A live-attenuated viral vector vaccine protects mice against lethal challenge with Kyasanur Forest disease virus

Kyasanur Forest disease virus (KFDV) is a tick-borne flavivirus endemic in India known to cause severe hemorrhagic and encephalitic disease in humans. In recent years, KFDV has spread beyond its original endemic zone raising public health concerns. Currently, there is no treatment available for KFDV but a vaccine with limited efficacy is used in India. Here, we generated two new KFDV vaccine candidates based on the vesicular stomatitis virus (VSV) platform. We chose the VSV-Ebola virus (VSV-EBOV) vector either with the full-length or a truncated EBOV glycoprotein as the vehicle to express the precursor membrane (prM) and envelope (E) proteins of KFDV (VSV-KFDV). For efficacy testing, we established a mouse disease model by comparing KFDV infections in three immunocompetent mouse strains (BALB/c, C57Bl/6, and CD1). Both vaccine vectors provided promising protection against lethal KFDV challenge in the BALB/c model following prime-only prime-boost and immunizations. Only prime-boost immunization with VSV-KFDV expressing full-length EBOV GP resulted in uniform protection. Hyperimmune serum derived from prime-boost immunized mice protected naïve BALB/c mice from lethal KFDV challenge indicating the importance of antibodies for protection. The new VSV-KFDV vectors are promising vaccine candidates to combat an emerging, neglected public health problem in a densely populated part of the world.

Development of an indirect ELISA for detection of anti-Mycoplasma hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera

Background Immunization of pigs with an inactivated Mycoplasma hyopneumoniae vaccine (bacterin) generates hyperimmune serum that contains high concentrations of anti-M. hyopneumoniae IgG. Commercially available IgG-ELISA kits cannot distinguish between anti-M. hyopneumoniae IgG in inactivated bacterin-induced hyperimmune sera and convalescent sera resulting from natural M. hyopneumoniae infection. Establishment of an ELISA to detect anti-M. hyopneumoniae IgG in convalescent sera will facilitate the evaluation of the M. hyopneumoniae status of pig farms. Results In this study, we expressed and purified recombinant Mhp366-N protein, which contains an epitope recognized by M. hyopneumoniae convalescent sera but not hyperimmune sera, for use as a coating antigen. For the M. hyopneumoniae convalescent serum IgG-ELISA, the optimal antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time and colorimetric reaction time were 0.25 µg/mL, 2.5 % skim milk, 1 h, 1:500, 0.5 h, 1:10,000, 1 h and 15 min, respectively. Validation of the M. hyopneumoniae convalescent serum IgG-ELISA showed a cut-off value of 0.323, the intra-assay CV ranged from 3.27 to 7.26 %, the inter-assay CV ranged from 3.46 to 5.93 %, and the assay was able to differentiate convalescent sera from antibodies to 7 other porcine respiratory pathogens. The convalescent serum IgG-ELISA detected no anti-M. hyopneumoniae IgG in hyperimmune serum samples while a commercial IgG-ELISA identified 95/145 of these sera as positive. The accuracy of the M. hyopneumoniae convalescent serum IgG-ELISA was comparable to the sIgA-ELISA but better than the commercial IgG-ELISA. Conclusions The convalescent serum IgG-ELISA is a reproducible, sensitive, and specific indirect ELISA to detect anti-M. hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera. This ELISA could be used to carry out large scale surveillance of M. hyopneumoniae infection in pig farms regardless of vaccination status.

Compassionate use of rectal Ozone (O3) in severe COVID-19 pneumonia: a case-control study.

