Identification of the glycopeptide epitope recognized by a protective Cryptosporidium monoclonal antibody.

Cryptosporidium species are a leading cause of pediatric diarrheal disease and death in low- and middle-income countries and pose a particular threat to immunocompromised individuals. As a zoonotic pathogen, Cryptosporidium can have devastating effects on the health of neonatal calves. Despite its impact on human and animal health, consistently effective drug treatments for cryptosporidiosis are lacking and no vaccine is available. We previously showed that C. parvum mucin-like glycoproteins, gp40, and gp900 express an epitope identified by a monoclonal antibody 4E9. 4E9 neutralized C. parvum infection in vitro as did glycan-binding proteins specific for the Tn antigen (GalNAc-α1-S/T). Here, we show that 4E9 ameliorates disease in vivo in a calf challenge model. The 4E9 epitope is present on C. hominis in addition to C. parvum gp40 and gp900 and localizes to the plasma membrane and dense granules of invasive and intracellular stages. To characterize the epitope recognized by 4E9, we probed a glycan array containing over 500 defined glycans together with a custom-made glycopeptide microarray containing glycopeptides from native mucins or C. parvum gp40 and gp15. 4E9 exhibited no binding to the glycan array but bound strongly to glycopeptides from native mucins or gp40 on the glycopeptide array, suggesting that the antibody epitope contains both peptide and glycan moieties. 4E9 only recognized glycopeptides with adjacent S or T residues in the motif S*/T*-X-S*/T* where X = 0 or 1. These data define the 4E9 epitope and have implications for the inclusion of the epitope in the development of vaccines or other immune-based therapies.

Molecular Basis of a Protective/Neutralizing Monoclonal Antibody Targeting Envelope Proteins of both Tick-Borne Encephalitis Virus and Louping Ill Virus.

Tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are members of the tick-borne flaviviruses (TBFVs) in the family Flaviviridae which cause encephalomeningitis and encephalitis in humans and other animals. Although vaccines against TBEV and LIV are available, infection rates are rising due to the low vaccination coverage. To date, no specific therapeutics have been licensed. Several neutralizing monoclonal antibodies (MAbs) show promising effectiveness in the control of TBFVs, but the underlying molecular mechanisms are yet to be characterized. Here, we determined the crystal structures of the LIV envelope (E) protein and report the comparative structural analysis of a TBFV broadly neutralizing murine MAb (MAb 4.2) in complex with either the LIV or TBEV E protein. The structures reveal that MAb 4.2 binds to the lateral ridge of domain III of the E protein (EDIII) of LIV or TBEV, an epitope also reported for other potently neutralizing MAbs against mosquito-borne flaviviruses (MBFVs), but adopts a unique binding orientation. Further structural analysis suggested that MAb 4.2 may neutralize flavivirus infection by preventing the structural rearrangement required for membrane fusion during virus entry. These findings extend our understanding of the vulnerability of TBFVs and other flaviviruses (including MBFVs) and provide an avenue for antibody-based TBFV antiviral development.IMPORTANCE Understanding the mechanism of antibody neutralization/protection against a virus is crucial for antiviral countermeasure development. Tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are tick-borne flaviviruses (TBFVs) in the family Flaviviridae They cause encephalomeningitis and encephalitis in humans and other animals. Although vaccines for both viruses are available, infection rates are rising due to low vaccination coverage. In this study, we solved the crystal structures of the LIV envelope protein (E) and a broadly neutralizing/protective TBFV MAb, MAb 4.2, in complex with E from either TBEV or LIV. Key structural features shared by TBFV E proteins were analyzed. The structures of E-antibody complexes showed that MAb 4.2 targets the lateral ridge of both the TBEV and LIV E proteins, a vulnerable site in flaviviruses for other potent neutralizing MAbs. Thus, this site represents a promising target for TBFV antiviral development. Further, these structures provide important information for understanding TBFV antigenicity.

Systematic review shows that immunising internationally adopted children is a major challenge for primary health care.

