Antigenic sites in SARS-CoV-2 spike RBD show molecular similarity with pathogenic antigenic determinants and harbors peptides for vaccine development

The spike protein of coronavirus is key target for drug development and other pharmacological interventions. In current study, we performed an integrative approach to predict antigenic sites in SARS-CoV-2 spike receptor binding domain and found nine potential antigenic sites. The predicted antigenic sites were then assessed for possible molecular similarity with other known antigens in different organisms. Out of nine sites, seven sites showed molecular similarity with 54 antigenic determinants found in twelve pathogenic bacterial species (Mycobacterium tuberculosis, Mycobacterium leprae, Bacillus anthracis, Borrelia burgdorferi, Clostridium perfringens, Clostridium tetani, Helicobacter Pylori, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, Vibrio cholera and Yersinia pestis), two malarial parasites (Plasmodium falciparum and Plasmodium knowlesi) and influenza virus A. Most of the bacterial antigens that displayed molecular similarity with antigenic sites in SARS-CoV-2 RBD (receptor binding domain) were toxins and virulent factors. Antigens from Mycobacterium that showed similarity were mainly involved in modulating host cell immune response and ensuring persistence and survival of pathogen in host cells. Presence of a large number of antigenic determinants, similar to those in highly pathogenic microorganisms, not merely accounts for complex etiology of the disease but also provides an explanation for observed pathophysiological complications, such as deregulated immune response, unleashed or dysregulated cytokine secretion (cytokine storm), multiple organ failure etc., that are more evident in aged and immune-compromised patients. Over-representation of antigenic determinants from Plasmodium and Mycobacterium in all antigenic sites suggests that anti-malarial and anti-TB drugs can prove to be clinical beneficial for COVID-19 treatment. Besides this, anti-leprosy, anti-lyme, anti-plague, anti-anthrax drugs/vaccine etc. are also expected to be beneficial in COVID-19 treatment. Moreover, individuals previously immunized/vaccinated or had previous history of malaria, tuberculosis or other disease caused by fifteen microorganisms are expected to display a considerable degree of resistance against SARS-CoV-2 infection. Out of the seven antigenic sites predicted in SARS-CoV-2, a part of two antigenic sites were also predicted as potent T-cell epitopes (KVGGNYNYL^444-452 and SVLYNSASF^366-374) against MHC class I and three (KRISNCVADYSVLYN^356-370, DLCFTNVYADSFVI^389-402, and YRVVVLSFELLHA^508-520) against MHC class II. All epitopes possessed significantly lower predicted IC50 value which is a prerequisite for a preferred vaccine candidate for COVID-19.     

Persistence of borrelial DNA in the joints of Borrelia burgdorferi‐infected mice after ceftriaxone treatment

Yrjänäinen H, Hytönen J, Hartiala P, Oksi J, Viljanen MK. Persistence of borrelial DNA in the joints of Borrelia burgdorferi‐infected mice after ceftriaxone treatment. APMIS 2010; 118: 665–73. We have earlier shown that Borrelia burgdorferi‐infected and ceftriaxone‐treated mice have viable spirochetes in their body, since immunosuppressive treatment allows B. burgdorferi to be detected by culture. However, the niche of the persisting spirochetes remained unknown. In the present study, we analyzed the tissues of B. burgdorferi‐infected and ceftriaxone‐treated mice by culture and PCR to reveal the foci of persisting spirochetes. C3H/HeN mice were infected via intradermal needle injection with B. burgdorferi s.s. N40. The mice were treated as follows: (i) short (5 days) and (ii) long (18 days) course of ceftriaxone at 2 weeks of infection and killed after either 10 or 30 weeks, or (iii) the mice received ceftriaxone for 5 days at 18 weeks of infection and were killed 21 weeks after the treatment. All samples of ceftriaxone‐treated mice were culture negative, whereas all untreated controls were culture positive. Importantly, B. burgdorferi DNA was detected in the joints of 30–100% of the treated mice. In conclusion, these results combined with earlier results suggest that the joint or a tissue adjacent to the joint is the niche of persisting B. burgdorferi in ceftriaxone‐treated mice.     

