Intrahost evolution of the HIV-2 capsid correlates with progression to AIDS.

HIV-2 infection will progress to AIDS in most patients without treatment, albeit at approximately half the rate of HIV-1 infection. HIV-2 capsid (p26) amino acid polymorphisms are associated with lower viral loads and enhanced processing of T cell epitopes, which may lead to protective Gag-specific T cell responses common in slower progressors. Lower virus evolutionary rates, and positive selection on conserved residues in HIV-2 env have been associated with slower progression to AIDS. In this study we analysed 369 heterochronous HIV-2 p26 sequences from 12 participants with a median age of 30 years at enrolment. CD4% change over time was used to stratify participants into relative faster and slower progressor groups. We analysed p26 sequence diversity evolution, measured site-specific selection pressures and evolutionary rates, and determined if these evolutionary parameters were associated with progression status. Faster progressors had lower CD4% and faster CD4% decline rates. Median pairwise sequence diversity was higher in faster progressors (5.7x10-3 versus 1.4x10-3 base substitutions per site, P<0.001). p26 evolved under negative selection in both groups (dN/dS=0.12). Median virus evolutionary rates were higher in faster than slower progressors - synonymous rates: 4.6x10-3 vs. 2.3x10-3; and nonsynonymous rates: 6.9x10-4 vs. 2.7x10-4 substitutions/site/year, respectively. Virus evolutionary rates correlated negatively with CD4% change rates (ρ = -0.8, P=0.02), but not CD4% level. The signature amino acid at p26 positions 6, 12 and 119 differed between faster (6A, 12I, 119A) and slower (6G, 12V, 119P) progressors. These amino acid positions clustered near to the TRIM5α/p26 hexamer interface surface. p26 evolutionary rates were associated with progression to AIDS and were mostly driven by synonymous substitutions. Nonsynonymous evolutionary rates were an order of magnitude lower than synonymous rates, with limited amino acid sequence evolution over time within hosts. These results indicate HIV-2 p26 may be an attractive therapeutic target.

State of the Art: Extracorporeal Cardiopulmonary Resuscitation for In-Hospital Arrest.

Extracorporeal membrane oxygenation has been used since the 1970s and recently has seen increased use for in-hospital arrest requiring extracorporeal cardiopulmonary resuscitation (ECPR). This paper provides an updated review of the ECPR literature and practical recommendations for implementation of an ECPR program.

Does smallpox vaccination modify HIV disease progression among ART-naive people living with HIV in Africa?

We examined the association between a history of smallpox vaccination and immune activation (IA) in a population of antiretroviral therapy-naïve people living with HIV (PLHIV). A cross-sectional study was conducted in Senegal from July 2015 to March 2017. Smallpox vaccination was ascertained by the presence of smallpox vaccine scar and IA by the plasma level of ß-2-microglobulin (ß2m). The association was analysed using logistic regression and linear regression models. The study population comprised 101 PLHIV born before 1980 with a median age of 47 years (interquartile range (IQR) = 42-55); 57·4% were women. Smallpox vaccine scar was present in 65·3% and the median ß2m level was 2·59 mg/l (IQR = 2·06-3·86). After adjustment, the presence of smallpox vaccine scar was not associated with a ß2m level ⩾2·59 mg/l (adjusted odds ratio 0·94; 95% confidence interval 0·32-2·77). This result was confirmed by the linear regression model. Our study does not find any association between the presence of smallpox vaccine scar and the ß2m level and does not support any association between a previous smallpox vaccination and HIV disease progression. In this study, IA is not a significant determinant of the reported non-targeted effect of smallpox vaccination in PLHIV.

Elevated hepcidin at HIV diagnosis is associated with incident tuberculosis in a retrospective cohort study.

Hepcidin inhibits ferroportin-mediated iron efflux, leading to intracellular macrophage iron retention, possibly favoring Mycobacterium tuberculosis iron acquisition and tuberculosis (TB) pathogenesis. Plasma hepcidin was measured at human immunodeficiency virus (HIV) diagnosis in a retrospective HIV-prevalent, antiretroviral-naïve African cohort to investigate the association with incident pulmonary and/or extra-pulmonary TB. One hundred ninety-six participants were followed between 5 August 1992 and 1 June 2002, with 32 incident TB cases identified. Greater hepcidin was associated with significantly increased likelihood of TB after a median time to TB of 6 months. Elucidation of iron-related causal mechanisms and time-sensitive biomarkers that identify individual changes in TB risk are needed.

