High Levels Of IgG Antibodies Research Articles

СТРАТЕГИЯ ДИАГНОСТИКИ ТОКСОПЛАЗМОЗА В РАЗЛИЧНЫХ ГРУППАХ НАСЕЛЕНИЯ

The analysis of laboratory examination of the population with latent and chronic toxoplasmosis has a number of complexities. Therefore, the search for accurate biomarkers of toxoplasmosis to differentiate the stage of invasion development is very important. From the group of 559 persons, the infection of T. gondii was found in 27,9%. It is shown that a person is more often infected in childhood, teenage years and adolescence, which is marked by the presence of IgM antibodies and high levels of IgG antibodies. It was confirmed that IgA antibodies is an effective marker of reactivation of latent toxoplasmosis. The laboratory determination of toxoplasmosis markers showed a significant heterogeneity of the study group in terms of the level of invasion, depending on age, as well as the possibility to identify the stage and duration of the disease, including reactivation. Keyword: toxoplasmosis, antibody detection, latent invasion, reactivation, immunological diagnostics

Оценка IgG-зависимого гуморального и клеточного иммунитета у лиц, перенесших Covid-19, и у здоровых добровольцев

43 individuals (medical workers) were examined, 15 of them suffered from COVID-19;7 persons had negative PCR tests, however, there were signs of a respiratory infection in the anamnesis;21 participants denied having had COVID-19 and any respiratory infections in the past year. The serum level of neutralizing IgG to S-Trimer Protein SARS-Cov-2 was determined. The number of antigen-recognizing receptors of B-lymphocytes represented by membrane IgG to S-Trimer Protein SARSCov-2 was also evaluated. It was found that in some of the subjects who consider themselves not sick, antibodies and antigen-recognizing receptors of B-lymphocytes are not detected. However, a significant proportion of healthy individuals (67 %) were found to have elevated levels of serum antibodies and a high number of antigen-recognizing receptors. Persons who recovered from COVID-19 had elevated levels of serum IgG antibodies and high levels of antigen-recognizing receptors of B-lymphocytes in peripheral blood. However, in patients with a more severe course of infection who were prescribed glucocorticosteroids during the treatment, the serum IgG and lymphocyte receptor levels were lower. Individuals who noted respiratory infection of varying severity during the epidemic period, but not confirmed by PCR for COVID-19 and did not receive glucocorticosteroids, there were high levels of serum neutralizing IgG antibodies and a high content of antigenrecognizing receptors of peripheral blood B-lymphocytes, represented by membrane IgG to S-Trimer Protein SARS-Cov-2.

Optimized production and immunogenicity of an insect virus-based chikungunya virus candidate vaccine in cell culture and animal models

ABSTRACT A chimeric Eilat/ Chikungunya virus (EILV/CHIKV) was previously reported to replicate only in mosquito cells but capable of inducing robust adaptive immunity in animals. Here, we initially selected C7/10 cells to optimize the production of the chimeric virus. A two-step procedure produced highly purified virus stocks, which was shown to not cause hypersensitive reactions in a mouse sensitization study. We further optimized the dose and characterized the kinetics of EILV/CHIKV-induced immunity. A single dose of 108 PFU was sufficient for induction of high levels of CHIKV-specific IgM and IgG antibodies, memory B cell and CD8+ T cell responses. Compared to the live-attenuated CHIKV vaccine 181/25, EILV/CHIKV induced similar levels of CHIKV-specific memory B cells, but higher CD8+ T cell responses at day 28. It also induced stronger CD8+, but lower CD4+ T cell responses than another live-attenuated CHIKV strain (CHIKV/IRES) at day 55 post-vaccination. Lastly, the purified EILV/CHIKV triggered antiviral cytokine responses and activation of antigen presenting cell (APC)s in vivo, but did not induce APCs alone upon in vitro exposure. Overall, our results demonstrate that the EILV/CHIKV vaccine candidate is safe, inexpensive to produce and a potent inducer of both innate and adaptive immunity in mice.