AIM: This systematic review aimed to provide an overview of the immunisation of internationally adopted children and to discuss possible vaccination strategies. METHODS: A literature search was performed covering papers published in English from 1988 to 15 June 2018 using the Ovid MEDLINE, EMBASE and Cochrane Library databases. This identified 749 studies and 41 full texts were evaluated. RESULTS: Overall, 19 studies conducted between 1988 and 2016 fulfilled our inclusion criteria. These covered 7663 children aged 1.1-5.7 years adopted from Asia, Eastern Europe, Africa and South and Central America. Tetanus protective antibody levels ranged from 35 to 95%, and similar data were reported for diphtheria. A higher percentage of adoptees had protective antibody levels for polio (50-93%) and measles (62-95%). More than a third (35%) did not have protective antibody titres for hepatitis B. Only one study investigated adoptees with protective antibodies against haemophilus influenza, and it reported that this was around 66%. CONCLUSION: The appropriate immunisation of internationally adopted children is a major challenge for primary health care and a number of different approaches have been suggested, with no clear conclusions. Further studies on the cost-effectiveness of different approaches should be performed to optimise screening strategies and develop recommendations.

Serum Dehydroepiandrosterone sulfate (DHEA-S) level and its potential impact on immune responses and symptom severity after Oxford-AstraZeneca COVID-19 vaccination.

BACKGROUND: Dehydroepiandrosterone sulfate (DHEA-S) has been associated with an immunomodulatory function. This study aims to explore the relationship between serum levels of DHEA-S and the immune responses triggered by the Oxford-AstraZeneca COVID-19 vaccine in individuals candidate for vaccination. METHODS: Serum levels of DHEA-S, cytokine release, antibody production and virus neutralization potential were assessed in 50 male and 50 female subjects before and 2 weeks after vaccination with Oxford-AstraZeneca COVID-19 vaccine. RESULTS: Level of DHEA-S before and 2 weeks after first and second dose of vaccination was not different significantly. Levels of Interleukin (IL)-2 and Interferon (IFN)-γ were significantly higher in the supernatant of peripheral blood mononuclear cells (PBMCs) obtained from subjects 2 weeks after both first and second dose of vaccination compared to before vaccination. Serum levels of IgM 2 weeks after first dose of vaccination was significantly higher compared to before first dose of vaccination. However, serum levels of IgG 2 weeks after first and second dose of vaccination were significantly higher compared to before first and second dose of vaccination. The 50 % focus reduction neutralization test (FRNT50) titer was significantly higher 2 weeks after both first and second dose of vaccination compared to before vaccination. Levels of DHEA-S did not have significant correlation with levels of IL-2, IFN-γ, IgM and IgG, and FRNT50 before and after first and second dose of vaccination. Vaccination did not result in intense unwanted clinical presentations. CONCLUSION: DHEA-S is not involved in the quality of protective immune response during Oxford-AstraZeneca COVID-19 vaccination.

Co-formulation of the rF1V plague vaccine with depot-formulated cytokines enhances immunogenicity and efficacy to elicit protective responses against aerosol challenge in mice.

This study evaluated a depot-formulated cytokine-based adjuvant to improve the efficacy of the recombinant F1V (rF1V) plague vaccine and examined the protective response following aerosol challenge in a murine model. The results of this study showed that co-formulation of the Alhydrogel-adsorbed rF1V plague fusion vaccine with the depot-formulated cytokines recombinant human interleukin 2 (rhuIL-2) and/or recombinant murine granulocyte macrophage colony-stimulating factor (rmGM-CSF) significantly enhances immunogenicity and significant protection at lower antigen doses against a lethal aerosol challenge. These results provide additional support for the co-application of the depot-formulated IL-2 and/or GM-CSF cytokines to enhance vaccine efficacy.

New Neutralizing Epitope Exposed on the Domain II of Tick-Borne Encephalitis Virus Envelope Glycoprotein E.

