Use of anti-gSG6-P1 IgG as a serological biomarker to assess temporal exposure to Anopheles’ mosquito bites in Lower Moshi

Background Malaria prevalence in the highlands of Northern Tanzania is currently below 1% making this an elimination prone setting. As climate changes may facilitate increasing distribution of Anopheles mosquitoes in such settings, there is a need to monitor changes in risks of exposure to ensure that established control tools meet the required needs. This study explored the use of human antibodies against gambiae salivary gland protein 6 peptide 1 (gSG6-P1) as a biomarker of Anopheles exposure and assessed temporal exposure to mosquito bites in populations living in Lower Moshi, Northern Tanzania. Methods Three cross-sectional surveys were conducted in 2019: during the dry season in March, at the end of the rainy season in June and during the dry season in September. Blood samples were collected from enrolled participants and analysed for the presence of anti-gSG6-P1 IgG. Mosquitoes were sampled from 10% of the participants’ households, quantified and identified to species level. Possible associations between gSG6-P1 seroprevalence and participants’ characteristics were determined. Results The total number of Anopheles mosquitoes collected was highest during the rainy season (n = 1364) when compared to the two dry seasons (n = 360 and n = 1075, respectively). The gSG6-P1 seroprevalence increased from 18.8% during the dry season to 25.0% during the rainy season (χ2 = 2.66; p = 0.103) followed by a significant decline to 11.0% during the next dry season (χ2 = 12.56; p = 0.001). The largest number of mosquitoes were collected in one village (Oria), but the seroprevalence was significantly lower among the residents as compared to the rest of the villages (p = 0.039), explained by Oria having the highest number of participants owning and using bed nets. Both individual and household gSG6-P1 IgG levels had no correlation with numbers of Anopheles mosquitoes collected. Conclusion Anti-gSG6-P1 IgG is a potential tool in detecting and distinguishing temporal and spatial variations in exposure to Anopheles mosquito bites in settings of extremely low malaria transmission where entomological tools may be obsolete. However studies with larger sample size and extensive mosquito sampling are warranted to further explore the association between this serological marker and abundance of Anopheles mosquito.

Transcriptomic Analysis of Salivary Glands of Ornithodoros brasiliensis Aragão, 1923, the Agent of a Neotropical Tick-Toxicosis Syndrome in Humans

Tick salivary glands produce and secrete a variety of compounds that modulate host responses and ensure a successful blood meal. Despite great progress made in the identification of ticks salivary compounds in recent years, there is still a paucity of information concerning salivary molecules of Neotropical argasid ticks. Among this group of ticks, considering the number of human cases of parasitism, including severe syndromes and hospitalization, Ornithodoros brasiliensis can be considered one of the major Neotropical argasid species with impact in public health. Here, we describe the transcriptome analysis of O. brasiliensis salivary glands (ObSG). The transcriptome yielded ~14,957 putative contigs. A total of 368 contigs were attributed to secreted proteins (SP), which represent approximately 2.5% of transcripts but ~53% expression coverage transcripts per million. Lipocalins are the major protein family among the most expressed SP, accounting for ~16% of the secretory transcripts and 51% of secretory protein abundance. The most expressed transcript is an ortholog of TSGP4 (tick salivary gland protein 4), a lipocalin first identified in Ornithodoros kalahariensis that functions as a leukotriene C4 scavenger. A total of 55 lipocalin transcripts were identified in ObSG. Other transcripts potentially involved in tick-host interaction included as: basic/acid tail secretory proteins (second most abundant expressed group), serine protease inhibitors (including Kunitz inhibitors), 5' nucleotidases (tick apyrases), phospholipase A2, 7 disulfide bond domain, cystatins, and tick antimicrobial peptides. Another abundant group of proteins in ObSG is metalloproteases. Analysis of these major protein groups suggests that several duplication events after speciation were responsible for the abundance of redundant compounds in tick salivary glands. A full mitochondrial genome could be assembled from the transcriptome data and confirmed the close genetic identity of the tick strain sampled in the current study, to a tick strain previously implicated in tick toxicoses. This study provides novel information on the molecular composition of ObSG, a Brazilian endemic tick associated with several human cases of parasitism. These results could be helpful in the understanding of clinical findings observed in bitten patients, and also, could provide more information on the evolution of Neotropical argasids.