Expression, purification, and characterization of pneumococcal PsaA-PspA fusion protein

Streptococcus pneumoniae is a gram-positive bacterial pathogen causing invasive pneumonia, meningitis, otitis media, and bacteremia. Owing to the current pitfalls of polysaccharide and polysaccharide-conjugate vaccines, protein vaccines are considered promising candidates against pneumonia. Pneumococcal surface protein A (PspA) and pneumococcal surface adhesin A (PsaA) are virulence proteins showing good immunogenicity and protective effects against S. pneumoniae strains in mice. In this study, we expressed the fusion protein PsaA-PspA, which consists of PsaA and the N-terminal region of PspA family 1 and 2, in Escherichia coli. We describe a novel and effective method to purify PsaA-PspA using hydroxyapatite and two-step chromatography. After determining the optimal induction conditions and a series of purification steps, we obtained PsaA-PspA fusion protein with over 95% purity at a final yield of 22.44% from the starting cell lysate. The molecular weight of PsaA-PspA was approximately 83.6 kDa and its secondary structure was evaluated by circular dichroism. Immunization with the purified protein induced high levels of IgG antibodies in mice. Collectively, these results demonstrate that our purification method can effectively produce high-purity PsaA-PspA fusion protein with biological activity and chemical integrity, which can be widely applied to the purification of other PspA subclass proteins.

Structural Insight into CVB3-VLP Non-Adjuvanted Vaccine.

Coxsackievirus B (CVB) enteroviruses are common pathogens that can cause acute and chronic myocarditis, dilated cardiomyopathy, aseptic meningitis, and they are hypothesized to be a causal factor in type 1 diabetes. The licensed enterovirus vaccines and those currently in clinical development are traditional inactivated or live attenuated vaccines. Even though these vaccines work well in the prevention of enterovirus diseases, new vaccine technologies, like virus-like particles (VLPs), can offer important advantages in the manufacturing and epitope engineering. We have previously produced VLPs for CVB3 and CVB1 in insect cells. Here, we describe the production of CVB3-VLPs with enhanced production yield and purity using an improved purification method consisting of tangential flow filtration and ion exchange chromatography, which is compatible with industrial scale production. We also resolved the CVB3-VLP structure by Cryo-Electron Microscopy imaging and single particle reconstruction. The VLP diameter is 30.9 nm on average, and it is similar to Coxsackievirus A VLPs and the expanded enterovirus cell-entry intermediate (the 135s particle), which is ~2 nm larger than the mature virion. High neutralizing and total IgG antibody levels, the latter being a predominantly Th2 type (IgG1) phenotype, were detected in C57BL/6J mice immunized with non-adjuvanted CVB3-VLP vaccine. The structural and immunogenic data presented here indicate the potential of this improved methodology to produce highly immunogenic enterovirus VLP-vaccines in the future.

Diagnosis of COVID-19 in a Dengue-Endemic Area.

.Emergence of SARS-CoV-2 in dengue virus (DENV)–endemic areas complicates the diagnosis of both infections. COVID-19 cases may be misdiagnosed as dengue, particularly when relying on DENV IgM, which can remain positive months after infection. To estimate the extent of this problem, we evaluated sera from 42 confirmed COVID-19 patients for evidence of DENV infection. No cases of SARS-CoV-2 and DENV coinfection were identified. However, recent DENV infection, indicated by the presence of DENV IgM and/or high level of IgG antibodies, was found in seven patients. Dengue virus IgM and/or high IgG titer should not exclude COVID-19. SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) testing is appropriate when dengue nonstructural protein 1 (NS1) or RT-PCR is negative. Given the possibility of coinfection, testing for both DENV and SARS-CoV-2 is merited in the setting of the current pandemic.

Effect of deletion of gra17 and gra23 genes on the growth, virulence, and immunogenicity of type II Toxoplasma gondii.

The protozoan parasite Toxoplasma gondii secretes a number of dense granule proteins (GRAs) from the dense granule organelle to manipulate the host cell. Two of these effector proteins (GRA17 and GRA23) are involved in the trafficking of molecules between the parasitophorous vacuole (PV) and the host cell cytoplasm. However, their roles in establishing chronic infection remain obscured. In this study, CRISPR-Cas9 was used to delete gra17 or gra23 gene in T. gondii Pru strain (type II). The growth, the virulence, the ability to establish chronic infection, and the immunogenicity of the constructed mutant strains were investigated in Kunming mice. Pru:Δgra17 and Pru:Δgra23 mutants developed PVs with abnormal morphology and exhibited reduced growth rate, compared with the wild-type Pru strain. Deletion of gra17 abrogated acute infection and blocked cyst formation. Although the deletion of gra23 caused slight attenuation of the parasite virulence in mice, it caused a significant reduction in cyst formation. Immunization with Pru:Δgra17 induced high levels of IgG (IgG1 and IgG2a) antibodies and cytokines (interleukin-2 [IL-2], IL-10, IL-12, and interferon gamma [IFN-γ]), which conferred significant protection in mice challenged with virulent type I (RH), ToxoDB#9 (PYS) strains, or less virulent type II (Pru) strain of T. gondii. These findings show that GRA17 and GRA23 play important roles in T. gondii chronic infection and that irreversible deletion of gra17 in T. gondii type II Pru strain can be a viable option for stimulating protective immunity to T. gondii infection.