Orthoflavivirus encephalitidis, formerly tick-borne encephalitis virus (TBEV), belongs to the Orthoflavivirus genus. TBEV is transmitted by tick bites and infection with TBEV can lead to serious disorders of the central nervous system. In this study, a new protective monoclonal mouse antibody (mAb) FVN-32, with high binding activity to glycoprotein E of TBEV, was selected and examined in post exposure prophylaxis in a mouse model of TBEV infection. BALB/c mice were injected mAb FVN-32 at doses of 200 µg, 50 µg, and 12.5 µg per mouse one day after a TBEV challenge. mAb FVN-32 showed 37.5% protective efficacy when administered at doses of 200 µg and 50 µg per mouse. The epitope for protective mAb FVN-32 was localized in TBEV glycoprotein E domain I+II, using a set of truncated fragments of glycoprotein E. Additionally, the target site recognized by mAb FVN-32 was defined using combinatorial libraries of peptides. Three-dimensional modeling revealed that the site is dspatially close to the fusion loop, but does not come into contact with it, and is localized in a region between 247 and 254 amino acid residues on the envelope protein. This region is conserved among TBEV-like orthoflaviviruses.

Anti-complement factor H (CFH) autoantibodies could delay pristane-induced lupus nephritis.

PURPOSE: Anti-complement factor H (CFH) autoantibodies could be detected in lupus and its significance remained to be elucidated. Herein, we aimed to explore the roles of anti-CFH autoantibodies based on pristane-induced lupus mice. METHODS: Twenty-four female Balb/c mice were randomly divided into four groups, with one group injected with pristane (pristane group), one group with pristane and then human CFH (hCFH) (pristane-CFH group) 3 times, and the other two as vertical controls, PBS group and PBS-CFH group. Histopathological analysis was performed six months after pristane administration. Levels of hCFH, anti-CFH autoantibodies and anti-dsDNA antibody were detected. Murine IgG (mIgG) were purified and cross-reactivity, epitopes, subclasses and functional analysis were further evaluated in vitro. RESULTS: Immunization with hCFH and subsequent development of anti-CFH autoantibodies significantly attenuated nephritis of pristane-induced lupus, including lower levels of urinary protein and serum creatinine, decreased levels of serum anti-dsDNA antibody, greatly ameliorated renal histopathologic damage, decreased IgG, complements (C1q, C3) deposits and lower inflammatory factor (IL-6) expression in glomerulus. Furthermore, the purified mIgG (contained anti-CFH autoantibodies) could recognize both hCFH and murine CFH, and the epitopes were predominantly located in hCFH short consensus repeats (SCRs) 1-4, 7 and 11-14. The IgG subclasses were predominant IgG1. The autoantibodies could enhance the binding between hCFH and C3b, and increase factor I mediated-C3b lysis in vitro. CONCLUSION: Our results suggested that anti-CFH autoantibodies could attenuate pristane-induced lupus nephritis by increasing bio-functions of CFH on regulating complement activation and controlling inflammation.

Immune persistence induced by three doses of 60 µg hepatitis B vaccine in non-responders following standard primary vaccination in Chinese adults.

This study consisted of two rounds of cross-sectional observations designed to evaluate the persistence of immune protection induced high antigen content hepatitis B (HB) vaccine at 60 µg/1.0 ml formulations administered at a three-dose schedule (Days 0, 28, and 56) in non-responders to routine HB vaccination. In the original phase 3 study, we enrolled 1091 healthy participants (16-60 years old) seronegative for antibody against HB surface antigen (anti-HBs) after primary vaccination. Participants were randomized (2:2:1) to receive three booster doses of HB vaccine containing 60 µg, 30 µg, or 10 µg of antigen per dose 28 days apart. In the group receiving the 60 µg HB vaccine, 428 participants' serum samples were available at pre-vaccination and 28 days after each vaccine dose and were included in immunogenicity analysis. With two written informed consents, we collected blood samples from 276 (67.2%) participants in 2014 and 239 (58.2%) in 2019, who had completed the full course of revaccination and reached the seropositive (anti-HBs≥10 mIU/ml) standard in the 60 µg vaccine group of the original phase 3 study. The HBV seropositive rate was found to decrease from 96.0% in 28 days after receiving the third dose of 60 µg HB vaccine, to 48.2% in 2014, and to 40.6% in 2019, with anti-HBs GMC of seropositive individuals was 584.0 mIU/ml, 142.4 mIU/ml, and 169.1 mIU/ml, respectively. Analysis of 181 vaccinees who had serologic test results available both in 2014 and in 2019, and results revealed a dynamic trend in anti-HBs titer similar to that for the whole immune persistence cohort. Of paramount importance, the serologic test results found that 24.9% (45/181) participants had higher anti-HBs concentrations in 2019 than in 2014, this could be interpreted as natural boosters, secondary to HBV exposure without infection because protected. In conclusion, protective antibody persists about 11 years after immunization of Chinese non-responders with 3 doses of 60 µg HB vaccine. Booster doses of vaccine do not seem necessary to ensure long-term protection.

