Directing an antigen to the endoplasmic reticulum (ER) improves the antigen-specific immune response, revealing a potentially useful strategy in cancer immunotherapy using tumor-associated antigens (TAAs). This can be achieved by fusing the antigen to an ER chaperone protein, such as calreticulin (CRT). We previously reported the antitumor response by fusing the CRT signal peptide (SP) and its ER retention sequence (KDEL) to full-length human papillomavirus type 16 (HPV-16) E6 and E7 antigens, obtaining a potent antitumoral effect. In this article, we compare the antitumor response due to the use of each signal (SP and/or KDEL) fused to HPV16 E6 and E7 antigens in a DNA vaccination model. Using both SP and KDEL signals promotes higher interferon (IFN)-γ production and a faster antitumor response than using only the SP, resulting in better tumor growth restraint and higher survival, indicating that the KDEL addition to an ER-directed antigen helps by shortening the time to response. Meanwhile, antigens without signals or only the KDEL signal showed no induction of antigen-specific IFN-γ or antitumor response. Our results indicate that directing the E6E7m antigen to the ER by the SP signal is sufficient to promote an efficient antitumor response. Importantly, this effect is stronger and faster when the antigen also has an ER retention sequence, such as the KDEL signal.
Population‐based immunoglobulin G (IgG) seroprevalence studies in asymptomatic individuals in Latin America are scarce. The objective of the study was to estimate the prevalence and geographic distribution of IgG antibodies induced by natural SARS‐CoV‐2 infection in asymptomatic adults, 5–8 months after the first case was reported in a northeastern state of Mexico. This was a population‐based cross‐sectional study carried out in Nuevo Leon during August–November 2020. Individuals ≥18 years with no previous diagnosis or symptoms suggestive of COVID‐19 were consecutively screened in one of the busiest subway stations. Also, a search for eligible individuals was done from house‐to‐house, after selecting densely populated geographic sectors of each of the municipalities of the metropolitan area ( n = 4495). The IgG antibodies to SARS‐CoV‐2 nucleocapsid protein were analyzed. The IgG antibody positivity rate was 27.1% (95% confidence interval [CI]: 25.8, 28.4); there were no differences by sex or age ( p > 0.05). Analysis by month showed a gradual increase from 11.9% (August) to 31.9% (November); Week 39 had the highest positivity rate (42.2%, 95% CI: 34.2, 50.7). Most people did not have evidence of previous SARS‐CoV‐2 infection. Preventive measures and promotion of the COVID‐19 vaccine should be strengthened.
<abstract> <p>The pH Low Insertion Peptide (pHLIP) has versatile applications in several diseases due to its differential behavior at slightly different pH values. pHLIP is an unstructured and peripheral membrane-associated peptide at neutral pH and an α-helical transmembrane peptide at acidic values. Similar to what happened to insulin and growth hormone, pHLIP´s expanding applications require high-yield production to further scale-up its usefulness. To date, synthesis of the pHLIP has not been reported in a prokaryotic platform, mainly relying on solid-phase synthesis. Bacterial production arises as an option for high-amount peptide generation and larger pHLIP fusion protein-synthesis; however, cell-based pH-responsive peptide production could be challenging due to intracellular peptide interactions or degradation due to unstructured conformations. An <italic>Escherichia coli</italic> (E. coli)-BL21 cell culture was induced with Isopropyl ß-D-1-thiogalactopyranoside (IPTG) in order to produce a Glutathione S-transferase-pHLIP (GST-pHLIP) fusion construct. Purification was done with Glutathione (GSH)-decorated magnetic beads using 4 ml of the induced cell culture. The production was quantified with Bradford reagent and characterized with SDS-PAGE and Western blot, contrasting Bradford results with densitometry analysis to obtain production approximate absolute values. A purified approximate total yield of ~26 µg with an apparent GSH-bead saturation and a total production of ~82 µg was obtained. Our Western Blot assay confirmed the presence of the GST-pHLIP construct in all the IPTG-induced fractions. Conclusion: A high-yield pHLIP production irrespective of its membrane affinity in acidic environments or its unstructured nature was achieved. Our study could be useful to scale up pHLIP synthesis for future applications.</p> </abstract>
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