Preventive Allergen-Specific Vaccination Against Allergy: Mission Possible?

Vaccines for infectious diseases have improved the life of the human species in a tremendous manner. The principle of vaccination is to establish de novo adaptive immune response consisting of antibody and T cell responses against pathogens which should defend the vaccinated person against future challenge with the culprit pathogen. The situation is completely different for immunoglobulin E (IgE)-associated allergy, an immunologically-mediated hypersensitivity which is already characterized by increased IgE antibody levels and T cell responses against per se innocuous antigens (i.e., allergens). Thus, allergic patients suffer from a deviated hyper-immunity against allergens leading to inflammation upon allergen contact. Paradoxically, vaccination with allergens, termed allergen-specific immunotherapy (AIT), induces a counter immune response based on the production of high levels of allergen-specific IgG antibodies and alterations of the adaptive cellular response, which reduce allergen-induced symptoms of allergic inflammation. AIT was even shown to prevent the progression of mild to severe forms of allergy. Consequently, AIT can be considered as a form of therapeutic vaccination. In this article we describe a strategy and possible road map for the use of an AIT approach for prophylactic vaccination against allergy which is based on new molecular allergy vaccines. This road map includes the use of AIT for secondary preventive vaccination to stop the progression of clinically silent allergic sensitization toward symptomatic allergy and ultimately the prevention of allergic sensitization by maternal vaccination and/or early primary preventive vaccination of children. Prophylactic allergy vaccination with molecular allergy vaccines may allow halting the allergy epidemics affecting almost 30% of the population as it has been achieved for vaccination against infectious diseases.

Type I pili, CsuA/B and FimA induce a protective immune response against Acinetobacter baumannii

BackgroundAcinetobacter baumannii, a nosocomial pathogen, is considered as a common cause of hospital and community-acquired infections. Emerging multidrug-resistance in this pathogen followed by subsequent problems in treatment has been increasing to alarming levels that warrant investigation of new therapeutic approaches. One strategy to reduce antibiotic resistance is to use of vaccines. Although there is no vaccine currently in development for this pathogen, different attempts have been made to develop one. MethodsIn this study, we used two different recombinant pilus proteins (CsuA/B and FimA) either singly or in combination to evaluate protective efficacy against A. baumannii in lethal and sub-lethal murine sepsis models. FindingsActive immunization with recombinant proteins in combination elicited high levels of IgG antibody after the first immunization and provided 62% (five of eight mice; p < 0·001) protection against a lethal dose (1·2 × 106 CFU) of A. baumannii along with efficient clearance of bacteria in internal organs viz. spleen, liver, and lungs at sub-lethal challenge. Immunization with CsuA/B alone conferred partial protection as demonstrated by low survival rate (three [37%] of eight mice; p < 0·05) after lethal challenge and reduction of bacteria in internal organs of the mice after 24 h post-sub-lethal infection. Immunization with FimA, in comparison to CsuA/B, showed better protection (four [50%] of eight mice; p < 0·01) and reduction in CFU after 14 h. InterpretationOur results showed that pilus proteins in combination as a single immunogen could potentially impart protection against A. baumannii. FundingShahed University.

