Distinct, IgG1-driven antibody response landscapes demarcate individuals with broadly HIV-1 neutralizing activity

Understanding pathways that promote HIV-1 broadly neutralizing antibody (bnAb) induction is crucial to advance bnAb-based vaccines. We recently demarcated host, viral, and disease parameters associated with bnAb development in a large HIV-1 cohort screen. By establishing comprehensive antibody signatures based on IgG1, IgG2, and IgG3 activity to 13 HIV-1 antigens in 4,281 individuals in the same cohort, we now show that the same four parameters that are significantly linked with neutralization breadth, namely viral load, infection length, viral diversity, and ethnicity, also strongly influence HIV-1-binding antibody responses. However, the effects proved selective, shaping binding antibody responses in an antigen and IgG subclass-dependent manner. IgG response landscapes in bnAb inducers indicated a differentially regulated, IgG1-driven HIV-1 antigen response, and IgG1 binding of the BG505 SOSIP trimer proved the best predictor of HIV-1 neutralization breadth in plasma. Our findings emphasize the need to unravel immune modulators that underlie the differentially regulated IgG response in bnAb inducers to guide vaccine development

Individuella utvecklingsplaner i ett lärarperspektiv

Idag ska alla skolor använda sig av individuella utvecklingsplaner för att hjälpa elever att nå de mål som finns uppsatta i läroplaner och kursplaner. Detta beslutades av politikerna och lades till i grundskoleförordningen 2005. Syftet med vår uppsats har varit att ta reda på hur lärarna uppfattar IUP och hur dessa uppfattningar förhåller sig till beslutsfattarnas avsikt med införandet av IUP. Som teoretisk utgångspunkt har vi haft ramfaktorteorin och då framför allt de centrala begreppen formuleringsarenan och realiseringsarenan. I bakgrunden visar vi på vad som historiskt föregått ett införande av individuella utvecklingsplaner samt summerar tidigare forskning om individuella utvecklingsplaner och närliggande ämnesområden. Därefter redovisar vi resultaten av vår undersökning som baseras på enkäter till ett antal lärare samt invervjuer av fyra lärare. Resultatet av vår analys visar att det övergripande syftet är detsamma för lärarna som för politikerna, att fler elever ska nå målen Däremot så avviker lärarnas uppfattningar om flera underliggande syften, som vad IUP:n ska innehålla och hur den bidrar till att stärka föräldrarnas intresse och delaktighet. Dessutom används inte IUP:n kontinuerligt i verksamheten på det sätt som är syftet - enligt vår mening. I diskussionen belyser vi hur det kan komma sig att dessa skillnader uppstår och hur de kan förklaras med att beslutet fattas på en nivå och sedan ska genomföras på en annan nivå. Dessutom är formuleringsarenan tudelad i sina uppfattningar i något fall, vilket gör att syftet blir svårdefinierbart för lärarna

Capacity of broadly neutralizing antibodies to inhibit HIV-1 cell-cell transmission is strain- and epitope-dependent

