Current State of Carbohydrate Recognition and C-Type Lectin Receptors in Pneumocystis Innate Immunity

Pneumocystis jirovecii is one of the most common fungal pathogens in immunocompromised individuals. Pneumocystis jirovecii pneumonia (PJP) causes a significant host immune response that is driven greatly by the organism’s cell wall components including β-glucans and major surface glycoprotein (Msg). These ligands interact with a number of C-type lectin receptors (CLRs) leading to downstream activation of proinflammatory signaling pathways. This minireview provides a brief overview summarizing known CLR/Pneumocystis interactions.

Persistently high antibody responses after AS03-adjuvanted H1N1pdm09 vaccine: Dissecting the HA specific antibody response

Current influenza vaccines have a suboptimal effectiveness. The introduction of a novel A/H1N1 influenza virus in 2009 (H1N1pdm09) provided a unique opportunity to study the humoral response to the AS03-adjuvanted H1N1pdm09 vaccine and repeated annual vaccination with the homologous virus in subsequent influenza seasons. Thirty-two HCWs immunized with the AS03-adjuvanted H1N1pdm09 vaccine in 2009 were divided into four groups based on the longevity of their antibody responses (persistently high or transient), and whether they were repeatedly annually vaccinated in the subsequent four influenza seasons or not. Serological assays were utilized to measure the quantity, quality and functionality of antibodies targeting the major surface glycoprotein hemagglutinin (HA). Persistent high responders (hemagglutination inhibition (HI) titre ≥ 80 at 12 months after H1N1pdm09 vaccination) had protective levels of HI antibodies throughout the study period. In addition, the quality and functionality of these antibodies were greater than the individuals who had a transient antibody response to the pandemic vaccine (HI titre < 40 at 12 months after H1N1pdm09 vaccination). All groups had similar levels of antibodies towards the conserved HA stalk domain. The level of HA head-specific antibodies gradually increased over time with annual vaccination in the transient responders. The AS03-adjuvanted H1N1pdm09 vaccine elicited a robust humoral response that persisted up to 5 years in some individuals. Seasonal annual vaccination boosted the HA-antibodies over time in individuals with a transient response to the pandemic H1N1pdm09 vaccine.

Engineered receptor binding domain immunogens elicit pan-coronavirus neutralizing antibodies

Effective countermeasures are needed against emerging coronaviruses of pandemic potential, similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Designing immunogens that elicit broadly neutralizing antibodies to conserved viral epitopes on the major surface glycoprotein, spike, such as the receptor binding domain (RBD) is one potential approach. Here, we report the generation of homotrimeric RBD immunogens from different sarbecoviruses using a stabilized, immune-silent trimerization tag. We find that that a cocktail of homotrimeric sarbecovirus RBDs can elicit a neutralizing response to all components even in context of prior SARS-CoV-2 imprinting. Importantly, the cross-neutralizing antibody responses are focused towards conserved RBD epitopes outside of the ACE-2 receptor-binding motif. This may be an effective strategy for eliciting broadly neutralizing responses leading to a pan-sarbecovirus vaccine.

Analisis filogenetik gen mitochondrial large subunit (mtLSU) Pneumocystis jirovecii pada Pasien Human Immunodeficiency Virus (HIV) Terduga Pneumoniae di Jakarta

Abstrak Pneumocystis jirovecii (P. jirovecii) merupakan patogen oportunistik yang penting pada pasien dengan gangguan kekebalan menurun khususnya human immunodeficiency virus (HIV). P. jirovecii tersebar dimanamana, menyebar melalui udara, dan menyerang sistem pernapasan atas. P. jirovecii mempunyai beberapa faktor virulensi, antara lain major surface glycoprotein (MSG) yang merupakan antigen yang paling banyak ditemukan di permukaan. Pendekatan biologi molekuler digunakan untuk mempelajari patogen ini karena hingga saat ini kultur belum dapat dilakukan. Penelitian ini bertujuan untuk memperoleh data genotip yang dapat dimanfaatkan sebagai dasar data demografi dan epidemiologi molekuler P. jirovecii di Indonesia. Dua puluh sampel sputum positif P. jirovecii pada real-time polymerase chain reaction (RT-PCR) dilakukan karakterisasi terhadap gen mitochondrial large subunit (mtLSU). Virulensi daerah hot spot gen mtLSU dianalisis dengan metode PCR dan sekuensing deoxyribonucleic acid (DNA). Diperoleh 30 strain dengan 7 varian didominasi oleh varian 3 yang bersirkulasi di Jakarta. Analisis filogenetik dengan strain negara lain menunjukkan strain Jakarta berkerabat dekat dengan strain Iran, India dan Korea. Kata kunci : Pneumocystis jirovecii, mtLSU, PCR, filogenetik Abstract Pneumocystis jirovecii (P. jirovecii) is an important opportunistic pathogen in immunocompromised patients especially human immunodeficiency virus (HIV). P. jirovecii is spread everywhere, spread through the air, and attacking the upper respiratory system. P. jirovecii has several virulence factors, including major surface glycoprotein (MSG) which is the most widely found on the surface antigen. The molecular biology approach is used to study this pathogen because until now culture cannot be done. This study aims to obtain genotype data that can be used as a basis for demographic and molecular epidemiological data of P. jirovecii in Indonesia. Twenty P. jirovecii positive sputum samples on real-time polymerase chain reaction (RT-PCR) were characterized by the mitochondrial large subunit (mtLSU) gene. Virulence of the mtLSU gene hot spot region was analyzed by PCR method and deoxyribonucleic acid (DNA) sequencing. Obtained 30 strains with 7 variants dominated by variant 3 circulating in Jakarta. Phylogenetic analyzed with strains of other countries shows that Jakarta strains are closely related to strains of Iran, India dan Korea. Keywords: Pneumocystis jirovecii, mtLSU, PCR, phylogenetic

