Edward P. Browne

Visualizing antibody affinity maturation in germinal centers

Diversity reigns in antibody responses During the course of an immune response, B cells specific for an invading pathogen divide. The antibodies they produce increase in affinity via somatic mutation in specialized lymph node structures called germinal centers. Tas et al. used multiphoton microscopy and sequencing to determine how different B cell clones compete with one another within mouse germinal centers. Multiple B cell clones can seed individual germinal centers, and germinal centers lose diversity at disparate rates. Such heterogeneity suggests that manipulating minor clonal populations to gain an advantage during vaccination may one day be possible. Science, this issue p. 1048 Germinal center B cells are clonally diverse, and the antibodies these cells express mature at different rates. Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with nonimmunodominant specificities must be elicited, as is the case for HIV-1 and influenza.

Toll-like Receptor 7 Controls the Anti-Retroviral Germinal Center Response

The development of vaccines that can enhance immunity to viral pathogens is an important goal. However, the innate molecular pathways that regulate the strength and quality of the immune response remain largely uncharacterized. To define the role of Toll-like receptor (TLR) signaling in control of a model retroviral pathogen, Friend virus (FV), I generated mice in which the TLR signaling adapter Myd88 was selectively deleted in dendritic cell (DC) or in B cell lineages. Deletion of Myd88 in DCs had little effect on immune control of FV, while B cell specific deletion of Myd88 caused a dramatic increase in viral infectious centers and a significantly reduced antibody response, indicating that B cell-intrinsic TLR signaling plays a crucial role, while TLR signaling in DCs is less important. I then identified the single-stranded RNA sensing protein TLR7 as being required for antibody-mediated control of FV by analyzing mice deficient in TLR7. Remarkably, B cells in infected TLR7-deficient mice upregulated CD69 and CD86 early in infection, but failed to develop into germinal center B cells. CD4 T cell responses were also attenuated in the absence of TLR7, but CD8 responses were TLR7 independent, suggesting the existence of additional pathways for detection of retroviral particles. Together these results demonstrate that the vertebrate immune system detects retroviruses in vivo via TLR7 and that this pathway regulates a key checkpoint controlling development of germinal center B cells.

Regulation of B-cell responses by Toll-like receptors

The discovery of host‐encoded gene products that sense molecular patterns in infectious microbes, and the demonstration of their role in triggering innate and adaptive immune responses, has been a key milestone in our understanding of immunology. Twenty‐three years after Janeway first outlined the fundamental concepts of the ‘pattern recognition’ model, and 15 years since the identification of Toll‐like receptors (TLRs) as pattern recognition receptors (PRRs), new insights continue to be revealed, and questions remain. For example, innate immune responses to microbes that are mediated by PRRs have historically been viewed as the domain of innate immune cell populations such as dendritic cells and macrophages. New evidence, however, has pointed to the role of B‐cell‐intrinsic TLR activation in shaping antibody responses. These studies have revealed that TLRs regulate a complex transcriptional network that controls multiple steps in the development of antigen‐specific antibodies. This review covers these recent developments regarding the role of TLRs in B‐cell gene expression and function in vitro and in vivo, and highlights the remaining challenges in the field, with particular emphasis on the role of TLRs in antibody responses to viral infection. A more complete understanding of how TLRs regulate antibody responses will lead to improved vaccine design.

Different modes of retrovirus restriction by human APOBEC3A and APOBEC3G in vivo.

The apolipoprotein B editing complex 3 (A3) cytidine deaminases are among the most highly evolutionarily selected retroviral restriction factors, both in terms of gene copy number and sequence diversity. Primate genomes encode seven A3 genes, and while A3F and 3G are widely recognized as important in the restriction of HIV, the role of the other genes, particularly A3A, is not as clear. Indeed, since human cells can express multiple A3 genes, and because of the lack of an experimentally tractable model, it is difficult to dissect the individual contribution of each gene to virus restriction in vivo. To overcome this problem, we generated human A3A and A3G transgenic mice on a mouse A3 knockout background. Using these mice, we demonstrate that both A3A and A3G restrict infection by murine retroviruses but by different mechanisms: A3G was packaged into virions and caused extensive deamination of the retrovirus genomes while A3A was not packaged and instead restricted infection when expressed in target cells. Additionally, we show that a murine leukemia virus engineered to express HIV Vif overcame the A3G-mediated restriction, thereby creating a novel model for studying the interaction between these proteins. We have thus developed an in vivo system for understanding how human A3 proteins use different modes of restriction, as well as a means for testing therapies that disrupt HIV Vif-A3G interactions.