An increasing number of broadly neutralizing antibodies (bnAbs) are considered leads for HIV-1 vaccine development and novel therapeutics. Here, we systematically explored the capacity of bnAbs to neutralize HIV-1 prior to and post-CD4 engagement and to block HIV-1 cell-cell transmission. Cell-cell spread is known to promote a highly efficient infection with HIV-1 which can inflict dramatic losses in neutralization potency compared to free virus infection. Selection of bnAbs that are capable of suppressing HIV irrespective of the transmission mode therefore needs to be considered to ascertain their in vivo activity in therapeutic use and vaccines. Employing assay systems that allow for unambiguous discrimination between free virus and cell-cell transmission to T cells, we probed a panel of 16 bnAbs for their activity against 11 viruses from subtypes A, B and C during both transmission modes. Over a wide range of bnAb-virus combinations tested, inhibitory activity against HIV-1 cellcell transmission was strongly decreased compared to free virus transmission. Activity loss varied considerably between virus strains and was inversely associated with neutralization of free virus spread for V1V2- and V3-directed bnAbs. In rare bnAb-virus combinations, inhibition for both transmission modes was comparable but no bnAb potently blocked cell-cell transmission across all probed virus strains. Mathematical analysis indicated an increased probability of bnAb resistance mutations to arise in cell-cell rather than free virus spread, further highlighting the need to block this pathway. Importantly, the capacity to efficiently neutralize prior to CD4 engagement correlated with the inhibition efficacy against free virus but not cell-cell transmitted virus. Pre-CD4 attachment activity proved strongest amongst CD4bs bnAbs and varied substantially for V3 and V1V2 loop bnAbs in a strain-dependent manner. In summary, bnAb activity against divergent viruses varied depending on the transmission mode and differed depending on the window of action during the entry process, underscoring that powerful combinations of bnAbs are needed for in vivo application

Phenotypic deficits in the HIV-1 envelope are associated with the maturation of a V2-directed broadly neutralizing antibody lineage

Broadly neutralizing antibodies (bnAbs) to HIV-1 can evolve after years of an iterative process of virus escape and antibody adaptation that HIV-1 vaccine design seeks to mimic. To enable this, properties that render HIV-1 envelopes (Env) capable of eliciting bnAb responses need to be defined. Here, we followed the evolution of the V2 apex directed bnAb lineage VRC26 in the HIV-1 subtype C superinfected donor CAP256 to investigate the phenotypic changes of the virus populations circulating before and during the early phases of bnAb induction. Longitudinal viruses that evolved from the VRC26-resistant primary infecting (PI) virus, the VRC26-sensitive superinfecting (SU) virus and ensuing PI-SU recombinants revealed substantial phenotypic changes in Env, with a switch in Env properties coinciding with early resistance to VRC26. Decreased sensitivity of SU-like viruses to VRC26 was linked with reduced infectivity, altered entry kinetics and lower sensitivity to neutralization after CD4 attachment. VRC26 maintained neutralization activity against cell-associated CAP256 virus, indicating that escape through the cell-cell transmission route is not a dominant escape pathway. Reduced fitness of the early escape variants and sustained sensitivity in cell-cell transmission are both features that limit virus replication, thereby impeding rapid escape. This supports a scenario where VRC26 allowed only partial viral escape for a prolonged period, possibly increasing the time window for bnAb maturation. Collectively, our data highlight the phenotypic plasticity of the HIV-1 Env in evading bnAb pressure and the need to consider phenotypic traits when selecting and designing Env immunogens. Combinations of Env variants with differential phenotypic patterns and bnAb sensitivity, as we describe here for CAP256, may maximize the potential for inducing bnAb responses by vaccination

Delineating CD4 dependency of HIV-1: Adaptation to infect low level CD4 expressing target cells widens cellular tropism but severely impacts on envelope functionality

A hallmark of HIV-1 infection is the continuously declining number of the virus' predominant target cells, activated CD4+ T cells. With diminishing CD4+ T cell levels, the capacity to utilize alternate cell types and receptors, including cells that express low CD4 receptor levels such as macrophages, thus becomes crucial. To explore evolutionary paths that allow HIV-1 to acquire a wider host cell range by infecting cells with lower CD4 levels, we dissected the evolution of the envelope-CD4 interaction under in vitro culture conditions that mimicked the decline of CD4high target cells, using a prototypic subtype B, R5-tropic strain. Adaptation to CD4low targets proved to severely alter envelope functions including trimer opening as indicated by a higher affinity to CD4 and loss in shielding against neutralizing antibodies. We observed a strikingly decreased infectivity on CD4high target cells, but sustained infectivity on CD4low targets, including macrophages. Intriguingly, the adaptation to CD4low targets altered the kinetic of the entry process, leading to rapid CD4 engagement and an extended transition time between CD4 and CCR5 binding during entry. This phenotype was also observed for certain central nervous system (CNS) derived macrophage-tropic viruses, highlighting that the functional perturbation we defined upon in vitro adaptation to CD4low targets occurs in vivo. Collectively, our findings suggest that CD4low adapted envelopes may exhibit severe deficiencies in entry fitness and shielding early in their evolution. Considering this, adaptation to CD4low targets may preferentially occur in a sheltered and immune-privileged environment such as the CNS to allow fitness restoring compensatory mutations to occur