Objectives: To evaluate effect of rectal Ozone in severe COVID-19 pneumonia and to compare to Standard-of-care (SOC). Material and Methods: In a case-control study, 14 patients with severe bilateral COVID-19 pneumonia (positive RT-PCR), treated with SOC and rectal Ozone, were evaluated before-and-after treatment and compared with SOC (14 patients) in a 10 day follow-up period. Ozone-protocol consisted of 8 sessions (1 session/day) of intra-rectal Ozone, (150mL volume, 35mg/ml concentration [5.25mg total dose]). The SOC-protocol included O 2- supply, antivirals (Remdesivir), corticosteroids (Dexamethasone/Metilprednisolone), monoclonal antibodies (Anakinra/Tocilizumab), antibiotics (Azytromicine), anticoagulants (Enoxaparine) and hyperimmune serum (if necessary). Primary outcome variables: a) clinical (O 2- saturation and O 2- supply); b) biochemical (Lymphocyte count, Fibrinogen, D-Dimer, Urea, Ferritin, LDH, IL-6 and CRP); c) radiological Taylor Scale. Secondary outcome variables: a) hospitalization length-of-stay, b) mortality-rate. Results: At baseline, Ozone/SOC-groups were not different on age, comorbidities, O 2 -saturation and O 2 -supply. Patients in Ozone-Group improved O 2- saturation and decrease O 2- supply. SOC maintained O 2- saturation and required more O 2- supply. Lymphocyte-count improved only in Ozone-group and with statistical difference (p<0.05). Biomarkers of inflammation (Fibrinogen, D-Dimer, Urea, LDH, CRP and IL-6) decreased in both groups, but only significantly in favor of Ozone-group (p<0.05). Ferritin showed a significant decrease in the Ozone-group but an increase on the SOC-Group. Radiological pneumonitis decreased on both groups but the decrease was only significant in the Ozone-Group (p<0.0001). Mortality and length-of-stay, although not significant, were inferior in Ozone-Group. Conclusion: Compassionate use of Rectal Ozone improved O 2 -saturation, reduced O 2 -supply, decreased inflammation biomarkers and improved Taylor’s radiological scale significantly when compared to SOC-Group. Mortality and length-of-stay was inferior in the Ozone-group, but this difference was not significant.

The Road towards Polyclonal Anti-SARS-CoV-2 Immunoglobulins (Hyperimmune Serum) for Passive Immunization in COVID-19

Effective treatments specific for COVID-19 are still lacking. In the setting of passive immunotherapies based on neutralizing antibodies (nAbs), randomized controlled trials of COVID-19 convalescent plasma (CCP) anti-SARS-CoV-2 Spike protein monoclonal antibodies (mAb), which have been granted emergency use authorization, have suggested benefit in early disease course (less than 72 hours from symptoms and seronegative). Meanwhile, polyclonal immunoglobulins (i.e., hyperimmune serum), derived either from CCP donations or from animals immunized with SARS-CoV-2 antigens, are likely to become the next nAb-derived candidate. We here discuss the pros and cons of hyperimmune serum versus CCP and mAb, and summarize the ongoing clinical trials of COVID-19 hyperimmune sera.

Natural Infection of Goats with Orf (Contagious ecthyma) and Its Diagnosis

Background: The Study was intended to evaluate some common diagnostics that could supplement the clinical and histological identification of orf in goats.Methods: Samples from suspected clinical cases of orf (contagious ecthyma) were collected from various organized and unorganized goat herds around Guwahati, Assam. Presumptive diagnosis was based on the typical signs and lesions. For confirmatory diagnosis, various molecular and immunoassays were employed for the detection of orf virus or circulating antibodies.Result: Cutaneous lesions observed included solitary to multifocal erythema, papules, vesicles, pustules and scab stages on the lips, ears, gums, tongue, udder and perineal region. No mortality was observed and the morbidity rate varied between 35-60%. Microscopic lesions in skin biopsies were typically marked by epidermal hyperplasia and ballooning degeneration of the keratinocytes along with other changes. Eosinophilic intracytoplasmic inclusion bodies were demonstrable within keratinocytes only in early papulo-vesicular stages. Dermis was infiltrated with polymorphonuclear and mononuclear cells in varying proportions. Initial screening of samples was done with PCR systems to specifically detect parapoxvirus DNA employing a semi-nested PCR targeted against the partial B2L gene (with reported primers yielding 594 bp and 235 bp amplified products) and an uniplex PCR targeting the whole B2L gene (with reported primer set to yield an amplified product of 1206 bp). Agar gel immuno-diffusion (AGID) failed to develop positive immunoprecipitation with hyperimmune serum against scab lysates; however, counter-immuno-electrophoresis (CIE) showed positive immunoprecipitation in the same samples. For rapid and in situ detection of ORFV antigen in tissues, immunofluorescent and immunoperoxidase techniques were successfully employed both on cryosections and formalin fixed skin biopsies. Immunofluorescent technique on cryosections was found to be easy, rapid and more specific. A dot-ELISA was also developed for successful confirmation of orf virus from clinical samples. For antibody detection in convalescing goats, an indirect-ELISA and a dot-ELISA was also successfully tested to demonstrate for antibody detection in convalescing goats, but protective titres later after infection was not addressed. Monoclonal antibody employed in the assays was found to be specific, sensitive and versatile for virus detection from direct clinical samples. It is contemplated that assays employing hyperimmune sera or detection of circulating antibody against orf virus may have limited diagnostic applications owing to its partial and transient humoral immunity and the inherent property of the virus to modulate and interfere with the host response and evade immune mechanisms.

