Corrigendum: Phenotypic alteration of low-density granulocytes in people with pulmonary post-acute sequelae of SARS-CoV-2 infection.

[This corrects the article DOI: 10.3389/fimmu.2022.1076724.].

Characterization of Circulating Fibrocytes in People Living with HIV on Stable Antiretroviral Therapy.

Highly effective combination antiretroviral therapy has reduced HIV infection to a manageable chronic disease, shifting the clinical landscape toward management of noninfectious comorbidities in people living with HIV (PLWH). These comorbidities are diverse, generally associated with accelerated aging, and present within multiple organ systems. Mechanistically, immune dysregulation and chronic inflammation, both of which persist in PLWH with well-controlled virally suppressive HIV infection, are suggested to create and exacerbate noninfectious comorbidity development. Persistent inflammation often leads to fibrosis, which is the common end point pathologic feature associated with most comorbidities. Fibrocytes are bone marrow-derived fibroblast-like cells, which emerged as key effector cells in tissue repair and pathologic fibrotic diseases. Despite their relevance to fibrosis, the circulating fibrocyte concentration in PLWH remains poorly characterized, and an understanding of their functional role in chronic HIV is limited. In this study, utilizing PBMCs from a cross-sectional adult HIV cohort study with matched uninfected controls (HIV-), we aimed to identify and compare circulating fibrocytes in blood. Both the percentage and number of fibrocytes and α-smooth muscle actin+ fibrocytes in circulation did not differ between the HIV+ and HIV- groups. However, circulating fibrocyte levels were significantly associated with increasing age in both the HIV+ and HIV- groups (the percentage and number; r = 0.575, p ≤ 0.0001 and r = 0.558, p ≤ 0.0001, respectively). Our study demonstrates that circulating fibrocyte levels and their fibroblast-like phenotype defined as collagen I and α-smooth muscle actin+ expression are comparable between, and strongly associated with, age irrespective of HIV status.

Pattern recognition receptor ligand-induced differentiation of human transitional B cells.

B cells represent a critical component of the adaptive immune response whose development and differentiation are determined by antigen-dependent and antigen-independent interactions. In this study, we explored the effects of IL-4 and pattern-recognition receptor (PRR) ligands on B cell development and differentiation by investigating their capacity to drive the in vitro maturation of human transitional B cells. In the presence of IL-4, ligands for TLR7/8, TLR9, and NOD1 were effective in driving the in vitro maturation of cord blood transitional B cells into mature, naïve B cells as measured by CD23 expression, ABCB1 transporter activation and upregulation of sIgM and sIgD. In addition, several stimulation conditions, including TLR9 ligand alone, favored an expansion of CD27+ IgM memory B cells. Transitional B cells stimulated with TLR7/8 ligand + IL-4 or TLR9 ligand, with or without IL-4, induced a significant subpopulation of CD23+CD27+ B cells expressing high levels of sIgM and sIgD, a minor B cell subpopulation found in human peripheral blood. These studies illustrate the heterogeneity of the B cell populations induced by cytokine and PRR ligand stimulation. A comparison of transitional and mature, naïve B cells transcriptomes to identify novel genes involved in B cell maturation revealed that mature, naïve B cells were less transcriptionally active than transitional B cells. Nevertheless, a subset of differentially expressed genes in mature, naïve B cells was identified including genes associated with the IL-4 signaling pathway, PI3K signaling in B lymphocytes, the NF-κB signaling pathway, and the TNFR superfamily. When transitional B cells were stimulated in vitro with IL-4 and PRR ligands, gene expression was found to be dependent on the nature of the stimulants, suggesting that exposure to these stimulants may alter the developmental fate of transitional B cells. The influence of IL-4 and PRR signaling on transitional B cell maturation illustrates the potential synergy that may be achieved when certain PRR ligands are incorporated as adjuvants in vaccine formulations and presented to developing B cells in the context of an inflammatory cytokine environment. These studies demonstrate the potential of the PRR ligands to drive transitional B cell differentiation in the periphery during infection or vaccination independently of antigen mediated BCR signaling.

Phenotypic alteration of low-density granulocytes in people with pulmonary post-acute sequalae of SARS-CoV-2 infection.