T-20 potently inhibits free virus and cell-cell transmission across diverse virus strains.

<p>Inhibition of free virus (black circles) and cell-cell (red circles) transmission of subtype A, B and C viruses by T-20 is shown. The graphs show means and standard error of means (SEM, error bars) of two to three independent experiments performed in duplicates and curve fits to sigmoid dose response curves (variable slope).</p

The capacity to neutralize free viruses prior to completion of CD4 attachment varies across bnAb classes.

<p><b>A:</b> Schematic of free virus infection of A3.01-CCR5 target cells to measure total and pre-attachment activity of bnAbs. Total activity of each bnAb-virus combination (where bnAbs were present throughout the infection process) was set to 100% and pre-attachment inhibition displayed relative to it. To assess pre-attachment activity, bnAbs were removed following attachment by spinoculation. <b>B: Pre-attachment neutralization activity is strain- and epitope-dependent.</b> Pre-attachment activity (relative, %) of the bnAb classes CD4bs, V3 glycans, V1V2 loop, MPER was assessed against the indicated virus strains. Data are means of two to three independent experiments. <b>C: Pre-attachment activity correlates with high potency against free virus, decreased post- attachment inhibition capacity and loss in activity during cell-cell transmission.</b> Summary of the interdependencies of pre-attachment, post-attachment inhibition, IC₅₀ for free virus inhibition, IC₅₀ for cell-cell inhibition and loss in activity during cell-cell inhibition. Correlations were determined by Spearman correlation based on the untransformed data sets and are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004966#ppat.1004966.s013" target="_blank">S8 Fig</a>. R and p values are depicted. n.s. indicates a non-significant correlation. Direct correlation is denoted in blue, inverse correlation in orange.</p

Evaluating A3.01-CCR5 T cell-based free virus and cell-cell transmission.