An adaptation of recombinant vaccinia-based ELISPOT and intracellular cytokine staining for a comparative measurement of cellular immune responses in HIV-1 and HIV-2 infections in West Africa.

An efficient and quantitative tool for rapid assessment of human immunodeficiency virus (HIV)-induced cellular immune responses is important for resource-limited settings, such as in sub-Saharan Africa. Modifications are required to previously reported methods for evaluating ex-vivo antigen-specific cellular responses based on direct recombinant vaccinia virus (rVV) stimulation of peripheral blood mononuclear cells (PBMCs) by enzyme linked immunosorbent assay (ELISPOT) and by flow cytometry intracellular cytokine assay (ICA). We made such modifications in order to detect specific responses and compared quantitative cellular immune responses in HIV-1 and HIV-2 infected Gambians. The sensitivity of the rVV-based ELISPOT assay was on average 1.25 interferon (IFN)-gamma spot forming cells (SFC) per 50 000 PBMCs specific for either infection, and 5 IFN-gamma-secreting CD8+ T cells/50 000 in the ICA. The level of IFN-gamma SFC detected by ELISPOT and by ICA were correlated (P < 0.02). ICA detected pol-specific responses in 88% and 67% of HIV-1 and HIV-2 subjects, respectively, and gag-specific responses in more than 80% of both infections. Lower proportions of responders were obtained with ELISPOT, for which pol responses were present in 60% of HIV-1 and 46% of HIV-2 infected patients, and gag responses in 55% and 69%, respectively. The assays did not show any significant difference in cellular immune responses between HIV-1 and HIV-2 infected subjects with CD4% >or= 20%. These outcomes are comparable with results obtained using standard techniques and thus this method is a suitable, rapid and less expensive assessment of cellular immunity.

Mycobacterium tuberculosis genome-wide screen exposes multiple CD8 T cell epitopes.

Mounting evidence suggests human leucocyte antigen (HLA) class I-restricted CD8(+) T cells play a role in protective immunity against tuberculosis yet relatively few epitopes specific for the causative organism, Mycobacterium tuberculosis, are reported. Here a total genome-wide screen of M. tuberculosis was used to identify putative HLA-B*3501 T cell epitopes. Of 479 predicted epitopes, 13 with the highest score were synthesized and used to restimulate lymphocytes from naturally exposed HLA-B*3501 healthy individuals in cultured and ex vivo enzyme-linked immunospot (ELISPOT) assays for interferon (IFN)-gamma. All 13 peptides elicited a response that varied considerably between individuals. For three peptides CD8(+) T cell lines were expanded and four of the 13 were recognized permissively through the HLA-B7 supertype family. Although further testing is required we show the genome-wide screen to be feasible for the identification of unknown mycobacterial antigens involved in immunity against natural infection. While the mechanisms of protective immunity against M. tuberculosis infection remain unclear, conventional class I-restricted CD8(+) T cell responses appear to be widespread throughout the genome.

Cellular immunity induced by the recombinant Plasmodium falciparum malaria vaccine, RTS,S/AS02, in semi-immune adults in The Gambia.

Vaccination of malaria-naive humans with recombinant RTS,S/AS02, which includes the C-terminus of the circumsporozoite protein (CS), has been shown to induce strong T cell responses to both the whole protein antigen and to peptides from CS. Here we show that strong T cell responses were also observed in a semi-immune population in The Gambia, West Africa. In a Phase I study, 20 adult male volunteers, lifelong residents in a malaria-endemic region, were given three doses of RTS,S/AS02 at 0, 1 and 6 months. Responses to RTS,S, hepatitis B surface antigen and peptides from CS were tested using lymphocyte proliferation, interferon (IFN)-gamma production in microcultures, and IFN-gamma ex vivo and cultured ELISPOT, before and after vaccination. Cytotoxic responses were tested only after vaccination and none were detected. Before vaccination, the majority of the volunteers (15/20) had detectable responses in at least one of the tests. After vaccination, responses increased in all assays except cytotoxicity. The increase was most marked for proliferation; all donors responded to RTS,S after the third dose and all except one donor responded to at least one peptide after the second or third dose. There was a lack of close association of peptide responses detected by the different assays, although in microcultures IFN-gamma responses were found only when proliferative responses were high, and responses by cultured ELISPOT and proliferation were found together more frequently after vaccination. We have therefore identified several peptide-specific T cell responses induced by RTS,S/AS02 which provides a mechanism to investigate potentially protective immune responses in the field.