Background: Low-density granulocytes (LDGs) are a distinct subset of neutrophils whose increased abundance is associated with the severity of COVID-19. However, the long-term effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on LDG levels and phenotypic alteration remain unexplored. Methods: Using participants naïve to SARS-CoV-2 (NP), infected with SARS-CoV-2 with no residual symptoms (NRS), and infected with SARS-CoV-2 with chronic pulmonary symptoms (PPASC), we compared LDG levels and their phenotype by measuring the expression of markers for activation, maturation, and neutrophil extracellular trap (NET) formation using flow cytometry. Results: The number of LDGs was elevated in PPASC compared to NP. Individuals infected with SARS-CoV-2 (NRS and PPASC) demonstrated increased CD10+ and CD16hi subset counts of LDGs compared to NP group. Further characterization of LDGs demonstrated that LDGs from COVID-19 convalescents (PPASC and NRS) displayed increased markers of NET forming ability and aggregation with platelets compared to LDGs from NP, but no differences were observed between PPASC and NRS. Conclusions: Our data from a small cohort study demonstrates that mature neutrophils with a heightened activation phenotype remain in circulation long after initial SARS-CoV-2 infection. Persistent elevation of markers for neutrophil activation and NET formation on LDGs, as well as an enhanced proclivity for platelet-neutrophil aggregation (PNA) formation in COVID-19 convalescent individuals may be associated with PPASC prognosis and development.

Building a Diverse Workforce and Thinkforce to Reduce Health Disparities.

The Research Centers in Minority Institutions (RCMI) Program was congressionally mandated in 1985 to build research capacity at institutions that currently and historically recruit, train, and award doctorate degrees in the health professions and health-related sciences, primarily to individuals from underrepresented and minority populations. RCMI grantees share similar infrastructure needs and institutional goals. Of particular importance is the professional development of multidisciplinary teams of academic and community scholars (the "workforce") and the harnessing of the heterogeneity of thought (the "thinkforce") to reduce health disparities. The purpose of this report is to summarize the presentations and discussion at the RCMI Investigator Development Core (IDC) Workshop, held in conjunction with the RCMI Program National Conference in Bethesda, Maryland, in December 2019. The RCMI IDC Directors provided information about their professional development activities and Pilot Projects Programs and discussed barriers identified by new and early-stage investigators that limit effective career development, as well as potential solutions to overcome such obstacles. This report also proposes potential alignments of professional development activities, targeted goals and common metrics to track productivity and success.

COVID-19 Special Column: Principles Behind the Technology for Detecting SARS-CoV-2, the Cause of COVID-19.

Nationwide shortages of tests that detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and diagnose coronavirus disease 2019 (COVID-19) have led the US Food and Drug Administration (FDA) to significantly relax regulations regarding COVID-19 diagnostic testing. To date the FDA has given emergency use authorization (EUA) to 48 COVID-19 in vitro diagnostic tests and 21 high complexity molecular-based laboratory developed tests, as well as implemented policies that give broad authority to clinical laboratories and commercial manufacturers in the development, distribution, and use of COVID-19 diagnostic tests. Currently, there are 2 types of diagnostic tests available for the detection of SARS-CoV-2: (1) molecular and (2) serological tests. Molecular detection of nucleic acid (RNA or DNA) sequences relating to the suspected pathogen is indicative of an active infection with the suspected pathogen. Serological tests detect antibodies against the suspected pathogen, which are produced by an individual's immune system. A positive serological test result indicates recent exposure to the suspected pathogen but cannot be used to determine if the individual is actively infected with the pathogen or immune to reinfection. In this article, the SARS-CoV-2 diagnostic tests currently approved by the FDA under EUA are reviewed, and other diagnostic tests that researchers are developing to detect SARS-CoV-2 infection are discussed.

The Research Centers in Minority Institutions (RCMI) Translational Research Network: Building and Sustaining Capacity for Multi-Site Basic Biomedical, Clinical and Behavioral Research.

The Research Centers in Minority Institutions (RCMI) program was established by the US Congress to support the development of biomedical research infrastructure at minority-serving institutions granting doctoral degrees in the health professions or in a health-related science. RCMI institutions also conduct research on diseases that disproportionately affect racial and ethnic minorities (ie, African Americans/Blacks, American Indians and Alaska Natives, Hispanics, Native Hawaiians and Other Pacific Islanders), those of low socioeconomic status, and rural persons. Quantitative metrics, including the numbers of doctoral science degrees granted to underrepresented students, NIH peer-reviewed research funding, peer-reviewed publications, and numbers of racial and ethnic minorities participating in sponsored research, demonstrate that RCMI grantee institutions have made substantial progress toward the intent of the Congressional legislation, as well as the NIH/NIMHD-linked goals of addressing workforce diversity and health disparities. Despite this progress, nationally, many challenges remain, including persistent disparities in research and career development awards to minority investigators. The continuing underrepresentation of minority investigators in NIH-sponsored research across multiple disease areas is of concern, in the face of unrelenting national health inequities. With the collaborative network support by the RCMI Translational Research Network (RTRN), the RCMI community is uniquely positioned to address these challenges through its community engagement and strategic partnerships with non-RCMI institutions. Funding agencies can play an important role by incentivizing such collaborations, and incorporating metrics for research funding that address underrepresented populations, workforce diversity and health equity.