A Lactococcus lactis-vectored oral vaccine induces protective immunity of mice against enterotoxigenic Escherichia coli lethal challenge

Enterotoxigenic Escherichia coli (ETEC) is a global primary pathogenic bacterium causing diarrhoea in human and a wide variety of neonatal animals. Lactococcus lactis as non-pathogenic and food-grade lactic acid bacteria has already been explored as a vector for mucosal vaccine. Here, the current study was undertaken to evaluate the live recombinant L. lactis (rL. lactis) vaccine expressing the trivalent enterotoxin protein STa-LTB-STb and the F5 fimbrial antigen (SLS-F5) with OmpH of Yersinia enterocolitica in protection against ETEC. Western blot confirmed the expression of fusion protein SLS-F5-OmpH in nisin-controlled expression (NICE) system. Mice orally immunized with rL. lactis-SLS-F5-OmpH were observed to produce high levels of mucosal SIgA and serum IgG antibodies, while also inducing increases in the production of CD4+ and CD8+ T cells, lymphocyte proliferation, and secretion of cytokines. Moreover, orally immunized mice produced complete protection after ETEC challenge. The above results suggested that rL. lactis-SLS-F5-OmpH has the potential as a candidate for oral vaccine against ETEC.

Immunostimulatory efficacy and protective potential of putative TgERK7 protein in mice experimentally infected by Toxoplasma gondii

The extracellular signal-regulated kinases (ERKs) serve as important determinants of cellular signal transduction pathways, and hence may play important roles during infections. Previous work suggested that putative ERK7 of Toxoplasma gondii is required for efficient intracellular replication of the parasite. However, the antigenic and immunostimulatory properties of TgERK7 protein remain unknown. The objective of this study was to produce a recombinant TgERK7 protein in vitro and to evaluate its effect on the induction of humoral and T cell-mediated immune responses against T. gondii infection in BALB/c mice. Immunization using TgERK7 mixed with Freund's adjuvants significantly increased the ratio of CD3e+CD4+ T/CD3e+CD8a+ T lymphocytes in spleen and elevated serum cytokines (IFN-γ, IL-2, IL-4, IL-10, IL-12p70, IL-23, MCP-1, and TNF-α) in immunized mice compared to control mice. On the contrary, immunization did not induce high levels of serum IgG antibodies. Five predicted peptides of TgERK7 were synthesized and conjugated with KLH and used to analyze the antibody specificity in the sera of immunized mice. We detected a progressive increase in the antibody level only against TgERK7 peptide A (DEVDKHVLRKYD). Antibody raised against this peptide significantly decreased intracellular proliferation of T. gondii in vitro, suggesting that peptide A can potentially induce a protective antibody response. We also showed that immunization improved the survival rate of mice challenged with a virulent strain and significantly reduced the parasite cyst burden within the brains of chronically infected mice. Our data show that TgERK7-based immunization induced TgERK7 peptide A-specific immune responses that can impart protective immunity against T. gondii infection. The therapeutic potential of targeting ERK7 signaling pathway for future toxoplasmosis treatment is warranted.

Intranasal immunization with outer membrane vesicle pertussis vaccine confers broad protection through mucosal IgA and Th17 responses

A vaccine based on outer membrane vesicles of pertussis (omvPV) is protective in a mouse-challenge model and induces a broad antibody and mixed Th1/Th2/Th17 response against multiple antigens following subcutaneous immunization. However, this route did not result in mucosal immunity and did not prevent nasopharyngeal colonization. In this study, we explored the potential of intranasal immunization with omvPV. Only intranasal immunization induced strong mucosal immune responses that encompasses enhanced pulmonary and nasal IgA antibody levels, mainly directed against Vag8 and LPS. Furthermore, high numbers of IgA- and IgG-producing plasma cells were detected as well as lung-resident IgA memory B-cells. Finally, only intranasal immunization induced pulmonary Th1/Th17-related cytokine responses. The magnitude and type of systemic immunity was comparable between both routes and included high systemic IgG antibody levels, strong IgG-producing plasma cell responses, memory B-cells residing in the spleen and systemic Th1/Th2/Th17-related cytokine responses. Importantly, only intranasal immunization prevented colonization in both the lungs and the nasal cavity. In conclusion, intranasal omvPV immunization induces mucosal IgA and Th17-mediated responses without influencing the systemic immunity profile. These responses resulted in prevention of Bordetella pertussis colonization in the respiratory tract, including the nasal cavity, thereby potentially preventing transmission.

Antibody response to lipopolysaccharides and recombinant proteins of Shiga toxin (STX)-producing Escherichia coli (STEC) in children with haemolytic uraemic syndrome in Poland.

