The first immunization in a protein prime-boost vaccination is likely to be critical for how the immune response unfolds. Using fine needle aspirates (FNAs) of draining lymph nodes (LNs), we tracked ...the kinetics of the primary immune response in rhesus monkeys immunized intramuscularly (IM) or subcutaneously (s.c.) with an eOD-GT8 60-mer nanoparticle immunogen to facilitate clinical trial design. Significant numbers of germinal center B (BGC) cells and antigen-specific CD4 T cells were detectable in the draining LN as early as 7 days post-immunization and peaked near day 21. Strikingly, s.c. immunization results in 10-fold larger antigen-specific BGC cell responses compared to IM immunization. Lymphatic drainage studies revealed that s.c. immunization resulted in faster and more consistent axillary LN drainage than IM immunization. These data indicate robust antigen-specific germinal center responses can occur rapidly to a single immunization with a nanoparticle immunogen and vaccine drainage substantially impacts immune responses in local LNs.
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•Rapid immunogen-specific lymph node responses detected at day 7 post-immunization•Lymph node immune response kinetics can be longitudinally tracked•Greater germinal center responses after subcutaneous versus intramuscular immunization•Immunization injection method substantially impacts immune responses in local LNs
The first immunization of protein prime-boost vaccination is likely critical but has been understudied in large animals and humans. Havenar-Daughton et al. use lymph node fine needle aspirates to determine primary germinal center response kinetics in rhesus monkeys immunized intramuscularly or subcutaneously with a clinical trial candidate nanoparticle immunogen.
Following immunization, high-affinity antibody responses develop within germinal centers (GCs), specialized sites within follicles of the lymph node (LN) where B cells proliferate and undergo somatic ...hypermutation. Antigen availability within GCs is important, as B cells must acquire and present antigen to follicular helper T cells to drive this process. However, recombinant protein immunogens such as soluble human immunodeficiency virus (HIV) envelope (Env) trimers do not efficiently accumulate in follicles following traditional immunization. Here, we demonstrate two strategies to concentrate HIV Env immunogens in follicles, via the formation of immune complexes (ICs) or by employing self-assembling protein nanoparticles for multivalent display of Env antigens. Using rhesus macaques, we show that within a few days following immunization, free trimers were present in a diffuse pattern in draining LNs, while trimer ICs and Env nanoparticles accumulated in B cell follicles. Whole LN imaging strikingly revealed that ICs and trimer nanoparticles concentrated in as many as 500 follicles in a single LN within two days after immunization. Imaging of LNs collected seven days postimmunization showed that Env nanoparticles persisted on follicular dendritic cells in the light zone of nascent GCs. These findings suggest that the form of antigen administered in vaccination can dramatically impact localization in lymphoid tissues and provides a new rationale for the enhanced immune responses observed following immunization with ICs or nanoparticles.
Conventional immunization strategies will likely be insufficient for the development of a broadly neutralizing antibody (bnAb) vaccine for HIV or other difficult pathogens because of the ...immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. We found that two independent methods of slow delivery immunization of rhesus monkeys (RMs) resulted in more robust T follicular helper (TFH) cell responses and GC B cells with improved Env-binding, tracked by longitudinal fine needle aspirates. Improved GCs correlated with the development of >20-fold higher titers of autologous nAbs. Using a new RM genomic immunoglobulin locus reference, we identified differential IgV gene use between immunization modalities. Ab mapping demonstrated targeting of immunodominant non-neutralizing epitopes by conventional bolus-immunized animals, whereas slow delivery-immunized animals targeted a more diverse set of epitopes. Thus, alternative immunization strategies can enhance nAb development by altering GCs and modulating the immunodominance of non-neutralizing epitopes.
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•Slow delivery immunization enhances HIV neutralizing antibody development in monkeys•Slow delivery immunization alters immunodominance of the responding B cells•Weekly longitudinal germinal center (GC) B and TFH analyses provides new GC insights•High-resolution rhesus immunoglobulin locus genomic reference sequence
An integrated immunological, bioinformatic and imaging approach demonstrates how slow delivery immunization enhances neutralizing antibody and germinal center reactions over conventional strategies in response to HIV Env protein immunization in non-human primates.
A major barrier to human immunodeficiency virus (HIV) eradication is the long-term persistence of latently infected CD4
T cells harboring integrated replication-competent virus. It has been proposed ...that the homeostatic proliferation of these cells drives long-term reservoir persistence in the absence of virus reactivation, thus avoiding cell death due to either virus-mediated cytopathicity or immune effector mechanisms. Here, we conducted an experimental depletion of CD4
T cells in eight antiretroviral therapy (ART)-treated, simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs) to determine whether the homeostatically driven CD4
T-cell proliferation that follows CD4
T-cell depletion results in reactivation of latent virus and/or expansion of the virus reservoir. After administration of the CD4R1 antibody, we observed a CD4
T cell depletion of 65 to 89% in peripheral blood and 20 to 50% in lymph nodes, followed by a significant increase in CD4
T cell proliferation during CD4
T cell reconstitution. However, this CD4
T cell proliferation was not associated with detectable increases in viremia, indicating that the homeostatic activation of CD4
T cells is not sufficient to induce virus reactivation from latently infected cells. Interestingly, the homeostatic reconstitution of the CD4
T cell pool was not associated with significant changes in the number of circulating cells harboring SIV DNA compared to results for the first postdepletion time point. This study indicates that, in ART-treated SIV-infected RMs, the homeostasis-driven CD4
T-cell proliferation that follows experimental CD4
T-cell depletion occurs in the absence of detectable reactivation of latent virus and does not increase the size of the virus reservoir as measured in circulating cells.