Antibody responses after documented COVID-19 disease in patients with autoimmune rheumatic disease.

Patients with autoimmune rheumatic diseases (AIRD) are suspected to have less robust immune responses during COVID-19 due to underlying immune dysfunction and the use of immune-suppressive drugs. Fifty consecutive patients with a diagnosis of AIRD on disease-modifying drugs were included at around 30 days after a confirmatory test for COVID-19. Fifty controls matched one to one for age, sex, and severity of COVID-19 were also included at around 30 days after testing positive for COVID-19. Antibody titers for anti-spike protein IgG and anti-nucleocapsid protein IgG were estimated. Cases (mean age 45.9 ± 13; 76% females) and controls (mean age 45.9 ± 13; 76% females) had similar proportion of comorbidities. Of the cases, 4 had moderate and 1 had severe COVID-19, while 3 and 1 of controls had moderate and severe COVID-19 respectively. Positivity of anti-N IgG was similar between patients (80%) and controls (90%) (p = 0.26). Similarly, anti-S IgG was positive in 82% of patients and 86% of controls (p = 0.79). Both the antibodies were negative in seven (14%) patients and five (10%) of controls (p = 0.76, Fischer exact test). Only anti-N IgG titers were lower in patients as compared to controls. In four patients with rheumatoid arthritis, two with spondyloarthritis and one with eosinophilic fasciitis both antibodies were not detectable. They did not differ from the rest of the cohort in clinical characteristics. The patients with AIRD had adequate protective antibody responses to COVID-19 at a median of 30 days post-infection. Thus, the presence of AIRD or the use of immunosuppressants does not seem to influence the development of humoral immune response against COVID-19. Key Points • Patients with autoimmune rheumatic diseases (AIRD) are suspected to have less robust immune responses. • In our cohort of 50 patients with AIRD with confirmed COVID-19, only seven did not have detectable protective antibodies at 30 days post infection. • Patients with AIRD on immunosuppressants have adequate protective antibodies post COVID-19 disease, at rates similar to that in health controls.

A serological survey of Bacillus anthracis reveals widespread exposure to the pathogen in free-range and captive lions in Zimbabwe.

Numerous unknown factors influence anthrax epidemiology in multi-host systems, especially at wildlife/livestock/human interfaces. Serology tests for anti-anthrax antibodies in carnivores are useful tools in identifying the presence or absence of Bacillus anthracis in a range. These were employed to ascertain whether the disease pattern followed the recognized high- and low-risk anthrax zonation in Zimbabwe and also to establish whether anthrax was absent from Hwange National Park in which there have been no reported outbreaks. African lions (Panthera leo) (n = 114) drawn from free-range protected areas and captive game parks located in recognized high- and low-risk zones across Zimbabwe were tested for antibodies to anthrax PA antigen using the ELISA immunoassay. A random selection of 27 lion sera samples comprising 17 seropositive and 10 seronegative sera was further tested in the species-independent toxin neutralization assay (TNA) in order to validate the former as a surveillance tool for anthrax in African lions. Using the ELISA-PA immunoassay, 21.9% (25/114) of the lions tested positive for antibodies to anthrax. Seropositivity was recorded in all study areas, and there was no significant difference (p = .852) in seropositivity between lions in high- and low-risk anthrax zones. Also, there was no significant difference (McNemar's chi-square test = 0.9, p = .343) in the proportion of lions testing positive to anti-PA anthrax antibodies on ELISA-PA immunoassay compared with the TNA, with fair agreement between the two tests [kappa (K) statistic = 0.30; 0.08 < K<0.613]. Results of this study indicate that anthrax could be more widespread than 42 currently realized in Zimbabwe, and present in recognized high- and low-risk zones, including 43 where it has not been reported in over 20 years such as Hwange National Park. This is also the 44 first report documenting the presence of anthrax lethal toxin-neutralizing antibodies in naturally 45 infected carnivores, further confirming exposure to B. anthracis. The research results point to a 46 need for revisiting the currently recognized anthrax risk zones in Zimbabwe. This should be based 47 on improved surveillance of the disease in both wild and domestic animals for better understanding and control of the disease.