PURIFIKASI PROTEIN IMUNOGENIK 31 DAN 56 kDa DARI KELENJAR SALIVA Aedes aegypti

Purification of 31 and 56 kDa Immunogenic Proteins from the Salivary Glands of Aedes aegyptiThe salivary gland of arthropod vector contains various bioactive compounds and plays a role in the transmission of pathogens to the host. The host develops anti-salivary antibodies against vector saliva exposure. Our previous research has identified two immunogenic proteins with molecular weights of 31 and 56 kDa from the Aedes aegypti salivary gland protein extract. However, the role of the 31 and 56 kDa immunogenic proteins from saliva Ae. aegypti is not fully known, so it is necessary to purify two immunogenic protein fractions to better specify the target of developing a dengue vaccine. This study aimed to purify the 31 and 56 kDa immunogenic protein fractions by electroelution and dialysis methods. The purification of the two protein fractions has been successful which were confirmed by the SDS-PAGE by the existence of single-band parallel to the positive control. These results were further supported by the dot blot analysis which showed a positive reaction in the form of dark spots in the two protein fractions which were reacted with dengue patients' serum, endemic healthy people, and neonates. These results indicated that the purified 31 and 56 kDa immunogenic protein fraction can be identified by specific antibodies.Keywords: dialysis, electroelution, immunogenic, purification, saliva  ABSTRAKKelenjar saliva vektor arthropoda mengandung berbagai senyawa bioaktif dan berperan dalam transmisi patogen ke tubuh inang. Tubuh inang mengembangkan antibodi anti-saliva terhadap paparan saliva vektor. Penelitian kami sebelumnya telah mengidentifikasi dua protein imunogenik dengan berat molekul 31 dan 56 kDa dari ekstrak protein kelenjar saliva Aedes aegypti. Namun demikian, peranan protein imunogenik 31 dan 56 kDa dari saliva Ae. aegypti belum diketahui sepenuhnya sehingga perlu dilakukan purifikasi dua fraksi protein imunogenik untuk lebih menspesifikkan target pengembangan vaksin dengue. Tujuan penelitian ini untuk melakukan purifikasi fraksi protein imunogenik 31 dan 56 kDa melalui metode elektroelusi dan dialisis. Keberhasilan purifikasi dua fraksi protein 31 dan 56 kDa terbukti dari hasil konfirmasi SDS-PAGE dengan terbentuknya pita tunggal sejajar dengan kontrol positif. Hasil tersebut diperkuat dengan analisis dot blot yang menunjukkan reaksi positif dengan munculnya noktah gelap pada dua fraksi protein tersebut ketika direaksikan dengan serum pasien DBD, penduduk sehat endemik dan neonatus. Hasil ini mengindikasikan bahwa fraksi protein imunogenik 31 dan 56 kDa hasil purifikasi dapat dikenali oleh antibodi spesifik.

Profiling Autoantibodies against Salivary Proteins in Sicca Conditions

Salivary gland dysfunction occurs in several autoimmune and immune-related conditions, including Sjögren syndrome (SS); immune checkpoint inhibitor-induced sicca (ICIS) that develops in some cancer patients and is characterized by severe, sudden-onset dry mouth; and autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Although subjects with these conditions present with oral dryness and often exhibit inflammatory infiltration of the salivary gland, little is known about the B-cell humoral responses directed against salivary gland protein targets. In this study, autoantibodies were evaluated against Ro52, Ro60, and La, as well as against a panel of 22 proteins derived from the salivary proteome. The tested cohort included healthy volunteers and subjects with SS, ICIS, and APECED without and with sicca. As expected, a high percentage of autoantibody seropositivity was detected against Ro52, Ro60, and La in SS, but only a few ICIS patients were seropositive for these autoantigens. A few APECED subjects also harbored autoantibodies to Ro52 and La, but only Ro60 autoantibodies were weakly associated with a small subset of APECED patients with sicca. Additional testing of the salivary panel failed to detect seropositive autoantibodies against any of the salivary-enriched proteins in the SS and ICIS subjects. However, APECED subjects selectively demonstrated seropositivity against BPI fold containing family A member 1 (BPIFA1), BPI fold containing family A member 2 (BPIFA2)/parotid salivary protein (PSP), and lactoperoxidase, 3 salivary-enriched proteins. Moreover, high levels of serum autoantibodies against BPIFA1 and BPIFA2/PSP occurred in 30% and 67% of the APECED patients with sicca symptoms, respectively, and were associated with an earlier age onset of oral dryness ( P = 0.001). These findings highlight the complexity of humoral responses in different sicca diseases and provide new insights and biomarkers for APECED-associated sicca (ClinicalTrials.gov: NCT00001196; NCT00001390; NCT01425892; NCT01386437).

Evaluation of Autoantibodies in Patients with Primary and Secondary Sjogren’s Syndrome

Background: Antibodies to salivary gland protein 1 (SP1), carbonic anhydrase 6 (CA6) and parotid secretory protein (PSP) were discovered in an animal model of Sjogren’s syndrome (SS). Their expression was noted in patients with SS, especially those with lower focus scores on lip biopsies and those with early disease lacking antibodies to Ro and La. Objective: The current studies evaluated these autoantibodies in patients with long-standing SS expressing high levels of anti-Ro antibodies and in patients with Sjogren’s syndrome secondary to systemic lupus erythematosus (SLE), systemic sclerosis (SSc) and mixed connective tissue disease (MCTD). Method: Sera were obtained from patients and evaluated by ELISA for IgG, IgA and IgM antibodies to SP1, CA6 and PSP. Results: IgA anti-CA6 antibodies were noted in 38% of these patients, but anti-SP1, CA6 and PSP IgM or IgG antibodies were identified only in a minority of patients. In patients with secondary SS, antibodies to SP1/CA6/PSP were more sensitive and specific than anti-Ro . Conclusion: While more studies are needed, antibodies to SP1, CA6 and PSP provide valuable markers for the diagnosis of primary and secondary SS, especially early in the course of the disease.