Global mass spectrometry based metabolomics profiling of erythrocytes infected with Plasmodium falciparum.

Malaria is a global infectious disease that threatens the lives of millions of people. Transcriptomics, proteomics and functional genomics studies, as well as sequencing of the Plasmodium falciparum and Homo sapiens genomes, have shed new light on this host-parasite relationship. Recent advances in accurate mass measurement mass spectrometry, sophisticated data analysis software, and availability of biological pathway databases, have converged to facilitate our global, untargeted biochemical profiling study of in vitro P. falciparum-infected (IRBC) and uninfected (NRBC) erythrocytes. In order to expand the number of detectable metabolites, several key analytical steps in our workflows were optimized. Untargeted and targeted data mining resulted in detection of over one thousand features or chemical entities. Untargeted features were annotated via matching to the METLIN metabolite database. For targeted data mining, we queried the data using a compound database derived from a metabolic reconstruction of the P. falciparum genome. In total, over one hundred and fifty differential annotated metabolites were observed. To corroborate the representation of known biochemical pathways from our data, an inferential pathway analysis strategy was used to map annotated metabolites onto the BioCyc pathway collection. This hypothesis-generating approach resulted in over-representation of many metabolites onto several IRBC pathways, most prominently glycolysis. In addition, components of the "branched" TCA cycle, partial urea cycle, and nucleotide, amino acid, chorismate, sphingolipid and fatty acid metabolism were found to be altered in IRBCs. Interestingly, we detected and confirmed elevated levels for cyclic ADP ribose and phosphoribosyl AMP in IRBCs, a novel observation. These metabolites may play a role in regulating the release of intracellular Ca(2+) during P. falciparum infection. Our results support a strategy of global metabolite profiling by untargeted data acquisition. Untargeted and targeted data mining workflows, when used together to perform pathway-inferred metabolomics, have the benefit of obviating MS/MS confirmation for every detected compound.

Production of the 42-kDa fragment of Plasmodium falciparum merozoite surface protein 1, a leading malaria vaccine antigen, in Arabidopsis thaliana seeds.

Malaria is widely associated with poverty, and a low-cost vaccine against malaria is highly desirable for implementing comprehensive vaccination programmes in developing countries. Production of malaria antigens in plants is a promising approach, but its development has been hindered by poor expression of the antigens in plant cells. In the present study, we targeted plant seeds as a low-cost vaccine production platform and successfully expressed the Plasmodium falciparum 42-kDa fragment of merozoite surface protein 1 (MSP142), a leading malaria vaccine candidate, at a high level in transgenic Arabidopsis seeds. We overcame hurdles of transcript and protein instabilities of MSP142 in plants by synthesizing a plant-optimized MSP142 cDNA and either targeting the recombinant protein to protein storage vacuoles or fusing it with a stable plant storage protein. An exceptional improvement in MSP142 expression, from an undetectable level to 5% of total extractable protein, was achieved with these combined strategies. Importantly, the plant-derived MSP142 maintains its natural antigenicity and can be recognized by immune sera from malaria-infected patients. Our results provide a strong basis for the development of a plant-based, low-cost malaria vaccine.

Shift in epitope dominance of IgM and IgG responses to Plasmodium falciparum MSP1 block 4.