A compromised specific humoral immune response against the SARS-CoV-2 receptor-binding domain is related to viral persistence and periodic shedding in the gastrointestinal tract.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been redetected after discharge in some coronavirus disease 2019 (COVID-19) patients. The reason for the recurrent positivity of the test and the potential public health concern due to this occurrence are still unknown. Here, we analyzed the viral data and clinical manifestations of 289 domestic Chinese COVID-19 patients and found that 21 individuals (7.3%) were readmitted for hospitalization after detection of SARS-CoV-2 after discharge. First, we experimentally confirmed that the virus was involved in the initial infection and was not a secondary infection. In positive retests, the virus was usually found in anal samples (15 of 21, 71.4%). Through analysis of the intracellular viral subgenomic messenger RNA (sgmRNA), we verified that positive retest patients had active viral replication in their gastrointestinal tracts (3 of 16 patients, 18.7%) but not in their respiratory tracts. Then, we found that viral persistence was not associated with high viral titers, delayed viral clearance, old age, or more severe clinical symptoms during the first hospitalization. In contrast, viral rebound was associated with significantly lower levels of and slower generation of viral receptor-binding domain (RBD)-specific IgA and IgG antibodies. Our study demonstrated that the positive retest patients failed to create a robust protective humoral immune response, which might result in SARS-CoV-2 persistence in the gastrointestinal tract and possibly in active viral shedding. Further exploration of the mechanism underlying the rebound in SARS-CoV-2 in this population will be crucial for preventing virus spread and developing effective vaccines.

Characterization of neutralizing monoclonal antibody against tick-borne encephalitis virus in vivo.

Tick-borne encephalitis virus (TBEV) is the most important tick-transmitted pathogen in the family Flaviviridae and causes one of the most severe human neuroinfections. In this study, a neutralizing mouse mAb 14D5, which was previously shown to have cross-reactive binding to several flaviviruses belonging to the TBEV group, was examined for its prophylactic and therapeutic effects in BALB/c mice infected with TBEV. Before and after infection, mice were administrated mAb 14D5 at doses 100 µg and 10 µg per mouse. mAb 14D5 showed clear protective efficacy when injected at the high dose one day after infection, with survival rates that were TBEV dose-dependent. Prophylactic administration of mAb 14D5 was more effective than post-exposure administration and complete protection was documented when the mAb was administered one day before infection. The protective efficacy of mAb 14D5 was significantly higher than that of the anti-TBE serum immunoglobulin. However, no protection was observed in mice received the low dose of mAb 14D5 independent of the timing of mAb injection and TBEV dose. The ability of species-matched mAb 14D5 to mediate TBEV infection in mice was also investigated, and the results indicated that mAb 14D5 did not augment TBEV infection independent of the time of mAb administration. The neutralizing epitope for mAb 14D5 was localized in domain III of glycoprotein E of TBEV in a region between residues 301-339, which is conserved among flaviviruses from the TBEV group.

An Unusual Case of Evolving Localized Tetanus Despite Prior Immunization and Protective Antibody Titer.