Despite successful suppression of HIV replication with antiretroviral therapy, current treatments are unable to eradicate the latent virus reservoir, and treatment interruption almost invariably results in the reactivation of HIV even after decades of virus suppression. Homeostatic proliferation of latently infected cells is one mechanism that could maintain the latent reservoir. To understand the impact of homeostatic mechanisms on virus reactivation and reservoir size, we experimentally depleted CD4
T cells in ART-treated SIV-infected rhesus macaques and monitored their homeostatic rebound. We find that depletion-induced proliferation of CD4
T cells is insufficient to reactivate the viral reservoir
Furthermore, the proportion of SIV DNA
CD4
T cells remains unchanged during reconstitution, suggesting that the reservoir is resistant to this mechanism of expansion at least in this experimental system. Understanding how T cell homeostasis impacts latent reservoir longevity could lead to the development of new treatment paradigms aimed at curing HIV infection.
Following immunization, high-affinity antibody responses develop within germinal centers (GCs), specialized sites within follicles of the lymph node (LN) where B cells proliferate and undergo somatic ...hypermutation. Antigen availability within GCs is important, as B cells must acquire and present antigen to follicular helper T cells to drive this process. However, recombinant protein immunogens such as soluble human immunodeficiency virus (HIV) envelope (Env) trimers do not efficiently accumulate in follicles following traditional immunization. Here, we demonstrate two strategies to concentrate HIV Env immunogens in follicles, via the formation of immune complexes (ICs) or by employing self-assembling protein nanoparticles for multivalent display of Env antigens. Using rhesus macaques, we show that within a few days following immunization, free trimers were present in a diffuse pattern in draining LNs, while trimer ICs and Env nanoparticles accumulated in B cell follicles. Whole LN imaging strikingly revealed that ICs and trimer nanoparticles concentrated in as many as 500 follicles in a single LN within two days after immunization. Imaging of LNs collected seven days postimmunization showed that Env nanoparticles persisted on follicular dendritic cells in the light zone of nascent GCs. These findings suggest that the form of antigen administered in vaccination can dramatically impact localization in lymphoid tissues and provides a new rationale for the enhanced immune responses observed following immunization with ICs or nanoparticles.
The translocator protein 18kDa (TSPO) is expressed in the outer mitochondrial membrane and is implicated in several functions, including cholesterol transport and steroidogenesis. Under normal ...physiological conditions, TSPO is present in very low concentrations in the human brain but is markedly upregulated in response to brain injury and inflammation. This upregulation is strongly associated with activated microglia. Therefore, TSPO is particularly suited for assessing active gliosis associated with brain lesions following injury or disease. For over three decades, TSPO has been studied as a biomarker. Numerous radioligands for positron emission tomography (PET) that target TSPO have been developed for imaging inflammatory progression in the brain. Although
CPK11195, the prototypical first-generation PET radioligand, is still widely used for in vivo studies, mainly now as its single more potent R-enantiomer, it has severe limitations, including low sensitivity and poor amenability to quantification. Second-generation radioligands are characterized by higher TSPO specific signals but suffer from other drawbacks, such as sensitivity to the TSPO single nucleotide polymorphism (SNP) rs6971. Therefore, their applications in human studies have the burden of needing to genotype subjects. Consequently, recent efforts are focused on developing improved radioligands that combine the optimal features of the second generation with the ability to overcome the differences in binding affinities across the population. This review presents essential principles in the design and development of TSPO PET ligands and discusses prominent examples among the main chemotypes.
In recent years, indolylglyoxylamide-based derivatives have received much attention due to their application in drug design and discovery, leading to the development of a wide array of compounds that ...have shown a variety of pharmacological activities. Combining the indole nucleus, already validated as a "privileged structure," with the glyoxylamide function allowed for an excellent template to be obtained that is suitable to a great number of structural modifications aimed at permitting interaction with specific molecular targets and producing desirable therapeutic effects. The present review provides insight into how medicinal chemists have elegantly exploited the indolylglyoxylamide moiety to obtain potentially useful drugs, with a particular focus on compounds exhibiting activity in in vivo models or reaching clinical trials. All in all, this information provides exciting new perspectives on existing data that can be useful in further design of indolylglyoxylamide-based molecules with interesting pharmacological profiles. The aim of this report is to present an update of collection data dealing with the employment of this moiety in the rational design of compounds that are able to interact with a specific target, referring to the last 20 years.
Correctly identifying and mapping the distribution patterns of passively dispersing freshwater organisms is an important task to understand the mechanisms through which these organisms move across ...the world and to counteract the introduction of invasive alien species in a timely way; moreover, in the light of the growing evidence of the pervasiveness of cryptic species and cryptic biological invasions among all animal groups, the use of molecular identification tools is strongly advisable. In this context, we present the first record of the freshwater jellyfish
Craspedacusta sowerbii
Lankester, 1880 in the Mediterranean island of Sicily, Italy. This invasive species, native to China, was found during a routine survey campaign aimed at assessing the freshwater species richness in the Sicilian region. Individuals were collected in November 2017 and identified to species level through the sequencing of fragments of both mitochondrial and nuclear genes, and their comparison to the sequences available on public databases. The analysis of molecular data proved that the Sicilian population belongs to
C. sowerbii
s.s., i.e. one of the two major
Craspedacusta
lineages which have been discovered to have invaded Europe and which were until recently erroneously jointly reported under the binomen
Craspedacusta sowerbii
. In order to get a clearer picture of the history and patterns of freshwater jellyfish invasion worldwide, the need for molecular identification of all the non-native
Craspedacusta
populations currently reported as
C. sowerbii
, and the opportunity of monitoring human-impacted ecosystems is stressed.
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