Selection and characterization of peptides mimetic to Campylobacter fetus subsp. venerealis using phage display

ABSTRACT: Bovine genital campylobacteriosis (BGC) is a venereal and subclinical disease that affects the fertility of cattle herds, and it is caused by Campylobacter fetus subsp. venerealis . This study selected peptides mimetic to the BGC-causing agent from a phage library. Phage display is a technique that applies bacteriophage libraries that reveal peptides fused to the viral capsid in biological selections against target proteins. Biopannings were performed for biological selection in the phage library using rabbit hyperimmune serum and C. fetus subsp. venerealis protein extract. Five selected heptapeptides were considered mimetic to Cfv-NCTC 10354 based on the results of bioinformatics analysis and assays with hyperimmune serum and cervicovaginal mucus obtained from heifers. ALASLPL and LSYLFPP were the most reactive peptides and considered promising as possible mimetic immunogens for C. fetus subsp. venerealis.

Porcine Reproductive and Respiratory Syndrome Virus Promotes SLA-DR-Mediated Antigen Presentation of Nonstructural Proteins To Evoke a Nonneutralizing Antibody Response In Vivo

ABSTRACT The humoral immune response against porcine reproductive and respiratory syndrome virus (PRRSV) infection is characterized by a rapid induction of nonneutralizing antibodies (non-NAbs) against nonstructural proteins (NSPs). Here, we systematically investigated the potential mechanism for the induction of PRRSV NSP-specific non-NAbs. Our data suggested that PRRSV NSP-specific antibodies appeared within 10 days after PRRSV infection in vivo. In the in vitro model, functional upregulation of swine leukocyte antigen (SLA)-DR was observed in bone marrow-derived dendritic cells (BMDCs) and porcine alveolar macrophages (PAMs), whereas remarkable inhibition at the mRNA level was observed after infection by both PRRSV-1 and PRRSV-2 isolates. Notably, the inconsistency in SLA-DR expression between the mRNA and protein levels resulted from deubiquitination of SLA-DR via the ovarian tumor (OTU) domain of PRRSV NSP2, which inhibited ubiquitin-mediated degradation. Moreover, mass spectrometry-based immunopeptidome analysis identified immunopeptides originating from multiple PRRSV NSPs within SLA-DR of PRRSV-infected BMDCs. Meanwhile, these PRRSV NSP-derived immunopeptides could be specifically recognized by serum from PRRSV-infected piglets. Notably, certain NSP-derived immunopeptides characterized in vitro could be identified from PAMs or hilar lymph nodes from PRRSV-infected piglets. More importantly, an in vitro neutralizing assay indicated that serum antibodies against NSP immunopeptides were unable to neutralize PRRSV in vitro. Conversely, certain structural protein (SP)-derived immunopeptides were identified and could be recognize by pig hyperimmune serum against PRRSV, which further indicates that the NSP-derived antibody response is nonprotective in vivo. In conclusion, our data suggested that PRRSV infection interferes with major histocompatibility complex class II (MHC-II) molecule-mediated antigen presentation in antigen-presenting cells (APCs) via promoting SLA-DR expression to present immunopeptides from PRRSV NSPs, which contributes to the induction of non-NAbs in vivo. IMPORTANCE PRRSV has haunted the swine industry for over 30 years since its emergence. Besides the limited efficacy of PRRSV modified live vaccines (MLVs) against heterogeneous PRRSV isolates, rapid induction of nonneutralizing antibodies (non-NAbs) against PRRSV NSPs after MLV immunization or wild-strain infection is one of the reasons why development of an effective vaccine has been hampered. By using in vitro-generated BMDCs as models to understand the antigen presentation process of PRRSV, we obtained data indicating that PRRSV infection of BMDCs promotes functional SLA-DR upregulation to present PRRSV NSP-derived immunopeptides for evoking a non-NAb response in vivo. Our work not only uncovered a novel mechanism for interference in host antigen presentation by PRRSV but also revealed a novel insight for understanding the rapid production of nonneutralizing antibodies against PRRSV NSPs, which may have benefit for developing an effective vaccine against PRRSV in the future.