Typical haemolytic uraemic syndrome (STEC-HUS), caused by Shiga toxin (Stx)-producing Escherichia coli (STEC), is a serious, life-threating disease that mainly affects children. Bacteriological and genetic tests are commonly used in the routine laboratory diagnosis of STEC-HUS; however, serological methods have emerged as useful and reliable diagnostic tools, especially when bacterial isolation fails. In this study, we present the results of the serological investigation of 72 paediatric patients suspected for HUS, hospitalized during 2011-2019 at the Department of Pediatrics and Nephrology of Children's Hospitals in Poland. During the routine laboratory investigation STEC strains were isolated only from nine stool samples. However, serological investigations confirmed 45 cases of STEC infections in children with HUS. In this study, 22 (48·9%) paediatric patients were infected by E. coli serotype O26, 11 (24·4%) by serotype O145, 9 (20·0%) by serotype O157, and 3 (6·7%) by E. coli serotype O111. In the majority of these patients, in addition to a high level of IgA, IgG and IgM antibodies to lipopolysaccharide of particular E. coli serotypes, antibodies to recombinant proteins Tir, Stx2b and intimin were detected. Our results confirm that serological tests are useful in the diagnosis of STEC-HUS. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that serological analysis greatly complements bacterial isolation and helps in the diagnosis and confirmation of Shiga toxin (verotoxin)-producing Escherichia coli (STEC) infections. Serological tests can be performed to qualify the patient for the typical haemolytic uraemic syndrome (STEC-HUS). In Poland, STEC-HUS in children is mostly caused by the E. coli serotype O26, which indicates that there is an increasing number of non-O157 STEC infections associated with human diseases in Europe.

Immunity and autoantibodies of a mouse strain with autistic-like behavior

Female and male mice of the BTBR T+Itpr3tf/J (BTBR) strain have behaviors that resemble autism spectrum disorder. In comparison to C57BL/6 (B6) mice, BTBR mice have elevated humoral immunity, in that they have naturally high serum IgG levels and generate high levels of IgG antibodies, including autoantibodies to brain antigens. This study focused on the specificities of autoantibodies and the immune cells and their transcription factors that might be responsible for the autoantibodies. BTBR IgG autoantibodies bind to neurons better than microglia and with highest titer to nuclear antigens. Two of the antigens identified were alpha-enolase (ENO1) and dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial (DLST). Surprisingly based on IgG levels, the blood and spleens of BTBR mice have more CD4+ and CD8+ T cells, but fewer B cells than B6 mice. The high levels of autoantibodies in BTBR relates to their splenic T follicular helper (Tfh) cell levels, which likely are responsible for the higher number of plasma cells in BTBR mice than B6 mice. BTBR mice have increased gene expression of interleukin-21 receptor (Il-21r) and Paired Box 5 (Pax5), which are known to aid B cell differentiation to plasma cells, and an increased Lysine Demethylase 6B (Kdm6b)/DNA Methyltransferase 1 (Dnmt1) ratio, which increases gene expression. Identification of gene expression and immune activities of BTBR mice may aid understanding of mechanisms associated with autism since neuroimmune network interactions have been posited and induction of autoantibodies may drive the neuroinflammation associated with autism.

Detection of antibodies to decorin-binding protein A (DbpA) and DbpB after infection of dogs with Borrelia burgdorferi by tick challenge.

We characterized the antibody response to decorin-binding protein A (DbpA) or DbpB from immune serum samples collected from 27 dogs infected with Borrelia burgdorferi by Ixodes scapularis ticks. Immunoglobulin M (IgM) antibodies to DbpA or DbpB were rarely detected, but high levels of IgG antibodies to DbpA were detected in 16 of 27 of the immune sera collected 1 mo after infection, 20 of 25 of the sera collected after 2 mo, and each of the 23, 17, or 11 serum samples evaluated after 3, 4, or 5 mo, respectively. In addition, IgG antibodies to DbpB were detected in 22 of 27 (p = 0.005) tested dogs after 1 mo, and the frequency of detecting the antibodies thereafter closely mimicked the antibody responses to DbpA. Moreover, antibodies to DbpA or DbpB were not produced by dogs vaccinated with a whole-cell B. burgdorferi bacterin; removing the antibodies to DbpA by adsorption to recombinant DbpA (rDbpA) did not affect the reactivity detected by a rDbpB ELISA. Therefore, the findings from our preliminary study showed that antigenically distinct antibodies to DbpA or DbpB are produced reliably during canine infection with B. burgdorferi, and the response is not confounded by vaccination with a Lyme disease bacterin. Larger studies are warranted to more critically evaluate whether detecting the antibody responses can improve serodiagnostic confirmation of canine Lyme disease.