A Gambian TNF haplotype matches the European HLA-A1,B8,DR3 and Chinese HLA-A33,B58,DR3 haplotypes.

Caucasians carry TNFA-308*2 in the 8.1 ancestral haplotype (AH) (HLA-A1,B8,DR3). In Gambians, TNFA-308*2 occurs without HLA-B8 or -DR3, suggesting an independent effect of TNFA-308 on disease. Hence we sought a segment of the 8.1 AH in Gambians. BAT1 (intron 10)*2 was selected as a specific marker of the haplotype and was found with TNFA-308*2 in Gambians. Samples homozygous at TNFA-308 and BAT1 (intron 10) demonstrated identity between the African TNFA-308*2 haplotype, the 8.1AH and the Asian diabetogenic 58.1AH (HLA-A33,B58,DR3) across a region spanning BAT1, ATP6G, IKBL, LTA, TNFA, LTB, LST-1 and AIF-1. Conservation of this block in geographically distinct populations suggests a common evolutionary origin and challenges current views of the role of TNFA-308*2 in disease.

Natural measles causes prolonged suppression of interleukin-12 production.

Among vaccine-preventable diseases, measles is the preeminent killer of children worldwide. Infection with measles virus (MV) is associated with prolonged suppression of cell-mediated immune responses, a phenomenon that is thought to underlie the susceptibility to secondary infections that accounts for most measles-related mortality. Interleukin (IL)-12 is critical for the orchestration of cellular immunity. MV specifically ablates IL-12 production by monocyte/macrophages in vitro through binding to CD46, a complement regulatory protein that is an MV receptor. To address the effect of MV on IL-12 responses in vivo, cytokine production was examined in Gambian patients with measles. IL-12 production by peripheral blood monocytes from such patients is markedly suppressed, which provides a unifying mechanism for many of the immunologic abnormalities associated with measles. This suppression is prolonged, with significant, stimulus-specific inhibition of IL-12 production demonstrable months after recovery from acute infection. However, despite this suppression, IL-12 responsiveness remains intact.

Cytotoxic T lymphocytes recognize structurally diverse, clade-specific and cross-reactive peptides in human immunodeficiency virus type-1 gag through HLA-B53.

Human immunodeficiency virus type-1 (HIV-1) cytotoxic T lymphocyte (CTL) epitopes have largely been defined in Caucasian populations infected with clade B virus. Identification of potentially protective CTL epitopes in non-B clade-infected African subjects is important for vaccine development. In a study of CTL responses in clade A-infected Gambians, using cytotoxicity, interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISpot) and HLA-B53-peptide tetramer assays, we identified three HLA-B53-restricted epitopes in HIV-1 gag p24. CTL specific for an epitope in a highly immunogenic region of the p24 protein showed no cross-reactivity to other HIV-1 clades. Two of the epitopes would not have been predicted from the peptide-binding motif due to the absence of a proline anchor at position 2. Structural analysis of HLA-B53 and its relative, HLA B35, enabled us to re-define the peptide-binding motif to include other P2 anchors. These results demonstrate the value of combined immunological and structural analyses in defining novel CTL epitopes and have implications for HIV-1 vaccine design.

HLA-B*35-restricted CD8 T cell epitopes in the antigen 85 complex of Mycobacterium tuberculosis.

Few target epitopes have been described for human CD8 T lymphocytes in antigens of Mycobacterium tuberculosis. By use of a reverse immunogenetics approach, 23 motif-bearing peptides of the Ag85 complex were tested for binding to HLA-B*35, one of the common B-types in West Africa. Three 9-mer peptides bound with high affinity to HLA-B*3501 and displayed low dissociation rates of peptide-major histocompatibility complexes (MHCs). IC(50) and half-life values of peptide-MHC class I complexes were in the same range as reported earlier for other immunogenic peptides. Immune responses against peptide Ag85C (aa 204-212) WPTLIGLAM were characterized in detail. Peptide-stimulated effector cells were able to kill macrophages infected with M. tuberculosis or bacille Calmette-Guérin. Peptide-specific CD8 T cells could be visualized by using HLA-B*3501 tetramers and were shown to produce interferon-gamma and tumor necrosis factor-alpha. Together with other published epitopes, these peptides can be used to study more closely the role of CD8 T cells in mycobacterial infection and tuberculosis.

Antigen-specific expansion of cytotoxic T lymphocytes in acute measles virus infection.

