-like receptors (KIRs) interacting with MHC class I molecules, including HLA-C. Retention of HLA-C despite downregulation of other MHC class I molecules on HIV infected cells might protect infected cells from NK cell recognition in vitro. To assess the role of inhibitory HLA-C ligands in the capacity of NK cells to recognize autologous infected T cells, we measured NK cell degranulation in vitro in viremic patients, controllers with low viremia, and healthy donors. No difference in NK cell response to uninfected compared to HIV-1(IIIB) infected targets was observed. Activation of NK cells was regulated by KIRs, because NK cell degranulation was increased by 1-7F9, a human antibody that binds KIR2DL1/L2/L3 and KIR2DS1/S2, and this effect was most pronounced in KIR haplotype B individuals.

Natural killer (NK) cells have been suggested to play a protective role in HIV disease progression. One potent effector mechanism of NK cells is antibody-dependent cellular cytotoxicity (ADCC) mediated by antiviral antibodies binding to the Fc gamma RIIIa receptor (CD16) on NK cells. We investigated NK cell-mediated ADCC function and the presence of ADCC antibodies in plasma from 20 HIV-1-infected patients and 10 healthy donors. The HIV-positive patients were divided into two groups: six who controlled viremia for at least 8 y without treatment (controllers), and 14 who were persistently viremic and not currently on treatment. Plasma from both patient groups induced NK cell IFN-gamma expression and degranulation in response to HIV-1 envelope (Env) gp140-protein-coated cells. Patient antibodies mediating ADCC were largely directed towards the Env V3 loop, as identified by a gp140 protein lacking the V3 loop. Interestingly, in two controllers ADCC-mediating antibodies were more broadly directed to other parts of Env. A high viral load in patients correlated with decreased ADCC-mediated cytolysis of gp140-protein-coated target cells. NK cells from both infected patients and healthy donors degranulated efficiently in the presence of antibody-coated HIV-1-infected Jurkat cells. In conclusion, the character of ADCC-mediating antibodies differed in some controllers compared to viremic patients. NK cell ADCC activity is not compromised in HIV-infected patients.

Aichi virus (AiV), genus Kobuvirus, family Picornaviridae, is associated with gastroenteritis in humans. Previous studies have shown high seroprevalence but low incidence (0.9-4.1%) in clinical samples. We report here the first detection of AiV in Sweden. Two hundred twenty-one specimens from hospitalized patients with diarrhea, who were negative for other enteric viruses, were included in the study. AiV were detected in three specimens, all from elderly patients. Phylogenetic analysis revealed that the three Swedish isolates belonged to genotype A and were genetically closest to European and Asian strains of AiV.

In this novel study, we have for the first time identified evolutionarily conserved capsid residues in an individual chronically infected with norovirus (GGII.3). From 2000 to 2003, a total of 147 P1-1 and P2 capsid sequences were sequenced and investigated for evolutionarily conserved and functionally important residues by the evolutionary trace (ET) algorithm. The ET algorithm revealed more absolutely conserved residues (ACR) in the P1-1 domain (47/53, 88 %) as compared with the P2 domain (86/133, 64 %). The capsid P1-1 and P2 domains evolved in time-dependent manner, with a distinct break point observed between autumn/winter of year 2000 (isolates P1, P3 and P5) and spring to autumn of year 2001 (isolates P11, P13 and P15), which presumably coincided with a change of clinical symptoms. Furthermore, the ET analysis revealed a similar receptor-binding pattern as reported for Norwalk and VA387 strains, with the CS-4 and CS-5 patch (Norwalk strain) including residues 329 and 377 and residues 306 and 310, respectively, all being ACR in all partitions. Most interesting was that residues 343, 344, 345, 374, 390 and 391 of the proposed receptor A and B trisaccharide binding site (VA387 strain) within the P2 domain remained ACR in all partitions, presumably because there was no selective advantage to alter the histo blood group antigens (HBGA) receptor binding specificity. In conclusion, this study provides novel insights to the evolutionary process of norovirus during chronic infection.