Centers for Disease Control and Prevention has reported that tetanus infection in a fully immunized person with the last dose within 10 years is extremely rare. The prevalence of localized tetanus in such a scenario is unknown. Only two case reports of localized tetanus in previously immunized patients have been reported so far, making this the third one. Also, this is the first case of its kind to demonstrate evolving localized tetanus. Our patient is a 19-year-old man who presented with shortness of breath, pain in right upper extremity, shoulder, and neck. His chest X-ray and creatine kinase were normal. The patient was sent home. He presented again to our hospital two days later with difficulty swallowing and speaking as well as chest tightness. Routine blood tests, electrocardiogram, CT angiography of the chest, and transthoracic echocardiogram were normal. He gave a history of a cut in the right middle finger while removing the carpet a week before his presentation. His immunization history was complete with documented last tetanus shot nine years and two months ago. He was treated with tetanus vaccine and penicillin. His tetanus antitoxoid titer came out protective.

Anti-complement factor H autoantibodies may be protective in lupus nephritis.

BACKGROUND: This study aimed to investigate the role of anti-CFH autoantibodies in lupus nephritis based on a well-defined cohort. METHODS: One hundred twenty patients with biopsy-proven active lupus nephritis were collected as the discovery cohort, sixty patients served as the validation cohort, thirty-four patients with SLE without renal involvement (NR-SLE) were as disease controls, and thirty healthy donors were also included. The anti-CFH autoantibodies and IgG subclasses were detected by ELISA, and epitopes were evaluated by western blot. Anti-CFH autoantibodies were purified by affinity chromatography column, and the interference on the biofunctions of CFH was further studied by the C3b binding assay and cofactor activity assay in vitro. RESULTS: The prevalence of anti-CFH autoantibodies in lupus nephritis was significantly higher than that in healthy controls (8.3% (10/120) vs. 0% (0/30), P = 0.017), and no significant difference was found between the discovery and the validation group (8.3% (10/120) vs. 11.7% (7/60), P = 0.268) or the discovery and the NR-SLE group (8.3% (10/120) vs. 11.8% (4/34), P = 0.231). The subclass was mainly IgG2 (7/10), and major epitopes were in the middle (8/10 in SCRs 11-14) and N-terminal (7/10 in SCRs 1-4) regions of CFH. Patients with anti-CFH autoantibodies had a significantly lower prevalence of acute kidney injury (0% (0/10) vs. 40.0%(4/10), P = 0.025), lower serum creatinine levels (0.76 (0.40, 1.06) vs. 1.43 (0.46, 11.15), mg/dL, P = 0.023), and higher hemoglobin levels (113.8 ± 24.63 vs. 90.0 ± 22.53, g/L, P = 0.037) than those who were negative after further stratified analysis. A functional study showed that anti-CFH autoantibodies purified from patients with lupus nephritis could improve the binding between CFH and C3b, and also enhance the cofactor activity of CFH in vitro. CONCLUSIONS: Anti-CFH autoantibodies were detected in patients with lupus nephritis in approximately 10% of patients with polyepitopes and IgG2 subclass predominance. Patients with anti-CFH autoantibodies presented with milder renal damage, and the purified autoantibodies could enhance the C3b binding and CFI cofactor activity of CFH in vitro, which suggested a protective role in the lupus nephritis.

Effect of complement Factor H on antibody repertoire and protection elicited by meningococcal capsular group B vaccines containing Factor H binding protein.

Bacteria produce surface ligands for host complement regulators including Factor H (FH), which allows the bacteria to evade immunity. Meningococcal Factor H binding protein (FHbp) is both a virulence factor and a vaccine antigen. Antibodies to FHbp can neutralize its function by inhibiting binding of FH to the bacteria and confer robust complement-mediated protection. However, in the presence of human or primate FH, antibodies to FHbp do not inhibit FH binding and the protective antibody responses are decreased. This immune suppression can be overcome by modification of the FHbp antigen to decrease FH binding, which modulates the antibody repertoire to inhibit FH binding and increase protection. When FHbp is present at sufficient density on the bacterial surface, two or more antibodies can synergize to activate the complement system. Thus, modification of FHbp antigens to decrease FH binding expands the anti-FHbp antibody repertoire and increases the potential for synergistic activity.

Structural Basis for Neutralization and Protection by a Zika Virus-Specific Human Antibody.