Pandemic influenza A(H1N1pdm09) vaccine induced high levels of influenza-specific IgG and IgM antibodies as analyzed by enzyme immunoassay and dual-mode multiplex microarray immunoassay methods

Influenza A viruses continue to circulate throughout the world as yearly epidemics or occasional pandemics. Influenza infections can be prevented by seasonal multivalent or monovalent pandemic vaccines. In the present study, we describe a novel multiplex microarray immunoassay (MAIA) for simultaneous measurement of virus-specific IgG and IgM antibodies using Pandemrix-vaccinated adult sera collected at day 0 and 28 and 180 days after vaccination as the study material. MAIA showed excellent correlation with a conventional enzyme immunoassay (EIA) in both IgG and IgM anti-influenza A antibodies and good correlation with hemagglutination inhibition (HI) test. Pandemrix vaccine induced 5–30 fold increases in anti-H1N1pdm09 influenza antibodies as measured by HI, EIA or MAIA. A clear increase in virus-specific IgG antibodies was found in 93–97% of vaccinees by MAIA and EIA. Virus-specific IgM antibodies were found in 90–92% of vaccinees by MAIA and EIA, respectively and IgM antibodies persisted for up to 6 months after vaccination in 55–62% of the vaccinees. Pandemic influenza vaccine induced strong anti-influenza A IgG and IgM responses that persisted several months after vaccination. MAIA was demonstrated to be an excellent method for simultaneous measurement of antiviral IgG and IgM antibodies against multiple virus antigens. Thus the method is well suitable for large scale epidemiological and vaccine immunity studies.

Hemagglutinin-Specific Non-neutralizing Antibody Is Essential for Protection Provided by Inactivated and Viral-Vectored H7N9 Avian Influenza Vaccines in Chickens.

Hemagglutination inhibition (HI) and virus neutralization antibody (nAb) do not always correlate with the protection of H7 avian influenza vaccines in mammals and humans. The contribution of different classes of antibodies induced by H7N9 vaccines to protection is poorly characterized in chickens. In this study, antibody responses induced by both inactivated and viral-vectored H7N9 vaccines in chickens were dissected. Chickens immunized with inactivated H7N9 vaccine showed 50% seroconversion rate and low HI and nAb titers at week 3 post immunization. However, inactivated H7N9 vaccine elicited 100% seroconversion rate in terms of high levels of HA-binding IgG antibody determined by ELISA. Despite inducing low levels of nAb, inactivated H7N9 vaccine conferred full protection against H7N9 challenge in chickens and markedly inhibited virus shedding. Similarly, Newcastle disease virus (NDV)-vectored H7N9 vaccine induced marginal HI and nAb titers but high level of IgG antibody against H7N9 virus. In addition, NDV-H7N9 vaccine also provided complete protection against H7N9 challenge. Chicken antisera had a high IgG/VN ratio, indicating that a larger proportion of serum antibodies were non-neutralizing antibody (non-nAb). More importantly, passive transfer challenge experiment showed that non-neutralizing antisera provided partial protection (37.5%) of chickens against H7N9 challenge, without significant difference from that provided by neutralizing antisera. In conclusion, our results suggest that antibodies measured by the traditional HI and virus neutralization assays do not correlate with the protection of inactivated and viral-vectored H7N9 vaccines in chickens, and HA-binding non-nAb also contributes to the protection against H7N9 infection. Total binding antibody can be used as a key correlate to the protection of H7N9 vaccine.

Next Generation Sequencing-Based Fetal ABO Blood Group Prediction by Analysis of Cell-Free DNA from Maternal Plasma

