Precursor Frequency and Affinity Determine B Cell Competitive Fitness in Germinal Centers, Tested with Germline-Targeting HIV Vaccine Immunogens.

How precursor frequencies and antigen affinities impact interclonal B cell competition is a particularly relevant issue for candidate germline-targeting HIV vaccine designs because of the in vivo rarity of naive B cells that recognize broadly neutralizing epitopes. Knowing the frequencies and affinities of HIV-specific VRC01-class naive human B cells, we transferred B cells with germline VRC01 B cell receptors into congenic recipients to elucidate the roles of precursor frequency, antigen affinity, and avidity on B cell responses following immunization. All three factors were interdependently limiting for competitive success of VRC01-class B cells. In physiological high-affinity conditions using a multivalent immunogen, rare VRC01-class B cells successfully competed in germinal centers (GC), underwent extensive somatic hypermutation, and differentiated into memory B cells. The data reveal dominant influences of precursor frequency, affinity, and avidity for interclonal GC competition and indicate that germline-targeting immunogens can overcome these challenges with high-affinity multimeric designs.

B Cells Carrying Antigen Receptors Against Microbes as Tools for Vaccine Discovery and Design.

Can basic science improve the art of vaccinology? Here, we review efforts to understand immune responses with the aim to improve vaccine design and, eventually, to predict the efficacy of human vaccine candidates using the tools of transformed B cells and targeted transgenic mice carrying B cells with antigen receptors specific for microbes of interest.

Glutamine mimicry suppresses tumor progression through asparagine metabolism in pancreatic ductal adenocarcinoma.

In pancreatic ductal adenocarcinoma (PDAC), glutamine is a critical nutrient that drives a wide array of metabolic and biosynthetic processes that support tumor growth. Here, we elucidate how 6-diazo-5-oxo-L-norleucine (DON), a glutamine antagonist that broadly inhibits glutamine metabolism, blocks PDAC tumor growth and metastasis. We find that DON significantly reduces asparagine production by inhibiting asparagine synthetase (ASNS), and that the effects of DON are rescued by asparagine. As a metabolic adaptation, PDAC cells upregulate ASNS expression in response to DON, and we show that ASNS levels are inversely correlated with DON efficacy. We also show that L-asparaginase (ASNase) synergizes with DON to affect the viability of PDAC cells, and that DON and ASNase combination therapy has a significant impact on metastasis. These results shed light on the mechanisms that drive the effects of glutamine mimicry and point to the utility of cotargeting adaptive responses to control PDAC progression.

Stopping the fat: Repurposing an antidepressant for cancer treatment.

In this issue of JEM, Chu and An et al. (2022. J. Exp. Med.https://doi.org/10.1084/jem.20221316) describe the role of the tricyclic antidepressant nortriptyline in inhibition of fatty acid uptake. Nortriptyline promotes cell acidification and suppresses macropinocytosis, providing a link between fatty acid uptake and tumor progression.

HIV-1 VACCINES. Priming a broadly neutralizing antibody response to HIV-1 using a germline-targeting immunogen.

A major goal of HIV-1 vaccine research is the design of immunogens capable of inducing broadly neutralizing antibodies (bnAbs) that bind to the viral envelope glycoprotein (Env). Poor binding of Env to unmutated precursors of bnAbs, including those of the VRC01 class, appears to be a major problem for bnAb induction. We engineered an immunogen that binds to VRC01-class bnAb precursors and immunized knock-in mice expressing germline-reverted VRC01 heavy chains. Induced antibodies showed characteristics of VRC01-class bnAbs, including a short CDRL3 (light-chain complementarity-determining region 3) and mutations that favored binding to near-native HIV-1 gp120 constructs. In contrast, native-like immunogens failed to activate VRC01-class precursors. The results suggest that rational epitope design can prime rare B cell precursors for affinity maturation to desired targets.

Cytoplasmic translocation of polypyrimidine tract-binding protein and its binding to viral RNA during Japanese encephalitis virus infection inhibits virus replication.

Japanese encephalitis virus (JEV) has a single-stranded, positive-sense RNA genome containing a single open reading frame flanked by the 5'- and 3'-non-coding regions (NCRs). The virus genome replicates via a negative-sense RNA intermediate. The NCRs and their complementary sequences in the negative-sense RNA are the sites for assembly of the RNA replicase complex thereby regulating the RNA synthesis and virus replication. In this study, we show that the 55-kDa polypyrimidine tract-binding protein (PTB) interacts in vitro with both the 5'-NCR of the positive-sense genomic RNA--5NCR(+), and its complementary sequence in the negative-sense replication intermediate RNA--3NCR(-). The interaction of viral RNA with PTB was validated in infected cells by JEV RNA co-immunoprecipitation and JEV RNA-PTB colocalization experiments. Interestingly, we observed phosphorylation-coupled translocation of nuclear PTB to cytoplasmic foci that co-localized with JEV RNA early during JEV infection. Our studies employing the PTB silencing and over-expression in cultured cells established an inhibitory role of PTB in JEV replication. Using RNA-protein binding assay we show that PTB competitively inhibits association of JEV 3NCR(-) RNA with viral RNA-dependent RNA polymerase (NS5 protein), an event required for the synthesis of the plus-sense genomic RNA. cAMP is known to promote the Protein kinase A (PKA)-mediated PTB phosphorylation. We show that cells treated with a cAMP analogue had an enhanced level of phosphorylated PTB in the cytoplasm and a significantly suppressed JEV replication. Data presented here show a novel, cAMP-induced, PTB-mediated, innate host response that could effectively suppress JEV replication in mammalian cells.

La protein can simultaneously bind to both 3'- and 5'-noncoding regions of Japanese encephalitis virus genome.

Japanese encephalitis virus (JEV) genome is a single-stranded, positive-sense RNA with noncoding regions (NCRs) of 95 and 585 bases at its 5' and 3' ends, respectively. These may interact with viral or host proteins important for viral replication. We have previously shown that La protein binds the 3'-stem-loop (SL) structure of JEV 3'-NCR. Using electrophoretic mobility shift and ultraviolet crosslinking assays, we now show that La protein binds both 3'-SL and 5'-NCR of JEV. La protein binding to 5'-NCR RNA was stable under high salt condition (300 mM KCl) and the affinity of RNA protein interaction was high; the dissociation constant (K(d)) for La binding with 5'-NCR RNA was 8.8 nM, indicating the physiological relevance of the interaction. RNA toe-printing assays showed that La protein interacted with nucleotides located in the top loop of the predicted structure of 5'-NCR RNA. Using competitive binding studies and 5'-3' coprecipitation assay, we have demonstrated that La protein could simultaneously bind both JEV 3'- and 5'-NCRs. This may help circularize the viral genome for its efficient transcription and translation.