Toll-Like Receptor 7 Inhibits Early Acute Retroviral Infection through Rapid Lymphocyte Responses

ABSTRACT Early events during retroviral infection play a critical role in determining the course of infection and pathogenesis, but the mechanisms that regulate this phase of infection are poorly understood. Toll-like receptor 7 (TLR7) is required for promoting germinal center reactions and virus-specific neutralizing antibodies at later time points postinfection, but TLR7s role in early acute infection has not been determined. By infecting TLR7-deficient mice with a retroviral pathogen, Friend virus (FV), I determined that TLR7 potently inhibits retroviral replication during the first 5 days of infection and is required for rapid secretion of virus-specific IgM and interleukin-10 (IL-10) in response to infection. Although the IgM response was nonneutralizing, plasmas from wild-type mice but not TLR7-deficient mice inhibited FV replication when passively transferred to infected mice, suggesting an indirect mechanism of antibody function. Interestingly, IL-10 was secreted primarily by CD4 T cells, and IL-10-deficient mice also exhibited accelerated early virus spread, demonstrating that this response inhibits acute infection. Surprisingly, TLR7-deficient mice exhibited normal or elevated secretion of proinflammatory cytokines during acute infection, revealing the existence of a TLR7-independent retrovirus-sensing pathway that drives inflammatory cytokine secretion. Together, these results establish a previously unappreciated role for lymphocytes in mediating rapid TLR7-dependent inhibition of early retroviral infection through nonneutralizing IgM and IL-10.

Removing human immunodeficiency virus (HIV) from human blood using immobilized heparin.

Heparin covalently attached to a water-insoluble resin suspended in HIV-infected aqueous buffer or whole blood captures the virus; subsequent physical separation of the immobilized heparin reduced the viral titers by over 80 and 50%, respectively. The detoxification concept has been validated by both circulating an HIV-1 solution through a column packed with the heparin–sepharose beads and successively mixing an HIV-1 solution with fresh beads.

A Computational Model of Inhibition of HIV-1 by Interferon-Alpha

Type 1 interferons such as interferon-alpha (IFNα) inhibit replication of Human immunodeficiency virus (HIV-1) by upregulating the expression of genes that interfere with specific steps in the viral life cycle. This pathway thus represents a potential target for immune-based therapies that can alter the dynamics of host-virus interactions to benefit the host. To obtain a deeper mechanistic understanding of how IFNα impacts spreading HIV-1 infection, we modeled the interaction of HIV-1 with CD4 T cells and IFNα as a dynamical system. This model was then tested using experimental data from a cell culture model of spreading HIV-1 infection. We found that a model in which IFNα induces reversible cellular states that block both early and late stages of HIV-1 infection, combined with a saturating rate of conversion to these states, was able to successfully fit the experimental dataset. Sensitivity analysis showed that the potency of inhibition by IFNα was particularly dependent on specific network parameters and rate constants. This model will be useful for designing new therapies targeting the IFNα network in HIV-1-infected individuals, as well as potentially serving as a template for understanding the interaction of IFNα with other viruses.

Prediction uncertainty and optimal experimental design for learning dynamical systems.

