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Keystone Symposium on HIV Vaccines: Adaptive Immunity and Beyond

The Keystone Symposium on HIV Vaccines: Adaptive Immunity and Beyond was held in Banff, Canada on 9-14 March, 2014.  The meeting’s main focus was the coordinated action of the humoral, cellular, and innate arms of the immune system in response to HIV vaccines or infection.  We bring you several stories that made a splash at the meeting and were discussed both in the conference center hallways and on the skiing lifts.

The game of tag and read

Next-generation sequencing of antibody genes is a powerful tool for assessing antibody repertoires and tracking how they change over time in response to infection or vaccination.  Dr. William Robinson from Stanford and his biotech company Atreca developed a technique that allows them to bar-code the cDNA on a single cell level before proceeding to the sequencing step.  This technique overcomes a number of shortcomings of the current technology.  Because barcodes change every day, it allows easy detection of contamination between runs.  It also allows correcting for amplification biases, when some genes are PCR-amplified more effectively than others distorting the relative frequencies of antibody variants in the sample.  Finally, because unique barcodes are used for each cell, this allows matching heavy and light chains of antibodies easily and, therefore, provides a more complete picture of the antibody landscape while greatly facilitating antibody cloning.  Dr. Robinson’s group is applying this technique to study antibody responses to various bacterial and viral infections, including HIV, where he is collaborating with the Ragon Institute to study antibody repertoires in elite controllers.

Lymph nodes: Special delivery

Kelly Moynihan from the Irvine lab at MIT described a novel approach to improve immunogenicity of peptide-based vaccines.  The approach is based on an observation that certain molecules, such as the indocyanine green dye, are effectively targeted to lymph nodes via binding to albumin and deposited on antigen-presenting cells.  The researchers screened for albumin binding a number of synthetic compounds that combined an albumin binding lipophilic tail with CpG oligonucleotides for improved solubility and adjuvant properties.  Lipo-CpG was identified as a promising candidate in mice as it accumulates in lymph nodes and remains there for more than 24 hours allowing the candidate molecule to retain CpG’s adjuvanting properties – enhanced antibody responses to ovalbumin, as well as CD8 T cell responses to SIV gag.  Importantly, fusing CpG to the carrier reduced the systemic inflammation that normally occurs after CpG administration and this effect was especially noticeable after multiple injections.  Moynihan and colleagues used a similar strategy, adding a diacyl tail via a PEG spacer, to target peptides to the lymph nodes.  As expected, this modification markedly improved immunogenicity as measured by CD8 T cell responses and by tumor reduction in a mouse model of cervical cancer (Liu, Nature, 2014).  This and similar targeting approaches might result in vaccines that are safer and more immunogenic.

The mysterious case of a shape-shifting virus

Dr. Mario Roederer from NIH’s Vaccine Research Center reported an interesting discovery that some SIV molecular clones existing in two antigenically distinct forms (Roederer, Nature, 2014).  The amino acids responsible for the phenotype were discovered during a sieve analysis of breakthrough viruses in macaques vaccinated with DNA/rAD5 and challenged with SIVsmE660.  In vitro neutralization assays showed that virions produced by a molecular clone carrying the mutation drastically differ in their neutralization sensitivity: approximately half of them have the regular Tier 1 neutralization phenotype, while the other half are extremely resistant and could not be neutralized even at the lowest dilution of sera from vaccinated macaques or with monoclonal antibodies to SIV Env.  They were, however, sensitive to soluble CD4 and CD4-Ig.  It’s not yet clear what causes this dichotomous phenotype, but Dr. Roederer proposes that rare protein folding or posttranslational modifications may play a role.  He also draws attention to the fact that envelope proteins produced from a molecular clone may in fact exist in very distinct forms, which has implications for immunogen design in HIV vaccines.

How MHC gets its peptides

It has been well established that MHC class I proteins are ubiquitously expressed in tissues and present peptides from self and foreign (pathogen) gene products for immune surveillance.  However, the mechanisms used to generate representative peptides of the entire self/non-self are not completely clear.  If class I molecules were loaded by peptides in proportion to the amounts of protein being synthesized at any given time, then high abundance proteins (e.g. chaperones, histones, ribosomes) would be predominantly presented to the immune system.  To circumvent the law of mass action and to ensure that each potential motif has equal ‘vote’ in the immune surveillance system, a more representative process needs to be in place.  Dr. Jonathan Yewdell, from NIAID, suggests that Defective Ribosomal Products (DRIPs) are the source of a more diverse panel of peptides for immune surveillance, both through canonical translation in the cytoplasm and non-canonical translation in the nucleus, potentially an important source of DRiPs (Apcher, PNAS, 2013).  Recent studies confirm that production of DRIPs is critical for both viral (Croft, PLoS, 2013), and self (Bourdetsky, PNAS, 2014) peptide presentation.  In this model, translation is compartmentalized to enable peptide generation from individual mRNAs in the absence of bulk competition from cytoplasmic peptides (Lev, PNAS, 2010).  Another benefit of the model is that it enables generation of stable clusters of class I molecules loaded with the same peptide to enhance the sensitivity of T cell recognition  (Lu, PNAS, 2012).