BACKGROUND: Plasmodium falciparum merozoite surface protein-1 (MSP1) has been extensively studied as a blood-stage malaria vaccine candidate, with most work focused on the conserved 19 kDa and semi-conserved 42 kDa C-terminal regions (blocks 16-17) and the hypervariable N-terminal repeat region (block 2). However, recent genotyping studies suggest that additional regions of MSP1 may be under selective pressure, including a locus of intragenic recombination designated as block 4 within the 3' region of the gene. METHODS: The current study examined the antibody response to the two parental and two recombinant forms of block 4 and to blocks 16-17 (3D7) in study populations from Colombia, Papua New Guinea and Cameroon that differ in malaria transmission intensity and ethnic composition. RESULTS: IgM and IgG antibodies were detected against parental and recombinant MSP1 block 4 peptides in all three populations. Overall, 32-44% of the individuals produced IgM to one or more of the peptides, with most individuals having IgM antibodies reactive with both parental and recombinant forms. In contrast, IgG seropositivity to block 4 varied among populations (range 15-65%), with the majority of antibodies showing specificity for one or a pair of block 4 peptides. The IgG response to block 4 was significantly lower than that to blocks 16-17, indicating block 4 is subdominant. Antibodies to block 4 and blocks 16-17 displayed distinct IgG subclass biases, with block 4 responses biased toward IgG3 and blocks 16-17 toward IgG1. These patterns of responsiveness were consistently observed in the three study populations. CONCLUSIONS: Production of antibodies specific for each parental and recombinant MSP1 block 4 allele in different populations exposed to P. falciparum is consistent with balancing selection of the MSP1 block 4 region by the immune response of individuals in areas of both low and high malaria transmission. MSP1 block 4 determinants may be important in isolate-specific immunity to P. falciparum.

Restricted genetic diversity of Plasmodium falciparum major merozoite surface protein 1 in isolates from Colombia.

The merozoite surface protein 1 (MSP-1) gene of Plasmodium falciparum encodes a major immune target under development as a malaria vaccine. In this study, we typed MSP-1 variable regions of parasites obtained from Buenaventura, Colombia. Four MSP-1 gene types were detected corresponding to prototype and recombinant K1 and MAD20 block 4 sequences. In contrast to variability within block 4, blocks 2, 6, and 16-17 corresponded exclusively to the MAD20 allelic type. Most (80%) blood samples contained multiple MSP-1 gene types. The presence of four MSP-1 variants within block 4 against a MAD20 background indicates that current P. falciparum populations in Buenaventura are derived from parasites expressing K1 and MAD20 alleles, some of which underwent two recombination events within or flanking block 4. Restricted MSP-1 diversity appears to be relatively stable in Buenaventura and suggests that selection has resulted in the dominance of the MAD20 type in most of the polymorphic blocks with the exception of block 4.

T-cell activation and cytokine production via a bispecific single-chain antibody fragment targeted to blood-stage malaria parasites.

A novel bispecific single-chain antibody fragment (biscFv) has been constructed to address the possibility of a new approach to malaria therapeutic drug development. The biscFv consists of 2 different single-chain antibody fragments linked by a flexible peptide linker (Gly(4)-Ser)(3). Of the 2 scFv fragments, one is directed against a conserved epitope of the 19-kDa C-terminal fragment of the major surface protein of human malignant malaria parasite, Plasmodium falciparum, and the other is directed against the CD3 antigen of human T cells. The biscFv expressed by a recombinant baculovirus retained the antigen-binding properties of the corresponding univalent single-chain antibody fragments and formed a bridge between P falciparum and T cells. In cooperation with T cells, the biscFv specifically induced not only interferon gamma and tumor necrosis factor alpha, but also a significant increase of merozoite phagocytosis and growth inhibition of P falciparum in vitro. Thus, the biscFv possesses highly selective malaria-targeting properties and stimulates T cells to induce cytokines, presumably resulting in activation of macrophages, neutrophils, and natural killer cells, and parasite killing in vivo.

Naturally acquired antibodies against the major merozoite surface coat protein (MSP-1) of Plasmodium falciparum acquired by residents in an endemic area of Colombia

A preliminary baseline epidemiological malaria survey was conducted in the village of Punta Soldado, Colombia. Parasite prevalence and density as well as serological data were obtained from 151 asymptomatic children and adults. Fifty individuals were infected with Plasmodium falciparum. The mean parasite density was 184 parasites/mm3. Greater than 90 of the sample population were P. falciparum antibody positive as detected by the indirect immunofluorescent antibody test (IFAT). The enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against the major merozoite surface protein (MSP-1) of P. falciparum. In this population, anti-MSP-1 antibody concentration is acquired in an age dependent manner with equal immunogenicity to both the N- and C-terminal regions of the molecule. Infection at the time of sampling was associated with a higher anti-MSP-1 antibody concentration than that found in non-infected individuals. Further studies are planned to assess the role of immune and non-immune factors in limiting the number of cases of severe malaria seen in this population.