We previously reported a human monoclonal antibody, ZK2B10, capable of protection against Zika virus (ZIKV) infection and microcephaly in developing mouse embryos. Here, we report the structural features and mechanism of action of ZK2B10. The crystal structure at a resolution of 2.32 Å revealed that the epitope is located on the lateral ridge of DIII of the envelope glycoprotein. Cryo-EM structure with mature ZIKV showed that the antibody binds to DIIIs around the icosahedral 2-fold, 3-fold, and 5-fold axes, a distinct feature compared to those reported for DIII-specific antibodies. The binding of ZK2B10 to ZIKV has no detectable effect on viral attachment to target cells or on conformational changes of the E glycoprotein in the acidic environment, suggesting that ZK2B10 functions at steps between the formation of the fusion intermediate and membrane fusion. These results provide structural and mechanistic insights into how ZK2B10 mediates protection against ZIKV infection.

Protective antibody response of Balb/c mice to Bali rabies virus isolate propagated in BHK-21 cells.

The protective antibody response of Balb/c mice to Bali rabies virus (RABV) in BHK-21 cells was studied. The virus was isolated from a rabid dog and was adapted to replicate in BHK-21 cell culture for seven passages. The BHK-21-adapted Bali RABV (BHK-Bali RABV) was inactivated with binary ethylenimine and 24 mice were immunized twice at 21-days intervals with the inactivated virus and Rabisin® vaccine. Virus replication was detected using indirect immunofluorescence, immunocytochemistry, and western blotting assays. Enzyme-linked immunosorbent assay examination 2 weeks after the first immunization revealed RABV antibody titers that were mostly below the minimum protective level (<0.5 equivalent unit, EU). Antibody titers increased sharply after the second immunization. Antibody titers in serum of mice induced by inactivated BHK-Bali RABV one week after the second immunization were slightly lower (0.8-3.8 EU) than those induced by Rabisin vaccine (0.9-6.3 EU). RABV antibody titers were stable for at least 6 weeks after the second immunization. Both Rabisin vaccine and inactivated BHK-Bali RABV induced neutralizing antibodies with neutralization titers (50% protective dose per ml) of 29.84 for 0.1 ml Rabisin, 211.41 for 0.2 ml Rabisin, 27.41 for 0.1 ml BHK-Bali RABV, and 28.25 for 0.2 ml BHK-Bali RABV. Thus, inactivated BHK-Bali RABV induces a protective immune response in Balb/c mice, but at lower levels compared to induction by Rabisin vaccine.

A virus-like particle vaccination strategy expands its tolerance to H3N2 antigenic drift by enhancing neutralizing antibodies against hemagglutinin stalk.

Seasonal influenza viruses impact public health annually due to their continual evolution. However, the current inactivated seasonal vaccines provide poor protection against antigenically drifted viruses and require periodical reformulation through hit-and-miss predictions about which strains will circulate during the next season. To reduce the impact caused by vaccine mismatch, we investigated the drift-tolerance of virus-like particles (VLP) as an improved vaccine candidate. The cross-protective humoral immunity elicited by the H3N2-VLP vaccine constructed for the 2011-2012 season was examined against viruses isolated from 2010 to 2015 in Taiwan evolving chronologically through clades 1, 4, 5, 3B and 3C, as well as viruses that were circulating globally in 2005, 2007 and 2009. Mouse immunization results demonstrated that H3N2-VLP vaccine elicited superior immunological breadth in comparison with the cognate conventional whole-inactivated virus (WIV) vaccine. Titers of neutralizing antibodies against heterologous strains representing each epidemic period in the VLP group were significantly higher than in the WIV group, indicating the antibody repertoire induced by the H3N2-VLPs was insensitive to viral antigenic drift over a span of at least 10 years. Noticeably, H3N2-VLP elicited higher levels of anti-stalk antibodies than H3N2-WIV, which offset the ineffectiveness caused by antigenic drift. This advantageous effect was attributed to the uncleaved precursor of their HA proteins. These results suggest a mechanism through which VLP-induced humoral immunity may better tolerate the evolutionary dynamics of influenza viruses and point to the possible use of a VLP vaccine as a method by which the requirement for annual updates of seasonal influenza vaccines may be diminished.