Introduction: ABO blood group incompatibility between a pregnant woman and her fetus as a cause of morbidity or mortality of the fetus or newborn remains an important, albeit rare, risk. When a pregnant woman has a high level of anti-A or anti-B IgG antibodies, the child may be at risk for hemolytic disease of the fetus and newborn (HDFN). Performing a direct prenatal determination of the fetal ABO blood group can provide valuable clinical information. Objective: Here, we report a next generation sequencing (NGS)-based assay for predicting the prenatal ABO blood group. Materials and Methods: A total of 26 plasma samples from 26 pregnant women were tested from gestational weeks 12 to 35. Of these samples, 20 were clinical samples and 6 were test samples. Extracted cell-free DNA was PCR-amplified using 2 primer sets followed by NGS. NGS data were analyzed by 2 different methods, FASTQ analysis and a grep search, to ensure robust results. The fetal ABO prediction was compared with the known serological infant ABO type, which was available for 19 samples. Results: There was concordance for 19 of 19 predictable samples where the phenotype information was available and when the analysis was done by the 2 methods. For immunized pregnant women (n = 20), the risk of HDFN was predicted for 12 fetuses, and no risk was predicted for 7 fetuses; one result of the clinical samples was indeterminable. Cloning and sequencing revealed a novel variant harboring the same single nucleotide variations as ABO*O.01.24 with an additional c.220C>T substitution. An additional indeterminable result was found among the 6 test samples and was caused by maternal heterozygosity. The 2 indeterminable samples demonstrated limitations to the assay due to hybrid ABO genes or maternal heterozygosity. Conclusions: We pioneered an NGS-based fetal ABO prediction assay based on a cell-free DNA analysis from maternal plasma and demonstrated its application in a small number of samples. Based on the calculations of variant frequencies and ABO*O.01/ABO*O.02 heterozygote frequency, we estimate that we can assign a reliable fetal ABO type in approximately 95% of the forthcoming clinical samples of type O pregnant women. Despite the vast genetic variations underlying the ABO blood groups, many variants are rare, and prenatal ABO prediction is possible and adds valuable early information for the prevention of ABO HDFN.

Low toxicity and high immunogenicity of an inactivated vaccine candidate against COVID-19 in different animal models

ABSTRACT The ongoing COVID-19 pandemic is causing huge impact on health, life, and global economy, which is characterized by rapid spreading of SARS-CoV-2, high number of confirmed cases and a fatality/case rate worldwide reported by WHO. The most effective intervention measure will be to develop safe and effective vaccines to protect the population from the disease and limit the spread of the virus. An inactivated, whole virus vaccine candidate of SARS-CoV-2 has been developed by Wuhan Institute of Biological Products and Wuhan Institute of Virology. The low toxicity, immunogenicity, and immune persistence were investigated in preclinical studies using seven different species of animals. The results showed that the vaccine candidate was well tolerated and stimulated high levels of specific IgG and neutralizing antibodies. Low or no toxicity in three species of animals was also demonstrated in preclinical study of the vaccine candidate. Biochemical analysis of structural proteins and purity analysis were performed. The inactivated, whole virion vaccine was characterized with safe double-inactivation, no use of DNases and high purity. Dosages, boosting times, adjuvants, and immunization schedules were shown to be important for stimulating a strong humoral immune response in animals tested. Preliminary observation in ongoing phase I and II clinical trials of the vaccine candidate in Wuzhi County, Henan Province, showed that the vaccine is well tolerant. The results were characterized by very low proportion and low degree of side effects, high levels of neutralizing antibodies, and seroconversion. These results consistent with the results obtained from preclinical data on the safety.

Fully Synthetic Invariant NKT Cell-Dependent Self-Adjuvanting Antitumor Vaccines Eliciting Potent Immune Response in Mice.

Constructing an effective therapeutic cancer vaccine is very attractive and promising for cancer immunotherapy. However, the poor immunogenicity of tumor antigens and suppression of the immune system in the tumor microenvironment are two major obstacles for developing effective cancer vaccines. Invariant NKT cells (iNKT cells), which are essential bridges between the innate and adaptive immune systems, can be rapidly activated by their agonists and, consequently, evoke whole immune systems. Herein, we conjugated a potent agonist of the iNKT cell, α-galactosylceramide (α-GalCer), with the tumor-associated MUC1 glycopeptide antigens as novel self-adjuvanting cancer vaccines through click chemistry. Immunological studies revealed that the mouse immune system was potently evoked and that high levels of tumor-specific IgG antibodies were elicited by vaccine conjugates without an external adjuvant. The produced antibodies could specifically recognize and bind to antigen-expressing cancer cells and, subsequently, induce cytotoxicity through complement-dependent cytotoxicity. Thus, the insertion of α-GalCer significantly improved the immunogenicity of the MUC1 glycopeptide and induced strong antigen-specific antitumor responses, indicating that α-GalCer is an effective built-in adjuvant for constructing potent chemical synthetic antitumor vaccines.