Toll-like receptors in Borrelia burgdorferi-induced inflammation

Lyme arthritis, the most common manifestation of late Lyme disease, has been associated with the presence of Borellia burgdorferi in the joint. However, it is still unclear whether the pathogen itself is able to elicit such a sustained inflammatory response, or whether an aberrant immunological reaction of the host is the main driving force. Borrelia antigens, including lipoproteins, flagellin and DNA, are ligands of Toll-like receptors, and can thus elicit a strong stimulation of host cells, such as neutrophils, mononuclear cells and resident tissue cells. Understanding the molecular basis of the signalling events caused by Borrelia lipoproteins will lead to a greater understanding of inflammation in Lyme arthritis and, hopefully, new treatment strategies for chronic antibiotic-resistant disease.     

Artificial-infection protocols allow immunodetection of novel Borrelia burgdorferi antigens suitable as vaccine candidates against Lyme disease

Vaccination with recombinant outer surface protein A (OspA) from Borrelia burgdorferi provides excellent antibody-mediated protection against challenge with the pathogen in animal models and in humans. However, the bactericidal antibodies are ineffective in the reservoir host, since OspA is expressed by spirochetes only in the vector, but rarely, if at all, in mammals. Using an artificially generated immune serum (anti-10(8) spirochetes) with high protective potential for prophylactic and therapeutic treatment, we have now isolated from an expression library of B. burgdorferi (strain ZS7) three novel genes, zs7.a36, zs7.a66 and zs7.a68. All three genes are located, together with ospA/B, on the linear plasmid lp54, and are expressed in vitro and in ticks. At least temporarily two of them, ZS7.A36 and ZS7.A66, are also expressed during infection. The respective natural antigens are poorly immunogenic ininfected normal mice but elicited antibodies in Lyme disease patients. We show that recombinant preparations of ZS7.A36, ZS7.A66 and ZS7.A68 induce functional antibodies in rabbits capable of protecting immunodeficient mice against subsequent experimental infection. These findings suggest that all three recombinant antigens represent potential candidates for a "second generation" vaccine to prevent and/or cure Lyme disease.     

新疆北部林区工作人员蜱传疾病的血清学调查研究

为了解新疆北部林区工作人员嗜吞噬细胞无形体病、森林脑炎、莱姆病3种蜱传疾病感染情况,采集新疆北部林区工作人员血清标本215份,采取间接免疫荧光法检测血清中嗜吞噬细胞无形体、森林脑炎病毒、伯氏疏螺旋体特异性IgG抗体.经检测嗜吞噬细胞无形体IgG抗体阳性率为40.47％ (87/215),森林脑炎病毒IgG抗体阳性率为20.00％ (43/215),莱姆病伯氏疏螺旋体IgG抗体阳性率为10.70％ (23/215).共检测出29例复合感染,复合感染率为13.49％ (29/215).其中嗜吞噬细胞无形体和森林脑炎病毒复合感染率6.05％ (13/215),嗜吞噬细胞无形体和莱姆病伯氏疏螺旋体复合感染率3.72％ (8/215),森林脑炎病毒和莱姆病伯氏疏螺旋体复合感染率1.40％ (3/215),三种病原体复合感染率2.33％ (5/215).新疆北部林区工作人员中存在嗜吞噬细胞无形体病、森林脑炎、莱姆病及其复合感染流行.     

莱姆病人工宿主动物模型的建立

为了建立莱姆病人工宿主模型进行实验传播研究,用从我国全沟硬蜱分离的莱姆病螺旋体Borrelia garinii CHNM4人工感染4种实验动物.结果表明,以0.1×106条/mL的剂量皮下注射3日龄昆明小鼠是比较理想的方案.由此建立的动物模型既能用作全沟硬蜱的血源动物,又能在15天内保持所感染螺旋体对全沟硬蜱的感染力,可作为人工宿主动物模型.     