Evaluation of the immunogenicity of the 13-valent conjugated pneumococcal vaccine in rheumatoid arthritis patients treated with etanercept.

OBJECTIVES: To prospectively evaluate the immunogenicity of a 13-valent conjugated pneumococcal vaccine (PCV13) in rheumatoid arthritis (RA) patients undergoing etanercept therapy. METHODS: Twenty-two RA patients treated with etanercept (ETA) in combination with methotrexate (MTX) (n=15) or monotherapy (n=7) for at least one year were included. Altogether 24 osteoarthritis patients not receiving biological or MTX therapy, treating only NSAIDs or analgesics served as controls. All subjects were vaccinated with a single dose (0.5ml) of the PCV13. Pneumococcal antibody levels at baseline, 4 and 8weeks were assessed by a VaccZyme™ Anti-PCP IgG Enzyme Immunoassay Kit. Based on recommendations of the American Academy of Allergy, Asthma & Immunology, an at least two-fold increase in antibody level, as the protective antibody response (pAR) was an indicator of responsiveness (i.e., ratio of postvaccination and prevaccination antibody levels). The antibody levels and their ratios were analysed in a variety of different ways, vaccine safety parameters (fever, infections, changes in regular antirheumatic treatments) were assessed at baseline, 4 and 8weeks after vaccination. RESULTS: Four weeks after vaccination, the anti-pneumococcal antibody levels significantly increased in both groups. At week 8, antibody levels somewhat decreased in both groups, however, still remained significantly higher compared to baseline. Compared with postvaccination levels at 4 and 8weeks between two groups, the mean protective antibody levels were higher in control group (1st month P=0.016; 2nd month: P=0.039). Possible predictors of pAR were analysed by logistic regression model. In RA, increases of antibody levels at week 8 compared to baseline exerted a negative correlation with age, (Spearman's R=-0,431; P=0.045). There were no clinically significant side effects or reaction after administration of vaccine observed in any of these patients after the 2-month follow-up period, all patients medical condition were stable. CONCLUSIONS: In RA patients treated with ETA, vaccination with PCV13 is effective and safe, resulting in pAR one and two months after vaccination. Higher age at vaccination was identified as predictors of impaired pAR. The efficacy of vaccination may be more pronounced in younger RA patients. The vaccine is safe in RA patients on ETA.

Ginsenoside Rd induces protective anti-Candida albicans antibody through immunological adjuvant activity.

The role of an antibody against candidiasis is controversial. However, a certain Candida albicans surface epitope produces a protective antibody. Yet, its isolation is difficult. In this study, we investigated if ginsenoside Rd from Panax ginseng has an immunoadjuvant ability to induce surface mannan extract (CASM) to produce a protective antibody. Mice were immunized twice i.p. with an emulsion form of CASM mixed with one of the following: IFA [CASM/IFA], or CFA [CASM/CFA] or Rd with IFA [CASM/Rd/IFA]. One week after the booster, these mice were challenged i.v. with live C. albicans and their survivability was measured. Results showed that four of five CASM/Rd/IFA-vaccinated mice survived during the entire 110 day-observation period, whereas CASM/IFA- or CASM/CFA-vaccinated mice died within 19 and 23 days (P<0.05). The antiserum from CASM/Rd/IFA-immunized mice transferred the protection to naïve mice, whereas antiserum from CASM/CFA-given mice was not protective although CASM/CFA induced an antibody four times greater than CASM/Rd/IFA. IgG isotyping revealed that CASM/Rd/IFA-vaccine produced the most abundant IgG and IgG2a-resulting in the highest ratio (1.32) of IgG2a to IgG, which is helpful in treating Th2-oriented candidiasis. In contrast, the formulae lacking Rd had these ratios less than 1. This strongly indicates that Rd could enhance Th1 immunity. Cytokine profiles and DTH further confirmed the Th1 dominance. Rd caused no hemolysis. Combining all of these data together, Rd can enhance Th1-response to CASM in mice. This protects mice against disseminated candidiasis by eliciting higher titers of Th1 type antibody and a Th1-dominant immune response.