<p><b>A to F:</b> As indicated, the Gaussia Luciferase reporter virus NLinGluc and the firefly luciferase reporter virus system NLlucAM were used to study free virus and cell-cell transmission. Infectivity was measured by assessing Gaussia and firefly luciferase reporter activity in the supernatant and from the lysed cells, respectively. The graphs show means and standard error of means (SEM, error bars) of two to three independent experiments performed in duplicates. Panels C-F depict curve fits to sigmoid dose response curves (variable slope). <b>A: DEAE dependency of the NLinGluc reporter virus.</b> JR-FL NLinGluc free virus was titrated on TZM-bl cells in 96-well plates in presence (black circles) or absence (green squares) of 10 µg/ml diethylaminoethyl (DEAE). <b>B: Absence of Gaussia luciferase activity in 293-T donor cells but high signal in 293-T-A3.01-CCR5 co-cultures allows for specific detection of cell-cell transmission.</b> 293-T cells transfected with JR-FL <i>env</i> and NLinGluc reporter virus were titrated and incubated in presence of 1.5*10⁴ A3.01-CCR5 target cells per 96 plate culture well (+A3.01, black circles) or in medium alone (-A3.01, green diamonds) in the absence of DEAE. <b>C: Assessing the sensitivity to antiretrovirals to validate free virus and cell-cell assays.</b> Testing the sensitivity of A3.01-CCR5 infection by free virus (JR-FL NLlucAM firefly luciferase reporter virus; left) and cell-cell (JR-FL NLinGluc transfected 293-T; right) transmission to the RT inhibitor Zidovudine (black circles) or the protease inhibitor Atazanavir (open circles) to verify that both pathways are sensitive to RT inhibition but only cell-cell transmission is affected by protease inhibition. <b>D: NLinGluc (Gaussia) and NLlucAM (firefly) luciferase reporter viruses yield comparable results in free virus inhibition assays.</b> Free virus inhibition of JR-FL NLinGluc (yellow circles) and JR-FL NLlucAM (black circles) reporter viruses by bnAbs VRC01 and 2F5 was compared. For both viruses, 100% infectivity was determined in cultures without inhibitor. <b>E: Equal neutralization sensitivity of free virus infection on A3.01-CCR5 and PBMC target cells.</b> Inhibition of free virus infection of PBMC (blue squares) or A3.01-CCR5 (black circles) by bnAbs VRC01 and 2F5 was studied using the firefly reporter virus JR-FL NLlucAM. For both cell types 100% infectivity was determined in cultures without inhibitor. <b>F: Equal sensitivity to neutralization in 293-T-A3.01-CCR5 and PBMC-PBMC cell-cell transmission.</b> The capacity of bnAbs VRC01 and 2F5 to block cell-cell transmission was assessed in co-cultures of JR-FL NLinGluc-transfected 293-T with A3.01-CCR5 (black circles) and JR-FL infected PBMC with rhTRIM5α-transduced PBMC (blue squares). Infectivity was assessed via determination of Gaussia luciferase activity in the 293-T-A3.01-CCR5 co-cultures or via intracellular p24 staining and flow cytometry analysis for the PBMC co-cultures. For both co-cultures, 100% infectivity was determined in cultures without inhibitor.</p

The capacity to neutralize post-CD4 receptor engagement differs between bnAbs and virus strains.

<p><b>A, B:</b> Schematic of free virus infection of A3.01-CCR5 target cells to measure total and post-attachment activity of bnAbs. <b>C:</b> Total activity of each bnAb-virus combination (where bnAbs were present throughout the infection process) was set to 100% and post-attachment inhibition displayed relative to it. Subtype A, B and C viruses are denoted in blue, black and orange, respectively. Mean values of two to three independent experiments are shown.</p

Decrease of bnAb activity during cell-cell transmission is variable and strain-dependent.

<p><b>A: Diverse pattern of bnAb activity loss within and across viral strains.</b> Inhibition of free virus (black circles) and cell-cell (red circles) transmission of subtype B virus strains PVO.4 and THRO by the indicated bnAbs was studied. The graphs show means and standard error of means (SEM, error bars) of two to three independent experiments performed in duplicates and curve fits to sigmoid dose response curves (variable slope). <b>B, C: Inhibitory activity of bnAbs in free virus and cell-cell transmission of divergent virus subtypes.</b> 50% inhibitory concentrations (IC₅₀ in µg/ml) of bnAbs against a panel of subtype A (blue), B (black) and C (orange) virus strains in free virus <b>(B)</b> and cell-cell <b>(C)</b> transmission. Graphs depict IC₅₀ values of all bnAb-virus pairs for which the virus proved sensitive. Non-sensitive combinations are recorded in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004966#ppat.1004966.s003" target="_blank">S3 Table</a>. Grey bars depict median IC₅₀ values for all virus strains sensitive to a given bnAb. <b>D: bnAbs predominantly lose activity in cell-cell transmission.</b> The change in inhibitory activity in cell-cell transmission compared to free virus inhibition is expressed as the ratio of IC₅₀ cell-cell/IC₅₀ free virus (fold change IC₅₀). Subtype A, B and C viruses are denoted in blue, black and orange, respectively. A fold change IC₅₀ of 0 indicates an equal activity during free virus and cell-cell transmission (black line). Median fold change IC₅₀ (grey bars) for all virus strains sensitive to a given bnAb are shown.</p