Heterogeneity in the complement-dependent bacteriolysis within the species of Borrelia burgdorferi

Sixteen Borrelia burgdorferi strains, including all three species, were compared in a colorimetric bactericidal assay for their ability to escape the complement-dependent bacteriolysis on incubation in normal human serum free of specific antibodies (NHS). The species B. afzelii was found to be serum resistant (EB1, EB3, FEM1, FEM2, Pko), whereas strains of the species B. garinii were found to be serum sensitive (1/B29, G1, G2, PSth, PBr, PTrob). Six strains, mainly B. burgdorferi sensu stricto, were only partially sensitive (Z25, 297, B31, PKa-I, PBi). All strains activated the complement cascade in NHS, whereas only four strains (G1, G2, PBr, PSth) could activate complement in the presence of EGTA-Mg. After complement activation, covalently bound C3 fragments (C3b, iC3b) were detected on serum-sensitive as well as serum-resistant borrelial strains. Heterogeneity, however, was observed between serum-resistant and serum-sensitive strains with respect to deposition of C6 and C9. Whereas serum-sensitive strains were strongly positive for C6 and C9 and were, therefore, killed by the terminal complement complex (TCC), serum-resistant strains were devoid of C6 and C9 on their cell surface. The serum resistance may, therefore, be due to an absent or only transient formation of TCC on the bacterial surface. Received: 17 September 1996     

中国莱姆病螺旋体鞭毛蛋白中央区的基因克隆和表达

目的　对中国莱姆病螺旋体Borreliagarinii基因种参照菌株PD91的鞭毛蛋白中央区的编码基因进行克隆表达 ,对重组鞭毛蛋白作为莱姆病血清学诊断抗原进行初步的研究 ,并进行基因序列和氨基酸序列分析。方法　设计引物 ,用PCR技术获得PD91的鞭毛蛋白中央区的编码基因片段 ,经酶切、连接 ,插入质粒pET 30a中 ,构成重组质粒pET30a mfla,转化到大肠杆菌BL2 1,提取质粒进行酶切、DNA测序和氨基酸序列分析鉴定 ,诱导表达 ,筛选高效表达株 ,应用SDS PAGE和Westernblot鉴定重组蛋白及其抗原性。结果　成功地获得了鞭毛蛋白中央区的编码基因片段和基因重组 ,重组蛋白在宿主菌BL2 1中高效表达。Westernblot结果显示重组鞭毛蛋白的中央区与PD91的鞭毛蛋白具有相同的抗原性。经测序显示该中央区基因片段为鞭毛蛋白基因的 4 0 9～ 786bp ,与北美莱姆病螺旋体标准株B31的DNA碱基序列比较分析 ,同源性 92 %。结论　成功地对中国莱姆病螺旋体Borreliagarinii基因种的鞭毛蛋白中央区进行了克隆表达 ,并证实具有抗原性 ,为我国莱姆病血清学诊断研究提供资料。     

Comparison of growth of Borrelia afzelii, B. garinii, and B. burgdorferi sensu stricto in MKP and BSK-II medium

Lyme borreliosis is a tick-borne disease caused by genetically diverse Borrelia strains including B. afzelii, B. garinii, and B. burgdorferi sensu stricto (s.s.). The aim of the present study was to assess and compare the growth of one strain per species of B. afzelii, B. garinii, and B. burgdorferi s.s. in modified Kelly-Pettenkofer (MKP) and Barbour-Stonner-Kelly-II (BSK-II) medium, and to check for the presence of the overgrowth after inoculating the media with a mixture of two different Borrelia species. All three Borrelia strains grew well in both media. In the majority of the experiments the number of B. afzelii cells was higher in MKP than in BSK-II medium while for B. garinii and B. burgdorferi s.s. a tendency for better growth in BSK-II than MKP was established. In a mixture of equivalent amounts of two species, B. burgdorferi s.s. as a rule overgrew the other two species while in the mixture of B. afzelii and B. garinii the latter was a "dominant" strain. Comparing the performance of the two media, B. burgdorferi s.s. usually overgrew either B. afzelii or B. garinii in MKP as well as in BSK-II medium, however, the results were found to be statistically significant only for MKP medium. In the mixture of B. afzelii and B. garinii the latter was the predominant species but significant differences were established only for BSK-II medium. It seems that the overgrowth is predominantly the result of the characteristics of the individual Borrelia species and most probably not a consequence of growth differences in the two culture media. Further work with a larger number of strains is needed to confirm these findings.