Dynamical systems are frequently used to model biological systems. When these models are fit to data, it is necessary to ascertain the uncertainty in the model fit. Here, we present prediction deviation, a metric of uncertainty that determines the extent to which observed data have constrained the models predictions. This is accomplished by solving an optimization problem that searches for a pair of models that each provides a good fit for the observed data, yet has maximally different predictions. We develop a method for estimating a priori the impact that additional experiments would have on the prediction deviation, allowing the experimenter to design a set of experiments that would most reduce uncertainty. We use prediction deviation to assess uncertainty in a model of interferon-alpha inhibition of viral infection, and to select a sequence of experiments that reduces this uncertainty. Finally, we prove a theoretical result which shows that prediction deviation provides bounds on the trajectories of the underlying true model. These results show that prediction deviation is a meaningful metric of uncertainty that can be used for optimal experimental design.

An Interleukin-1 Beta-Encoding Retrovirus Exhibits Enhanced Replication In Vivo

ABSTRACT Interleukin-1 beta (IL-1β) is an inflammatory cytokine that is secreted in response to inflammasome activation by innate microbe-sensing pathways. Although some retroviruses can trigger IL-1β secretion through the DNA-sensing molecule IFI16, the effect of IL-1β on the course of infection is unknown. To test whether IL-1β secretion affects retroviral replication in vivo, I constructed a novel murine leukemia virus strain (FMLV-IL-1β) that encodes the mature form of IL-1β. This virus replicated with kinetics similar to that of wild-type virus in tissue culture but caused a dramatically more aggressive infection of both C57BL/6 and BALB/c mice. By 7 days postinfection (PI), mice infected with FMLV-IL-1β exhibited splenomegaly and viral loads 300-fold higher than those in mice infected with wild-type FMLV. Furthermore, the enlarged spleens of FMLV-IL-1β-infected mice correlated with a large expansion of Gr-1+ CD11b+ myeloid-derived suppressor cells, as well as elevated levels of immune activation. Although FMLV-IL-1β infection was controlled by C57BL/6 mice by 14 days p.i., FMLV-IL-1β was able to establish a significant persistent infection and immune activation in BALB/c mice. These results demonstrate that IL-1β secretion is a powerful positive regulator of retroviral infection and that FMLV-IL-1β represents a new model of proinflammatory retroviral infection. IMPORTANCE Interleukin-1 beta (IL-1β) is an inflammatory cytokine released in response to activation of innate pathogen-sensing pathways during microbial infection. To examine the potential impact of IL-1β on retroviral replication in vivo, I constructed a novel mouse retrovirus strain (FMLV-IL-1β) that encodes IL-1β and promotes abundant IL-1β secretion from infected cells. This virus replicates with normal kinetics in cultured cells but displays a dramatically enhanced ability to replicate in mice and caused persistent infection and immune activation in the BALB/c strain of mice. These results establish IL-1β as a positive regulator of retroviral replication and suggest that targeting this pathway may have therapeutic benefits in infections with proinflammatory retroviruses. This virus can also be used to further study the impact of inflammatory pathways on retroviral infection.

The effect of HIV-1 Vif polymorphisms on A3G anti-viral activity in an in vivo mouse model

Humans encode seven APOBEC3 proteins (A–H), with A3G, 3F and 3H as the major factors restricting HIV-1 replication. HIV-1, however, encodes Vif, which counteracts A3 proteins by chaperoning them to the proteasome where they are degraded. Vif polymorphisms found in HIV-1s isolated from infected patients have varying anti-A3G potency when assayed in vitro, but there are few studies demonstrating this in in vivo models. Here, we created Friend murine leukemia viruses encoding vif alleles that were previously shown to differentially neutralize A3G in cell culture or that were originally found in primary HIV-1 isolates. Infection of transgenic mice expressing different levels of human A3G showed that these naturally occurring Vif variants differed in their ability to counteract A3G during in vivo infection, although the effects on viral replication were not identical to those seen in cultured cells. We also found that the polymorphic Vifs that attenuated viral replication reverted to wild type only in A3G transgenic mice. Finally, we found that the level of A3G-mediated deamination was inversely correlated with the level of viral replication. This animal model should be useful for studying the functional significance of naturally occurring vif polymorphisms, as well as viral evolution in the presence of A3G.