Psp140: an immunodominant antigen in the supernatant of Streptococcus pneumoniae culture

Background and Objectives: Streptococcus pneumoniae causes many lethal infections. Due to its reduced sensitivity to commonly used antibiotics, development of new strategies against pneumococcal infections seems to be necessary. We aimed to investigate immunodominant antigens in S. pneumoniae culture supernatant in order to develop novel targets for pneumococcal vaccines. Materials and Methods: In this study S. pneumoniae ATCC49619 was sub-cultured into BHI broth from overnight culture at 37°C for 4 h. The supernatant proteins were precipitated using acetone precipitation method. A rabbit was intramuscularly immunized with alum adjuvant and 100 μg pneumococcal supernatant proteins, 6 times at 14 days' intervals to produce hyperimmune serum. ELISA assay was performed to determine the antibody level response to pneumococcal secretory proteins. Then dot blot applied for rapid evaluation of hyperimmune serum reactivity to pneumococcus supernatant proteins. The western blot was also used to determine the interaction of supernatant proteins with immunogenic rabbit's hyperimmune-serum. Results: According to the western blot analysis, the immunodominant protein had 140KDa molecular weight and designated as pneumococcal secretory protein140 (Psp140). Conclusion: The Psp140 protein in the supernatant of S. pneumoniae culture is an immunodominant protein and it is likely related to pneumococcal secretory protein or surface exposed protein which released into culture supernatant during bacterial growth.

Current research into snake antivenoms, their mechanisms of action and applications

Snakebite is a major public health issue in the rural tropics. Antivenom is the only specific treatment currently available. We review the history, mechanism of action and current developments in snake antivenoms. In the late nineteenth century, snake antivenoms were first developed by raising hyperimmune serum in animals, such as horses, against snake venoms. Hyperimmune serum was then purified to produce whole immunoglobulin G (IgG) antivenoms. IgG was then fractionated to produce F(ab) and F(ab′)2 antivenoms to reduce adverse reactions and increase efficacy. Current commercial antivenoms are polyclonal mixtures of antibodies or their fractions raised against all toxin antigens in a venom(s), irrespective of clinical importance. Over the last few decades there have been small incremental improvements in antivenoms, to make them safer and more effective. A number of recent developments in biotechnology and toxinology have contributed to this. Proteomics and transcriptomics have been applied to venom toxin composition (venomics), improving our understanding of medically important toxins. In addition, it has become possible to identify toxins that contain epitopes recognized by antivenom molecules (antivenomics). Integration of the toxinological profile of a venom and its composition to identify medically relevant toxins improved this. Furthermore, camelid, humanized and fully human monoclonal antibodies and their fractions, as well as enzyme inhibitors have been experimentally developed against venom toxins. Translation of such technology into commercial antivenoms requires overcoming the high costs, limited knowledge of venom and antivenom pharmacology, and lack of reliable animal models. Addressing such should be the focus of antivenom research.