Paneth cells make a debut in SIV pathogenesis

Alterations of gastro-intestinal mucosal barriers, accompanied by pathogen translocation and on-going immune activation, are believed to be the cause of sustained inflammation during HIV or SIV infection.  Several presentations focused on the examination of mucosal tissues during HIV or SIV infection and the associated events leading to immune dysfunction.

Dr. Satya Dandekar’s lab in UCLA is using a ligated intestinal loop model in Rhesus Macaques (RM) to focus on the very early stages of SIV infection in the gut mucosa, the consequences for later responses to microbiota and pathogens, and the adaptation to chronic infection.  In a ligated loop model, a section of an intestine in a live animal is isolated from the rest of the intestine with silk ligatures, which allows application of a test solution specifically to that region.  A neighboring section of an intestine injected with a saline may be used as a control.  The advantage of the ligated intestinal loops is that it preserves all cell types including cells that cannot be maintained in tissue culture, and allow in vivo host-pathogen interactions in real time.  To observe very early infection events, RMs were injected with SIV and intestinal loop were harvested 2.5 days post infection, a time when mucosal CD4+T cells are not yet depleted and viral load is low, corresponding to a window of acute infection.  In this acute group, SIV infection led to induction of chemokines involved in monocyte trafficking and inflammatory cytokines as well as a strong up-regulation of IL-1 beta production.  The source of IL-1 beta was attributed to a specialized cell type in intestinal crypts: the Paneth cells.  Paneth cells are known to express TLRs and are involved in sensing and responding to pathogens through production of defensins and anti-microbial peptides. They also participate in the maintenance of the epithelial stem cell niches.  This is the first time that these cells have been shown to have a role in SIV pathogenesis and to be a source of IL-1 beta.  Since IL-1 beta has been shown to regulate IL17 production and the polarization of TH17 cells, this effect reveals very early interactions of epithelial and immune cells in SIV pathogenesis.

Intestinal samples were also collected from RMs at 10 weeks post SIV infection, corresponding to a chronic infection stage.  In these samples CD4 T cells were greatly depleted, IFN gamma responses were dominant, and the main source of IL-1 beta was no longer Planeth cells, but macrophages.

The same intestinal model was used to test the gut immune responses to pathogenic and commensal bacteria in the context of acute SIV infection in RMs.  Intestinal loops of acutely infected RMs and controls were inoculated with either Salmonella typhimurium (a pathogenic bacterial strain) or Lactobacillus plantarum (a non-pathogenic strain) and samples were captured 5 hours after inoculation.  At this early stage, mucosal responses to Salmonella were not significantly altered in SIV-infected animals as compared to control animals.  In contrast, the commensal Lactobacilli induced an abnormal and strong recruitment of IL17+ CD4 cells (Th17) in intestinal loops of SIV+ animals.  These results clearly show that SIV has an early effect on immune homeostasis in the intestine.

In conclusion, Paneth cells are involved in the very early events of SIV infection, and the infection quickly leads to changes in the intestinal immune system, including responses to commensal bacteria.

Tfh cells are free to come and go

Dr. Gabriel Victora, from the Whitehead institute at MIT, investigated the cellular interactions between B and Tfh cells and the movement of these cells within and outside of GCs (Shulman, Science, 2013).  Using a variety of approaches, he and colleagues found that:

  • Tfh cells control high-affinity B cell selection even before the cells form GCs. Few pre-immune B cell access GCs and then are further selected in the GCs.  There is a harsh competition for Tfh cells binding and activation.
  • Unlike B cells, which come from 1-3 initial clones in a single germinal center, Tfh in GCs are not clonally restricted.
  • Tfh cells are not limited to one GC, but can circulate to different GCs within lymph nodes; migrating T cells are CXCR5high PD1+.
  • Newly activated T cells can enter established GCs and possibly influence ongoing B cell selection and differentiation and enhance the maturation of high-affinity antibodies.

The improved understanding of the interplay between B and T cells in germinal centers can greatly inform future vaccine design, especially as the researchers will try to focus antibody affinity maturation on specific sites of the virus using sequential immunizations with rationally-designed immunogens.

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