Skewing of the T-cell receptor repertoire of CD8(+) T cells has been shown in some persistent infections with viruses, such as human immunodeficiency virus, simian immunodeficiency virus, and Epstein-Barr virus. We have demonstrated that similar distortions also occur in nonpersistent measles virus infection. In addition, two of four children immunized with live, attenuated measles virus showed larger and more persistent CD8(+) T-cell expansions than their naturally infected counterparts. The expanded lymphocyte populations were monoclonal or oligoclonal and lysed target cells infected with recombinant vaccinia virus expressing measles virus protein. These results demonstrate that the expansions of CD8(+) T lymphocytes are antigen driven.

Ex vivo analysis of cytotoxic T lymphocytes to measles antigens during infection and after vaccination in Gambian children.

The study of cytotoxic T cell responses to measles antigens during infection and after vaccination may provide insight into the immunopathology of the infection. It will also provide a knowledge of the immunity conferred by wild or attenuated virus, which will help in the design of new vaccines. Direct cytotoxic T cell responses, which did not require in vitro restimulation, were measured from peripheral blood by a standard 51Cr-release assay in 35 patients with acute measles, using HLA class I matched allogeneic B cells as targets. 77% showed specific responses to measles fusion protein, 69% to the hemagglutinin, and 50% to the nucleoprotein. These responses, which were related to severity of disease and history of previous vaccination, had waned by 14-24 wk after measles when memory responses to the same antigens could be elicited by restimulation in 71% of the 13 patients tested. A similar pattern followed vaccination: direct cytotoxic responses to fusion and hemagglutinin proteins were shown in 70% of the 20 children tested while 50% responded to the nucleoprotein. These responses, which were mediated by both CD8(+) and CD4(+) cells, faded over 6 wk when memory responses could be restimulated. Thus, a vigorous cytotoxic T lymphocyte response to fusion, hemagglutinin, and nucleoproteins is important in both natural and vaccine-induced immunity to measles.

Human leukocyte antigen class I- and class II-restricted cytotoxic T lymphocyte responses to measles antigens in immune adults.

The study of cytotoxic T cell (CTL) responses to measles polypeptides in persons with different HLA frequencies will provide information for the design of new vaccines. Peripheral blood mononuclear cells derived from African blacks and Caucasians were stimulated with measles virus-infected autologous cells and tested in a standard 51Cr-release assay against autologous B cells infected with vaccinia virus recombinants expressing measles virus antigens. The proportion of subjects who generated CTL to the fusion, hemagglutinin, and nucleoprotein antigens was 43%, 38%, and 28%, respectively. The use of HLA-mismatched targets showed killing to be restricted by both HLA class I and class II antigens, although CD8-mediated class I cytotoxicity predominated. Measles vaccine boosted CTL responses in subjects with low initial activity. These data suggest that the fusion and hemagglutinin proteins are important targets for the measles CTL response.

Clustering of haemagglutinin gene sequences of measles viruses isolated in the Gambia.

Partial nucleotide sequences of the haemagglutinin (H) gene of 18 measles viruses isolated in the Gambia during 1994 and 1995 show a high degree of homology (> 98.5%) when compared with each other. These sequences form a cluster distinct from measles viruses isolated from other geographic regions and from the sequences of vaccine strains and two isolates from the Gambia from earlier years. Despite the low level of genetic heterogeneity observed, a common feature of the Gambian isolates is the presence of three nucleotide changes (at positions 7963, 8649 and 8653) not observed in isolates from other locations.

Electrophoresis of bovine erythrocyte sialic acids: existence of additional band in trypanotolerant Ndama cattle.

Mild acid-hydrolysis of erythrocyte surface sialic acids of the trypanotolerant Ndama and the trypanosusceptible White/Fulani Zebu breeds of cattle was performed. The cleaved sialic acids from the two breeds of cattle were simultaneously subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE), along with commercial standard N-acetylneuraminic acid (MW 309.28), blank gel and plasma proteins. The cleaved sialic acids migrated in the globulin fractions, as shown by the plasma protein electrophoresis. While the Ndama and the Zebu erythrocytes had one migrating band each of which coincided with the standard N-acetylneuraminic acid, the Ndama had another trailing band of sialic acid, of an estimated molecular weight of 30 kDa, which may account for the higher erythrocyte sialic acid concentrations of the Ndama. This additional band was absent in the Zebu. All these bands were readily reproducible.