Pneumocystis carinii Major Surface Glycoprotein Dampens Macrophage Inflammatory Responses to Fungal β-Glucan

Background Pneumocystis major surface glycoprotein (Msg) is a 120-kD surface protein complex on the organism with importance in adhesion and immune recognition. In this study, we show that Msg significantly impairs tumor necrosis factor (TNF)-α secretion by macrophages induced by Saccharomyces cerevisiae and Pneumocystis carinii (Pc) β-glucans. Methods Major surface glycoprotein was shown to greatly reduce β-glucan-induced Dectin-1 immunoreceptor tyrosine-based activating motif (ITAM) phosphorylation. Major surface glycoprotein also down regulated Dectin-1 receptor messenger ribonucleic acid (mRNA) expression in the macrophages. It is interesting that Msg incubation with macrophages resulted in significant mRNA upregulation of both C-type lectin receptors (CLR) Mincle and MCL in Msg protein presence alone but to even greater amounts in the presence of Pc β-glucan. Results The silencing of MCL and Mincle resulted in TNF-α secretions similar to that of macrophages treated with Pneumocystis β-glucan alone, which is suggestive of an inhibitory role for these 2 CLRs in Msg-suppressive effects on host cell immune response. Conclusions Taken together, these data indicate that the Pneumocystis Msg surface protein complex can act to suppress host macrophage inflammatory responses to the proinflammatory β -glucan components of the organisms.

Detection of Opportunistic Fungus Pneumocystis jirovecii Major Surface Glycoprotein (MSG) gene in HIV-AIDS Patients with Pneumoniae in Jakarta

Pneumocystis jirovecii is known to cause opportunistic infections in the lower respiratory tract in individuals with low immune systems, especially patient with HIV infection. The prevalence of P. jirovecii pneumonia (PjP) in various countries show varying numbers. In Indonesia, HIV cases continue to rise. However, the data in Indonesia concerning the case of PjP is very limited. Until now the prevalence of PjP in Indonesia is only based on clinical symptoms of the patient. Currently, diagnosis of PjP relies on microscopic examination. The disadvantage of this examination is not easy to do and has a high negative predictive value. Thus, this study was conducted to develop a molecular test to diagnose PjP infection in HIV-AIDS suspected pneumonia. Molecular diagnostic test aimed for Major Surface Glycoprotein (MSG) gene of P. jirovecii detection was done through real-time PCR against 100 sputum samples. Demographic data show that the prevalence of PjP infection in HIV-AIDS suspected pneumonia patients in Jakarta is 20.0%, male 75% within 31-40 y.o (35%), dominant (80%) from patients with CD4+ T-lymphocytes of 200-349 cells/µL. Molecular real-time PCR methods were shown to give five times sensitivity higher than Giemsa stain. Keywords: P. jirovecii, HIV, real-time PCR

The Fungal PCR Initiative's evaluation of in-house and commercial Pneumocystis jirovecii qPCR assays: Toward a standard for a diagnostics assay

Quantitative real-time PCR (qPCR) is increasingly used to detect Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia (PCP), but there are differences in the nucleic acids targeted, DNA only versus whole nucleic acid (WNA), and also the target genes for amplification. Through the Fungal PCR Initiative, a working group of the International Society for Human and Animal Mycology, a multicenter and monocenter evaluation of PCP qPCR assays was performed. For the multicenter study, 16 reference laboratories from eight different countries, performing 20 assays analyzed a panel consisting of two negative and three PCP positive samples. Aliquots were prepared by pooling residual material from 20 negative or positive- P. jirovecii bronchoalveolar lavage fluids (BALFs). The positive pool was diluted to obtain three concentrations (pure 1:1; 1:100; and 1:1000 to mimic high, medium, and low fungal loads, respectively). The monocenter study compared five in-house and five commercial qPCR assays testing 19 individual BALFs on the same amplification platform. Across both evaluations and for all fungal loads, targeting WNA and the mitochondrial small sub-unit (mtSSU) provided the earliest Cq values, compared to only targeting DNA and the mitochondrial large subunit, the major surface glycoprotein or the beta-tubulin genes. Thus, reverse transcriptase-qPCR targeting the mtSSU gene could serve as a basis for standardizing the P. jirovecii load, which is essential if qPCR is to be incorporated into clinical care pathways as the reference method, accepting that additional parameters such as amplification platforms still need evaluation.